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Originally published In Press as doi:10.1074/jbc.M310078200 on October 21, 2003

J. Biol. Chem., Vol. 279, Issue 1, 142-151, January 2, 2004
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C5a Mutants Are Potent Antagonists of the C5a Receptor (CD88) and of C5L2

POSITION 69 IS THE LOCUS THAT DETERMINES AGONISM OR ANTAGONISM*

Magnus Otto{ddagger}§, Heiko Hawlisch§, Peter N. Monk||, Melanie Müller{ddagger}, Andreas Klos{ddagger}, Christopher L. Karp¶, and Jörg Köhl¶**

From the {ddagger}Institute of Medical Microbiology, Medical School Hannover, 30625 Hannover, Germany, the Division of Molecular Immunology, Cincinnati Children's Hospital Research Foundation, Cincinnati, Ohio 45229, and the ||Department of Neurology, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, United Kingdom

Received for publication, September 10, 2003 , and in revised form, October 15, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The anaphylatoxin C5a exerts a plethora of biologic activities critical in the pathogenesis of systemic inflammatory diseases. Recently, we reported on a C5a mutant, jun/fos-A8, as a potent antagonist for the human and mouse C5a receptor (CD88). Addressing the molecular mechanism accounting for CD88 receptor antagonism by site-directed mutagenesis, we found that a positively charged amino acid at position 69 is crucial. Replacements by either hydrophobic or negatively charged amino acids switched the CD88 antagonist jun/fos-A8 to a CD88 agonist. In addition to CD88, the seven-transmembrane receptor C5L2 has recently been found to provide high affinity binding sites for C5a and its desarginated form, C5adesArg74. A jun/fos-A8 mutant in which the jun/ fos moieties and amino acids at positions 71–73 were deleted, A8{Delta}71–73, blocked C5a and C5adesArg74 binding to CD88 and C5L2. In contrast, the cyclic C5a C-terminal analog peptide AcF-[OP-D-ChaWR] inhibited binding of the two anaphylatoxins to CD88 but not to C5L2, suggesting that the C5a core segment is important for high affinity binding to C5L2. Both receptors are coexpressed on human monocytes and the human mast cell line HMC-1; however, C5L2 expression on monocytes is weaker as compared with HMC-1 cells and highly variable. In contrast, no C5L2 expression was found on human neutrophils. A8{Delta}71–73 is the first antagonist that blocks C5a and C5adesArg74 binding to both C5a receptors, CD88 and C5L2, making it a valuable tool for studying C5L2 functions and for blocking the biological activities of C5a and C5adesArg74 in mice and humans.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The anaphylatoxin C5a is generated by proteolytic cleavage of the fifth component of complement (C5). C5a and its degradation product, C5adesArg74, exert their various biological activities through interaction with all classes of leukocytes (1), parenchymal cells of the brain (2), kidney (3, 4), liver (5), and lung (5), as well as with endothelial cells (6) and smooth muscle cells (7). C5adesArg74 has a different spectrum of bioactivity than C5a; for example, stimulation of human basophils by C5a mediates secretion of leukotriene C4, interleukin-4, and interleukin-13, whereas C5adesArg74 only stimulates the production of interleukin-4 and interleukin-13. Interestingly, C5adesArg74 is a superagonist for interleukin-13 release (8).

Until recently, only one C5a receptor (C5aR)1 had been described (CD88) (9, 10). CD88 belongs to the large group of seven-transmembrane receptors that signal through both G protein-dependent and -independent pathways (11). CD88 is able to bind two different classes of G proteins: the pertussis toxin-sensitive G{alpha}i and the pertussis toxin-insensitive G{alpha}16 subunits. Many but not all of the proinflammatory properties of C5a are sensitive to pertussis toxin treatment (1). Genetic deletion of CD88 protects mice from development of immune complex diseases such as rheumatoid arthritis (12) and immune complex alveolitis (13), as well as from delayed type hypersensitivity reactions (14). Interestingly, CD88 deficiency failed to protect against experimental allergic encephalomyelitis (15), suggesting that in addition to CD88, C5a may signal through other receptors.

Recently, a second C5aR was cloned, C5L2, which also belongs to the family of seven-transmembrane receptors (16, 17). In contrast to CD88, C5L2 binds both C5a and C5adesArg74 with high affinity, is uncoupled from G proteins, and lacks receptor internalization (18). These data were confirmed in a recent publication (19). Tissue expression of mRNA for human (16, 17, 19) and mouse (19) C5L2 is broad and corresponds well to the expression of CD88. The missing G protein coupling of C5L2 suggests that this receptor may signal through G protein-independent pathways (11).

C5aR antagonists (C5aRA) that block the interaction of C5a and C5adesArg with CD88 and C5L2 may prove useful to prevent deleterious biologic activities of C5a without broadly impairing the complement system. Several antagonists for CD88 have been described, including peptides (20–22), a nonpeptidic compound (23), C5a mutants (24–26), and anti-C5aR antibodies (27, 28). All of these compounds are potent CD88 antagonists in vitro; however, only the C5a mutants C5aRAM (24) and jun/fos-A8 (25), the cyclic peptide AcPhe[L-ornithine-Pro-D-cyclohexylalanine-Trp-Arg] (AcF-(OpdChaWR)) (29), and a nonpeptidic antagonist (23) have been proven useful in vivo. Administration of these inhibitors protects animals from inflammatory responses in immune complex disease (25, 29, 30), ischemia/reperfusion injury (25, 31, 32), and C5a-induced neutropenia (23, 24). The molecular mechanisms underlying the C5aR antagonism are largely unknown. All of these molecules are potent C5aRA for human CD88; however, only the C5a mutant jun/fos-A8 is also a potent antagonist for mouse, rat, and guinea pig CD88. C5aRAM acts as an antagonist for rabbit and micropig CD88 (24), AcF-(OpdChaWR) acts as an antagonist for rat and dog CD88 (29), and the nonpeptidic compound acts as an antagonist for gerbil and cynomolgus monkey CD88 (23).

The ability of these CD88 antagonists to interact with C5L2 remains unknown. The CD88 antagonist jun/fos-A8 was selected from a phage library in which the C terminus of hC5a had been randomly mutated (25). This C5aRA has been widely used in vitro (25, 33) and in vivo (25, 30, 32–35). Six positions are mutated in jun/fos-A8 as compared with C5a: C27A, H67F, D69R, M70S, Q71L, and G73R. In addition, Arg74 is deleted. Further, parts of the leucine zipper of transcription factor fos are fused N-terminally to A8, forming a heterodimer with parts of the leucine zipper of transcription factor jun, resulting in jun/fos-C5aC27A-(1–66)-FKRSLLR.

We assessed the molecular mechanism that renders jun/fos-A8 a C5aRA by site-directed mutagenesis. Our results provide evidence that position 69 is crucial for CD88 antagonism, whereas both the N-terminal jun/fos moieties and the three C-terminal residues 71–73 are not. Further, we demonstrate that jun/fos-A8 and the deletion mutant A8{Delta}71–73 are potent inhibitors of C5a and C5adesArg74 binding to C5L2, whereas the peptidic CD88 antagonist AcF-(OPdChaWR) is not. In addition, we present data that CD88 and C5L2 are coexpressed on human monocytes and the human mast cell line HMC-1 but that PMN exclusively express CD88.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Generation of C5a Mutants—The C5a mutants depicted in Table I were constructed by PCR using the pJuFo-{Delta} pIII-A8 mutant as template (25). To obtain fos-C5aC27A-(1–66)-FKRSLLR (fos-A8), the jun cassette was deleted by digesting the vector with XhoI and BamHI with 15 units/µg DNA at 37 °C for 1 h. The digested, linearized vector fragment was run on an agarose gel, and a band at the expected size was cut out and purified using glass milk beads. The purified fragment was religated. The 50-µl ligation reaction included 200 ng of digested vector and was incubated overnight at 15 °C with 640 units of T4-DNA ligase (New England Biolabs) to yield the new vector fragment, pFo-{Delta}pIII-A8. The deletion was confirmed by DNA sequencing.


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  		TABLE I
Primer sequences that were used to construct the different A8 mutants

 
To obtain C5aC27A-(1–66)-FKRSLLR (A8), the fos cassette was deleted by digesting the pFo-{Delta}pIII-A8 vector with SacI and KpnI with 15 units/µg DNA at 37 °C for 1 h. This vector (p{Delta}JuFo-{Delta}pIII-A8=pA8B) was then used as a template to construct all other C5a mutants. The primer combinations depicted in Table I were used to generate the different A8 mutants.

PCRs were performed in 100-µl volumes containing 10 pg of template DNA pA8B, 200 µM of each dNTP, 20 pmol of each primer, and reaction buffer supplied by the manufacturer (Stratagene). The reaction mixtures were overlaid with mineral oil and kept at 94 °C for 5 min (hot start) in a thermocycler. After adding 5 units of Pfu polymerase (Stratagene), the mixtures were cycled 30 times (94 °C for 90 s, 52 °C for 120 s, and 72 °C for 120 s), followed by incubation at 72 °C for 10 min.

After phenol chloroform extraction and ethanol precipitation, the DNA fragments were digested with SacI and KpnI (New England Biolabs) using 60 units/µg DNA at 37 °C for 1 h. The ligation mix was heat inactivated at 70 °C for 30 min, purified by phenol chloroform extraction and ethanol precipitation, followed by resuspension in 5 µl of TE (10 mM Tris, 1 mM EDTA). Two portions of 2.5 µl each were electroporated into Escherichia coli TG1 cells (Stratagene) using a Bio-Rad pulser set at 25 microfarads, 2.5 kV, and 200 {Omega}. Immediately after the pulse, 1 ml of freshly prepared SOC medium (0.5% yeast extract, 2% Tryptone, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 20 mM MgSO4, 20 mM glucose) was added, and the cells were grown for 1 h at 37 °C and plated on TYE plates (15 g/liter Bacto agar, 10 g/liter Tryptone, 10 g/liter yeast extract, 5 g/liter NaCl) supplemented with 100 µg/ml ampicillin and 1% glucose (TYE+amp+gluc) as described (26). To score the total number of independent transformants, 100 µl of appropriate dilutions were plated onto TYE+amp+gluc plates.

Purification of C5a Mutants by Immune Affinity Chromatography— TG1 bacteria transfected with pA8 were grown in 2x TY+amp medium containing 100 µg/ml ampicillin and 0.1% glucose. At an A600 of 0.9, isopropyl-{beta}-D-thiogalactopyranoside 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 as described (26). CNBr-activated Sepharose (Amersham Biosciences) was coated with C5a-specific mAb 561 (36) according to the manufacturer's instructions. A polyethylene-column (Biognosis) was loaded with 100 µl of gel matrix. The periplasmic fraction (400 µl diluted 1:5 with PBS) prepared from TG1 cells transformed with one of the C5a mutants was applied to the column for 1 h at 4 °C. The column was washed with 2 ml of PBS and subsequently loaded with a pre-elution buffer (10 mM phosphate buffer, pH 6.8). Shifting the pH to 2.5 using 100 mM glycin/HCl buffer eluted the bound C5a mutant. Ten 50-µl aliquots were collected and subsequently tested in a dot-blot with mAb 561. Positive fractions were pooled. The concentration of purified C5a mutants was determined by enzyme-linked immunosorbent assay. Purified C5a mutants appeared as a single band after separation on 15% SDS-PAGE and subsequent silver staining (data not shown).

Enzyme-linked Immunosorbent Assay to Determine the Concentration of Purified C5a Mutants—The enzyme-linked immunosorbent assay was performed as described previously (36). Briefly, anti-hC5a-specific mAb 561 was diluted to 10 µg/ml in PBS and was coated overnight at 22 °C to a polystyrene microtiter plate (Greiner). Then the plates were rinsed three times in PBS and saturated with PBS containing 2% non-fat dry milk for 60 min at 37 °C. After washing three times with 50 mM Tris buffer, 0.15 M NaCl, pH 7.5 (Tris), 50 µl of the purified C5a mutants diluted in Tris supplemented with 10% non-fat dry milk powder (Tris-milk) were added. After incubation for 90 min at 22 °C, the plates were washed four times with Tris and subsequently incubated with 50 µl of biotinylated anti C5a mAb 557 (10 µg/ml) diluted in Tris-milk for 90 min at 22 °C. The plates were washed four times with 50 mM Tris. Subsequently, 50 µl of avidin-alkaline-phosphatase diluted in Tris was added and incubated for 30 min at 22 °C. The plates were developed with p-nitrophenolphosphate (1 mg/ml diluted in diethanolamine, 5 mM MgCl2, pH 9).

Competitive Binding Study—Competitive binding studies were performed as described previously (25). Briefly, 40 µl of a reaction mixture containing a constant amount of 125I-rhC5a as tracer (0.1 nM; {approx}17,000 cpm) and increasing concentrations of unlabeled C5a mutants were incubated in a microtiter plate on ice. The binding reaction was started by adding 25 µl of ice-cold CD88-transfected RBL-2H3 (CD88-RBL) cell suspension (37). After incubation on ice for 30 min, cell-bound and free 125I-rhC5a were separated by filtration of 60 µl through a microtitration membrane plate (MultiscreenTM HV; 0.45 µm, Millipore) using a vacuum manifold (Millipore). The wells were washed out once with 100 µl of HAG-CM buffer (20 mM HEPES, pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 0.25% bovine serum albumin, and 0.5 mM glucose), dried by a heat lamp, and punched out with a membrane punch assembly. The punched out membranes were counted in a {gamma}-counter (Packard). The plot of cell-bound 125I-rhC5a versus the concentration of unlabeled competitor yielded the half-maximal inhibitory concentrations (IC50).

Cellular Activation and Inhibition Assays—The release of N-acetyl-{beta}-D-glucosaminidase from CD88-transfected RBL cells was determined as described (26). The ED50 of rhC5a (Sigma-Aldrich) and C5a mutants was determined as the concentration leading to half-maximal enzyme release. The antagonistic potency of the different C5aRAs was tested as their ability to inhibit the release of N-acetyl-{beta}-D-glucosaminidase from CD88-transfected RBL cells induced by 10–9 M C5a. Briefly, 75 µl/well of CD88-transfected RBL cells (2 x 106/ml) were suspended in HAG-CM buffer and equilibrated for 5 min at 37 °C. Meanwhile, 20 µl of serial dilutions of the purified C5a mutants were transferred to a microtiter plate (Greiner) and incubated for 5 min at 37 °C. After 5 min of preincubation, cytochalasin B (20 µg/ml) was added to the cells and incubated for 3 min at 37 °C. Subsequently, the cells were transferred to the C5aRA and incubated for 10 min at 37 °C. Then rhC5a was added at a final concentration of 10–9 M, and the reaction was allowed to proceed for 5 min at 37 °C. The reaction was stopped by centrifugation at 1157 x g for 3 min at 4 °C. Subsequently, 75 µl of the supernatant were mixed with 100 µl of substrate (p-nitrophenyl-N-acetyl-{beta}-D-glucosaminide). The reaction was stopped by adding 75 µl of glycine/NaOH, pH 10.4/well. The absorbance (405 nm) was determined in an enzyme-linked immunosorbent assay reader. The ID50 of the C5a mutants was determined as the concentration leading to inhibition of half-maximal enzyme release induced by 10–9 M rhC5a.

Fluorescent Labeling of jun/fos-C5aAla27—jun/fos-C5aC27A was labeled with FLUOS (Roche Applied Science) at a molar ratio of 1:10 (ligand to FLUOS) for 1 h at room temperature according to the manufacturer's recommendations. Labeled ligand was separated from free FLUOS on a Sephadex G25 M column. As demonstrated previously, jun/fos-C5aC27A exhibits the same binding properties and functional potency as rhC5a (38).

Generation of FLUOS-jun/fos-C5aC27A-desArg74—To cleave off the C-terminal arginine, FLUOS jun/fos-C5aC27A was treated with carboxypeptidase B (Calbiochem) (5 units/mg) for 15 min at 37 °C as we have described previously (39).

Flow Cytometric Analysis of Agonist and Antagonist Binding to CD88 and C5L2—To assess the specific binding of rhC5a, the different A8 mutants, and AcF-(OpdChaWR) to CD88-RBL cells or C5L2-transfected CHO (C5L2-CHO) cells (18), 5 x 105 cells (in 50 µl) were incubated with FLUOS-jun/fos-C5aC27A (1 x 10–8 M) in the presence of increasing concentrations of the respective molecules for 12h at 4 °C in PBS supplemented with bovine serum albumin (1%). The ability of the C5a mutant A8{Delta}71–73 and AcF-(OpdChaWR) to inhibit binding of C5adesArg74 to CD88 or C5L2 was tested by incubating CD88-RBL-2H3 cell and C5L2-CHO cells with jun/fos-C5aC27A-desArg74 (5 x 10–8 M) in the presence of increasing concentrations of the respective C5aRA. Subsequently, the cells were pelleted, washed three times with 200 µl of PBS, and resuspended in 4% paraformaldehyde in PBS for fluorescence-activated cell scanning analysis.

Flow Cytometric Analysis of CD88 and C5L2 Expression on Human PMN, Monocytes, and HMC-1 Cells—Human monocytes were isolated by countercurrent elutriation (40) from healthy volunteers. Human neutrophils were isolated from healthy volunteers by density centrifugation from EDTA plasma using PolymorphprepTM (Nycomed) according to the manufacturer's recommendations. Purity of PMN was >95% as determined microscopically using Diff-Quick staining (Baxter). PMN, monocytes, or HMC-1 cells (1 x 106 cells) were incubated with either anti-CD88 mAb 5S/1 (10 µg/ml) (kindly provided by Dr. O. Götze, Göttingen, Germany) (27) or the protein A-purified polyclonal rabbit-anti-C5L2 antibody (5 µg/ml) described earlier (41) for 60 min at 4 °C in 100 µl of PBS-bovine serum albumin (1%). The purified polyclonal rabbit-anti-C5L2 antibody does not bind to untransfected RBL cells or RBL cells transfected with CD88, hC3aR, human formyl peptide receptor-like 1/lipoxin A4 receptor (FPRL1/LXA4R), or ChemR23 (42).2 Isotype-matched control antibodies (IgG2a (10 µg/ml) or protein A-purified rabbit nonimmune polyclonal antibody (5 µg/ml)) were used as controls. In some experiments, the specificity of C5L2 binding by polyvalent rabbit anti-C5L2 antibody binding was assessed in the presence of the immunizing peptide (1 µg/ml) as described (41). The cells were washed twice in 200 µl of PBS and incubated with either fluorescein isothiocyanate-labeled anti-mouse or anti-rabbit antibody (PharMingen) for 60 min at 4 °C. After two additionally washes, the cells were resuspended in 200 µl of PBS (4% paraformaldehyde) for fluorescence-activated cell scanning analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
N-terminal Deletion of the jun/fos Leucine Zipper Improves Binding and Functional Activity—The jun/fos-A8 mutant is expressed from a bicistronic vector that encodes the leucine zipper of transcription factor jun and the leucine zipper of transcription factor fos fused to C5aC27A-(1–66)-FKRSLLR, i.e. fos-C5aC27A-(1–66)-FKRSLLR (38). When expressed in E. coli, jun and fos form a heterodimer. To assess the impact of the jun-leucine zipper and the N-terminally fused fos-zipper moiety on CD88 binding and function, jun as well as jun/fos were deleted, resulting in the two C5a mutants fos-A8 and A8, respectively. Binding potency of fos-A8 to CD88-transfected RBL-2H3 cells as well as its ability to inhibit C5a-induced enzyme release slightly increased as compared with jun/fos-A8 (Fig. 1 and Table II). However, deletion of both jun and fos significantly increased C5a receptor binding (IC50 from 150.8 ± 8.7 to 35.4 ± 3.4 nM; p < 0.001, ANOVA) and antagonistic potency (ID50 from 129.9 ± 6.4 nM to 22.3 ± 2.1 nM; p < 0.001, ANOVA) in comparison with jun/fos-A8 (Fig. 1), suggesting that the fos moiety negatively impacts the interaction of the C5a mutant with CD88.



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  		FIG. 1.
Inhibition of 125I-rhC5a binding to CD88-RBL cells and inhibition of enzyme release from these cells by the C5a mutants jun/fos-A8, fos-A8, and A8. A, CD88-RBL cells were incubated with 0.1 nM 125I-rhC5a in the presence or the absence of the indicated concentrations of the C5a mutants, and specific binding was determined as outlined under "Experimental Procedures." B, CD88-RBL cells were incubated for 10 min at 37 °C with the indicated concentrations of the different C5a mutants. Subsequently the cells were challenged with rhC5a (1 x 10–9 M) to secrete N-acetyl-{beta}-D-glucosaminidase. C, half-maximal concentrations of the different C5a mutants to block 125I-rhC5a binding (IC50; left panel) or to block enzyme release (ID50; right panel) from CD88-RBL cells are depicted. The data are means ± S.E. of multiple experiments (n = 3–4). **, p < 0.001 jun/fos A8 versus A8 (ANOVA).

 


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  		TABLE II
Binding and functional properties of C5a mutants and the cyclic C-terminal derived peptide AcF-(OPdChaWR)

Binding studies were performed using 125I-rhC5a as a tracer (0.1 nM) as described under "Experimental Procedures."

 
Impact of C-terminal Mutations or Deletions on CD88 Binding and Function—The A8 mutant has a hydrophobic cluster at positions 71–72 consisting of two leucine residues. To assess whether the enlargement of this cluster may impact binding and/or antagonistic function, the neutral but polar serine-residue at position 70 was replaced by leucine (A8S70L). This replacement decreased the IC50 from 35.4 ± 3.4 to 23.1 ± 2.6 nM (p < 0.05, ANOVA) and the ID50 from 22.3 ± 2.1 to 7.9 ± 0.5 nM (p < 0.001, ANOVA; Fig. 2 and Table II).



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  		FIG. 2.
Effect of C-terminal amino acid replacements on the ability of A8 to inhibit 125I-rhC5a binding to CD88 or to block enzyme release from CD88-RBL cells. A, CD88-transfected RBL cells were incubated with different concentrations of the A8 mutants A8S70L, A8S70L,R73Y, A8R73C, A8R73C,{Delta}72–73, or A8{Delta}71–73. The IC50 values are depicted to compare the different potencies of the A8 mutants to inhibit 125I-rhC5a binding to CD88. B, CD88-transfected RBL cells were incubated with the same A8 mutants as depicted in A for 10 min at 37 °C. The cells were challenged with C5a (1 x 10–9 M) and tested for their ability to block C5a-induced enzyme release. The ID50 values are depicted to assess the effects of the amino acid replacements on the antagonistic potency. The data are the means ± S.E. of multiple experiments (n = 3–5). **, p < 0.001 A8S70L and A8S70L,R73Y versus A8; ++, p < 0.001 A8R73C,{Delta}72–73 versus A8 (ANOVA).

 
Next, we tested whether the C-terminal arginine residue contributes to CD88 receptor binding and/or antagonistic potency by replacing it by either a tyrosine (A8S70L,R73Y) or cysteine residue (A8R73C). These replacements affected neither the IC50 nor the ID50, suggesting that position 73 is not necessary for CD88 binding and antagonistic properties of A8 (Fig. 2 and Table II).

To assess the impact of the two leucine residues at positions 71 and 72, we constructed two C-terminal deletion mutants. In the first mutant, positions 72 and 73 were deleted, and the leucine residue at position 71 was replaced by a cysteine residue resulting in A8L71C,{Delta}72–73. The rationale for the L71C replacement were data obtained by Pellas et al. (24) suggesting that a C-terminal cysteine residue at position 71 is important for antagonistic properties of C5a mutants. Interestingly, the IC50 and the ID50 of this mutant (A8L71C,{Delta}72–73) were 4-fold higher as compared with A8 (p < 0.001, ANOVA; Fig. 2 and Table II). To determine whether the deletion of residue 72 or the L71C replacement accounted for the loss in binding and antagonistic potency, the cysteine residue at position 71 was deleted resulting in A8{Delta}71–73. Interestingly, the IC50 and the ID50 of A8{Delta}71–73 were indistinguishable from A8, suggesting that positions 71–73 are not necessary for CD88 binding and antagonism (Fig. 2 and Table II).

Positively Charged Amino Acids at Position 69 Are Crucial for CD88 Antagonism—After ruling out positions 70–73 as being critical for CD88 antagonism, positions 67–69 were considered candidates contributing to the antagonistic activity of A8. However, our previous data and data from literature suggested that amino acids at positions 67 and 68 contribute to CD88 binding and agonist activity (43–46). Thus, we focused on position 69 and evaluated whether the arginine residue might account for the observed C5aR antagonism of A8. First, the cationic arginine residue was replaced by the cationic lysine residue. This replacement increased the IC50 3-fold from 35.4 ± 3.4 nM (in A8) to 114.1 ± 4.7 nM (in A8R69K) (p < 0.001, ANOVA) and the ID50 2.5-fold from 22.3 ± 2.1 to 53.8 ± 4.4 nM (p < 0.05, ANOVA), suggesting that the guanidinium function of Arg69 may be of importance (Table II). Next, we replaced Arg69 by the hydrophobic alanine residue. Intriguingly, this exchange destroyed the antagonistic property of A8, suggesting that the positive charge at position 69 is crucial. In fact, A8R69A was a potent C5aR agonist with an ED50 of 36.8 ± 2.9 nM (Fig. 3, A and C, and Table II). However, binding to CD88 was essentially unchanged as compared with A8 (Table II). These data suggest that position 69 is not a binding residue; however, a positive charge at that position prevents C-terminal amino acids interacting with CD88 sites that trigger signaling and/or formation of CD88 dimers or oligomers (47), which may be important for CD88 signaling. Finally, the positive charge at position 69 was replaced by the negatively charged aspartic acid residue (which is native to C5a). This mutant (A8R69D) was a pure C5aR agonist (Fig. 3, B and C) with ID50 and ED50 values almost identical to those of rhC5a (Table II).



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  		FIG. 3.
Effect of amino acid replacements at position 69 on CD88 antagonism of A8. A, CD88-transfected RBL cells were incubated with different concentrations of rhC5a, the CD88 antagonist A8, or three different A8 mutants in which Arg69 was replaced by either Asp69 (A8R69D), Ala69 (A8R69A), or Lys69 (A8R69K) and tested for their ability to induce enzyme release. The enzyme release is given in percentages. B, CD88-transfected RBL cells were incubated with different concentrations of A8 or the A8 mutant A8R69K for 10 min at 37 °C and subsequently challenged with rhC5a (1 x 10–9 M). The inhibition of the C5a-induced enzyme release is given in percentages. C, effect of amino acid replacements at position 69 on CD88 antagonism and agonism. A positively charged amino acid at position 69 (either Arg or Lys; black bars) renders A8 a pure C5aR antagonist with an ID50 in the low nanomolar range; replacement of the positive charge by a hydrophobic or a negatively charged amino acid transforms A8 from a CD88 antagonist into a CD88 agonist (black versus white bars). The data are the means ± S.E. of multiple experiments (n = 3–4). **, p < 0.001 A8R69K versus A8 (t test); ++, p < 0.001 A8R69A versus rhC5a (ANOVA).

 
The C5aRA A8{Delta}71–73 but Not AcF-(OpdChaWR) Inhibits Binding of C5a/C5adesArg74 to C5L2—In addition to CD88, C5a and C5adesArg74 bind to C5L2 (18, 19). Because all of the highly conserved binding residues in the core region of C5a are preserved in the C5a mutants, we wondered whether A8 or A8{Delta}71–73 might bind to C5L2 as well. Because A8 and A8{Delta}71–73 showed the same behavior in the binding experiments outlined below, only data obtained with A8{Delta}71–73 are shown. A8{Delta}71–73 dose-dependently blocked FLUOS-labeled jun/fos-C5aC27A binding to CD88-RBL cells and to C5L2-CHO cells (Fig. 4, A and B). In contrast, no inhibition of C5a binding to C5L2 was found using the peptidic CD88 antagonist AcF-(OpdChaWR) (Fig. 4A), which, however, potently blocked C5a binding to and signaling through CD88 in CD88-RBL cells (Fig. 4A and Table II).



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  		FIG. 4.
Effect of A8 and AcF-(OpdChaWR) on binding of C5a and C5desArg74 to CD88 and to C5L2. A and B, CD88-RBL cells (A) or C5L2-CHO cells (B) were incubated with FLUOS-jun/fos-C5aC27A (1 x 10–8 M) in the presence or the absence of different concentrations of the C5a mutant A8{Delta}71–73 or the cyclic C5a analog peptide AcF-(OpdChaWR), and specific binding was determined. C, to test the ability of A8 and AcF-(OpdChaWR) to block C5adesArg74 binding to C5L2, C5L2-CHO cells were incubated with FLUOS-jun/fos-C5aC27AdesArg74 (5 x 10–8 M) in the presence or the absence of rhC5a (1 x 10–6 M), A8 (5 x 10–6 M), or AcF-(OpdChaWR) (5 x 10–6 M), and specific binding was determined. The data are the means ± S.E. of multiple experiments (n = 3).

 
The binding affinity of hC5adesArg74 to CD88 is 20–100-fold lower as compared with hC5a (18, 19). In contrast, C5L2 provides a high affinity binding site for hC5adesArg74, the IC50 of which is only 2–5-fold lower as compared with hC5a (18, 19). These data imply that in the presence of both receptors, C5adesArg74 preferentially binds to C5L2. Thus, to target bioactivities induced by C5adesArg74, blocking of C5adesArg74 binding to C5L2 appears to be required. Consequently, we tested whether A8{Delta}71–73 could block the binding of FLUOS-C5aC27A-desArg74 to C5L2. In fact, A8D71–73 (5 x 10–6 M) decreased binding of FLUOS-C5aC27A-desArg (5 x 10–8 M) to C5L2 by 75%, whereas AcF-(OpdChaWR) (5 x 10–6 M) had no effect (Fig. 4C).

Expression of CD88 and C5L2 on Human PMN, Monocytes, and the Human Mast Cell Line HMC-1—CD88 and C5L2 mRNAs are abundantly expressed in several tissues and in peripheral blood leukocytes (16, 17, 19), suggesting coexpression of both receptors. To determine whether mRNA expression corresponds to protein expression on the cell surface, three different immunocompetent leukocyte populations were studied by flow cytometry: PMN, monocytes, and the human mast cell line HMC-1. PMN from three different healthy donors stained highly positive for CD88; however, we did not find any positive staining for C5L2 (Fig. 5A). On monocytes and HMC-1 cells, we found a homogeneous expression of both CD88 and C5L2 (Fig. 5, B and C). However, the signal intensity of C5L2 staining on monocytes was much lower as compared with that of CD88 (Fig. 5B). This may be due to lower receptor numbers or may simply reflect different affinities of CD88 and C5L2 antibodies. Further, C5L2 (but not CD88) expression on monocytes of four different healthy donors was highly variable (Fig. 5B; median fluorescence intensities were 7.3–24.3). In contrast, we found strong C5L2 and CD88 staining on HMC-1 cells, suggesting expression of high receptor numbers of both CD88 and C5L2 (Fig. 5C).



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  		FIG. 5.
Expression of CD88 and C5L2 on PMN, monocytes, and HMC-1 cells. Flow cytometric analysis of CD88 and C5L2 expression on: PMN purified by density centrifugation (A); monocytes purified by countercurrent elutriation (B); and HMC-1 cells (C). To assess CD88 expression, the cells were stained with CD88-specific antibody 5S/1 (10 µg/ml; gray histogram) or an isotype-matched control IgG (white histogram). To assess C5L2 expression, PMNs were stained with purified polyclonal rabbit anti-C5L2 antibody (5 µg/ml), in the presence (white histogram) or absence (gray histogram) of the C5L2-derived immunizing peptide (1 µg/ml) or with a purified polyclonal rabbit antibody taken before immunization (data not shown). On monocytes (B), the following median fluorescence intensities were obtained from the four different donors after C5L2 staining: 24.3 (donor 1), 11.4 (donor 2), 7.3 (donor 3), and 22.6 (donor 4). The curves of donors 1 and 3 are depicted.

 
An almost identical intensity of CD88 and C5L2 staining was found on CD88-transfected RBL-2H3 cells and C5L2-transfected CHO cells (data not shown), suggesting that the differences in C5L2 and CD88 staining on monocytes rather reflect lower receptor numbers than different antibody affinities and/or limiting concentrations of anti-C5L2 antibodies.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Recent studies modeling pulmonary infection, sepsis, autoimmune disease, and ischemia/reperfusion injury in mice and rats suggest that agents blocking C5a effector functions may provide potent therapeutics in humans. Several agents have been developed that block the interaction of C5a with CD88, most of which, however, lack in vivo activity (48), suffer from being partial agonists, and/or appear to be cytotoxic (49). Recently, C5a mutants (24, 25), a cyclic peptide (22, 29), and a nonpeptidic compound (23) have been described that block C5a activity in vivo. We previously selected the C5a mutant jun/fos-A8 from a phage library, in which the C-terminal positions 69–73 had been randomly mutated (25). jun/fos-A8 turned out to be a potent and specific C5aRA, blocking C5a activities in vitro (25, 33) and in vivo (25, 30, 32–35). However, the mechanisms underlying the C5aR antagonism remained unclear.

C5a and C5adesArg74 binding to CD88 is complex. Site-directed mutagenesis studies in which either C5a (44, 50, 51), CD88 (52, 53), or both (54, 55) were mutated provide evidence that the C-terminal amino acids from positions 65–74 confer binding and agonist activity. The core segment, comprising the remainder of the C5a molecule, contributes significantly to high affinity binding but not to biologic activity. In search of the mechanism that renders jun/fos-A8 a CD88 antagonist, we focused primarily on the N-terminal jun/fos heterodimer. Knowledge of the structure-activity relationships for C5a suggested an interaction of the N-terminal jun/fos heterodimer (comprising 80 amino acids) with the C-terminal tail of jun/fos-A8 as a means to disrupt its interaction with CD88. In fact, several aspartic acid residues within the leucine zippers could potentially interact with the two positively charged residues, Arg69 and Arg73, in jun/fos-A8. However, deletion of one or both leucine zippers did not negatively impact the antagonistic potential of the A8 mutant. Rather, deletion of jun and fos improved antagonism and binding to CD88, suggesting some impact of the C terminus of A8.

The facts that the C terminus confers C5a activity and that only amino acids 69–71 had been mutated in the phage library from which A8 was selected led to experiments that tested alternative C-terminal amino acids. Neither amino acid substitutions at position 70 or 73 nor deletion of amino acids at positions 71–73 had a negative impact on CD88 antagonism or CD88 binding. A cysteine residue at position 70, as in C5aM70C,{Delta}71–74, or at position 71, as in C5aQ71C,{Delta}72–74, had previously been reported as being crucial for C5aR antagonism of C5a mutants (24). Thus, we assessed whether deletion of amino acids 72 and 73 combined with a L71C replacement improves the antagonistic potency of A8. Surprisingly, we found a 4-fold decrease in antagonistic and binding potency, suggesting that the interaction of C71 with CD88 acted contrary to the mechanisms that renders A8 a CD88 antagonist. Further, it is noteworthy that A8 (C5aC27A, H67F,D69R,M70S) is an antagonist for human and murine CD88, whereas C5aQ71C,{Delta}72–74 and C5aM70C,{Delta}71–73 (24) are antagonists for human but not for murine CD88 (but for rabbit and micropig CD88). These data point to the importance of C-terminal amino acid composition for species-specific CD88 binding because they provide evidence that subtle changes in the composition of C-terminal amino acids (positions 67, 69, and/or 70) are sufficient to change the species specificity of C5a mutants.

After ruling out both N-terminal addition of jun/fos and C-terminal amino acids at positions 70–73, the D69R replacement at position 69 remained as a candidate responsible for CD88 antagonism of A8. Of note, position 69 is not conserved between species and was not considered as a binding residue in site-directed mutagenesis studies (44, 50). However, the positive charge at position 69 turned out to be essential for CD88 antagonism. In fact, replacement of Arg69 by the hydrophobic alanine residue or the negatively charged glutamic acid residue switched antagonism to agonism. Interestingly, the R69A replacement did not change the binding properties of A8 (35.4 + 3.4 versus 30.1 + 0.7 nM), suggesting that Arg69 does not contribute directly to the binding of A8 to CD88 but rather prevents amino acids known to confer activity and binding, such as Phe67, Lys68, and Leu72, to interact with CD88 and to initiate signaling. Alternatively, Arg69 may prevent CD88 disulfide-linked dimer formation, which may be important in eliciting signaling events, as has been demonstrated for some chemokine receptors (reviewed in Ref. 56). Further, the critical importance of a positive charge for receptor antagonism appears to depend on the composition of the neighboring amino acids, because the D69K replacement within C5a results in a pure agonist in which both binding and biologic activity are essentially unchanged (44).

The pleiotropic functions of C5a, such as modulation of cytokine release (1), regulation of apoptosis (57, 58) and tissue regeneration (59), along with different spectra of C5a and C5adesArg74 bioactivities, suggest that CD88 couples to different signaling pathways and/or that C5a and C5adesArg74 bind and signal through an additional receptor. Coupling of CD88 to G{alpha}i and G{alpha}16 subunits has been described; however, knowledge of CD88 signaling and the linkage between signaling and effector functions is rather sketchy. Recently, a second C5a/C5adesArg74-binding protein was discovered, C5L2, the mRNA expression of which is remarkably similar to that of CD88. Of note, C5L2 receptors are expressed on immature (but not mature) dendritic cells, and abundant C5L2 mRNA expression occurs in the spleen (16). These data suggest that C5L2 may have immunomodulatory functions on immature dendritic cells. Interestingly, C5L2 has high affinity binding sites for both C5a and C5adesArg74, whereas C5adesArg74 binding to CD88 is poor (20–100-fold lower as compared with C5a). Under circumstances in which the complement system is activated in the circulation (e.g. during sepsis), C5a is rapidly converted to C5adesArg74 by carboxypeptidases R and N (60) and will preferentially bind to C5L2. C5L2 does not follow the classical scheme of seven-transmembrane receptor activation, because it does not couple to G proteins and is not internalized on ligand binding (18, 19). However, C5a induces receptor phosphorylation of C5L2, although the signal intensity is much weaker compared with that of CD88 (19). Unfortunately, C5adesArg74, which behaves as a superagonist for some bioactivities (8), was not tested by these authors. Altogether these data suggest that C5L2 may use alternative, G protein-independent pathways for signaling as recently described for a panel of seven-transmembrane receptors (reviewed in Ref. 11). Consequently, it may prove useful to block binding of both C5a (and C5adesArg74) to CD88 and of C5adesArg74 (and C5a) to C5L2. We tested the ability of the two most widely used C5aRAs, i.e. A8 and the cyclic C5a C-terminal analog peptide AcF-(OPdChaWR), to block binding of C5a or C5adesArg74 to CD88 and C5L2. In particular we focused on A8{Delta}71–73, because among all mutants, A8{Delta}71–73 is most similar to C5a differing only at four positions (C27A, H67F, D69R, and M70S). As shown in Fig. 4, A8{Delta}71–73, but not AcF-(OPdChaWR), inhibits both C5a and C5adesArg74 binding to CD88 and C5L2. Interestingly, the agonistic C5a analog hexapeptide FKAdChaChadR has recently been described to displace C5a from CD88 and C5L2 with an almost identical affinity (19). However, the apparent affinities of FKAdChaChadR to CD88 (1.8 + 0.7 x 10–5 M) and C5L2 (2.7 + 0.9 x 10–5 M) calculated by these authors are 1000-fold lower as compared with the IC50 values that we determined for AcF-(OPdChaWR) to CD88 (2.21 + 0.18 x 10–8 M; Table II). Competitive binding studies with FKAdChaChadR performed in our laboratory revealed IC50 values that are in very good agreement with the data obtained by Okinaga et al. (19) (IC50 for CD88, 1.02 x 10–5 M; IC50 for C5L2, 5.84 x 10–5 M). Thus, the C5a analog peptide FKAdChaWR binds to CD88 and C5L2; however, its binding potency is very low. These data suggest that the core segment of C5a (amino acids 1–64) provides most of the binding energy to both CD88 and C5L2, whereas the contribution of the C5a C terminus is only marginal. Further, amino acid substitutions that improve binding affinity toward CD88 (FKAdChaChadR versus AcF-(OPdChaWR)) have no measurable effect on C5L2 binding, suggesting that the interactions of the C5a C terminus with CD88 and C5L2 are different.

C5L2 mRNA expression appears to be most abundant in peripheral leukocytes. PMN and monocytes represent two important cell types of this population mediating bacterial defense and inflammation. Both populations express CD88 in high numbers (1). We assessed whether these cells express C5L2 as well. Interestingly, we found no expression of C5L2 on PMN of three different healthy donors. The missing expression of C5L2 on PMN is in contrast to data obtained in a recent publication. These authors found coexpression of CD88 and C5L2 in approximately equal numbers (19). These differences may be explained by the regulation of C5L2 expression through different PMN purification protocols or by different donors. On monocytes, CD88 and C5L2 were coexpressed, although the expression level of C5L2 was lower as compared with CD88 and varied substantially from donor to donor (Fig. 5B). It will be interesting to see in further studies whether C5L2 expression is modulated by local or systemic inflammation. In addition to peripheral leukocytes, CD88 is expressed on skin, cardiac, and synovial mast cells and the human mast cell line HMC-1 (61, 62). Surprisingly, and in contrast to PMN and monocytes, we found strong and homogeneous C5L2 expression on HMC-1 cells (Fig. 5C). These data fit well with results by Werfel et al. (63) showing that C5a but not C5adesArg74 induced transient mobilization of intracellular calcium in HMC-1 cells, whereas both C5a and C5adesArg74 induce transient mobilization of intracellular calcium in PMN. Because PMN express only CD88 but not C5L2, C5a and C5adesArg74 exclusively signal through CD88, resulting in mobilization of intracellular calcium. However, in HMC-1 cells C5adesArg74 will preferentially bind to C5L2, which does not to trigger calcium mobilization (18, 19).

In summary, we have identified a positively charged amino acid at position 69 as being crucial for the CD88 antagonism of the C5a mutant A8. Further, we have found that A8 and A8{Delta}71–73 (but not the cyclic peptidic AcF-(OPdChaWR)) are potent inhibitors of C5a and C5adesArg74 binding to C5L2. Finally, we demonstrate differential expression of CD88 and C5L2 on human PMN, monocytes, and HMC-1 cells. The fact that A8{Delta}71–73 is structurally very similar to C5a, blocks C5a and C5adesArg74 binding to the two known C5aRs, and is a powerful C5aRA in animal models of inflammation suggests that A8{Delta}71–73 may be a useful therapeutic agent to target C5a/C5adesArg74 bioactivities in humans.


   FOOTNOTES
 
* This work was supported by Cincinnati Children's Hospital Research Foundation funding and by Deutsche Forschungsgemeinschaft Grant KO 1245/1-1 (to J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work. Back

** To whom correspondence should be addressed: Div. of Molecular Immunology, Cincinnati Children's Hospital Research Foundation, MLC 7021, Cincinnati, OH 45229. Tel.: 513-636-1219; Fax: 513-636-5355; E-mail: Joerg.Koehl{at}chmcc.org.

1 The abbreviations used are: C5aR, C5a receptor; C5aRA, C5aR antagonist; CHO, Chinese hamster ovary cell line; FLUOS, 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester; PBS, phosphate-buffered saline; PMN, polymorphonuclear neutrophil granulocytes; AcF-(Opd-ChaWR), AcPhe[L-ornithine-Pro-D-cyclohexylalanine-Trp-Arg]; mAb, monoclonal antibody; ANOVA, analysis of variance. Back

2 P. N. Monk, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ember, J. A., Jagels, M. A., and Hugli, T. E. (1998) in The Human Complement System in Health and Disease (Volanakis, J. E., and Frank, M. M., eds) pp. 241–284, Marcel Dekker Inc., New York
  2. Gasque, P., Neal, J. W., Singhrao, S. K., McGreal, E. P., Dean, Y. D., Van, B. J., and Morgan, B. P. (2002) Mol. Neurobiol. 25, 1–17[CrossRef][Medline] [Order article via Infotrieve]
  3. Fayyazi, A., Scheel, O., Werfel, T., Schweyer, S., Oppermann, M., Götze, O., Radzun, H. J., and Zwirner, J. (2000) Immunology 99, 38–45[CrossRef][Medline] [Order article via Infotrieve]
  4. Zahedi, R., Braun, M., Wetsel, R. A., Ault, B. H., Khan, A., Welch, T. R., Frenzke, M., and Davis, A. E. (2000) Clin. Exp. Immunol. 121, 226–233[CrossRef][Medline] [Order article via Infotrieve]
  5. Haviland, D. L., McCoy, R. L., Whitehead, W. T., Akama, H., Molmenti, E. P., Brown, A., Haviland, J. C., Parks, W. C., Perlmutter, D. H., and Wetsel, R. A. (1995) J. Immunol. 154, 1861–1869[Abstract]
  6. Laudes, I. J., Chu, J. C., Huber-Lang, M., Guo, R. F., Riedemann, N. C., Sarma, J. V., Mahdi, F., Murphy, H. S., Speyer, C., Lu, K. T., Lambris, J. D., Zetoune, F. S., and Ward, P. A. (2002) J. Immunol. 169, 5962–5970[Abstract/Free Full Text]
  7. Drouin, S. M., Kildsgaard, J., Haviland, J., Zabner, J., Jia, H. P., McCray, P. B., Tack, B. F., and Wetsel, R. A. (2001) J. Immunol. 166, 2025–2032[Abstract/Free Full Text]
  8. Eglite, S., Pluss, K., and Dahinden, C. A. (2000) J. Immunol. 165, 2183–2189[Abstract/Free Full Text]
  9. Gerard, N. P., and Gerard, C. (1991) Nature 349, 614–617[CrossRef][Medline] [Order article via Infotrieve]
  10. Boulay, F., Mery, L., Tardif, M., Brouchon, L., and Vignais, P. (1991) Biochemistry 30, 2993–2999[CrossRef][Medline] [Order article via Infotrieve]
  11. Marinissen, M. J., and Gutkind, J. S. (2001) Trends Pharmacol. Sci. 22, 368–376[CrossRef][Medline] [Order article via Infotrieve]
  12. Ji, H., Ohmura, K., Mahmood, U., Lee, D. M., Hofhuis, F. M., Boackle, S. A., Takahashi, K., Holers, V. M., Walport, M., Gerard, C., Ezekowitz, A., Carroll, M. C., Brenner, M., Weissleder, R., Verbeek, J. S., Duchatelle, V., Degott, C., Benoist, C., and Mathis, D. (2002) Immunity 16, 157–168[CrossRef][Medline] [Order article via Infotrieve]
  13. Bozic, C. R., Lu, B., Hopken, U. E., Gerard, C., and Gerard, N. P. (1996) Science 273, 1722–1725[Abstract/Free Full Text]
  14. Tsuji, R. F., Kawikova, I., Ramabhadran, R., Akahira-Azuma, M., Taub, D., Hugli, T. E., Gerard, C., and Askenase, P. W. (2000) J. Immunol. 165, 1588–1598[Abstract/Free Full Text]
  15. Reiman, R., Gerard, C., Campbell, I. L., and Barnum, S. R. (2002) Eur. J. Immunol. 32, 1157–1163[CrossRef][Medline] [Order article via Infotrieve]
  16. Ohno, M., Hirata, T., Enomoto, M., Araki, T., Ishimaru, H., and Takahashi, T. A. (2000) Mol. Immunol. 37, 407–412[CrossRef][Medline] [Order article via Infotrieve]
  17. Lee, D. K., George, S. R., Cheng, R., Nguyen, T., Liu, Y., Brown, M., Lynch, K. R., and O'Dowd, B. F. (2001) Brain Res. Mol. Brain Res. 86, 13–22[Medline] [Order article via Infotrieve]
  18. Cain, S. A., and Monk, P. N. (2002) J. Biol. Chem. 277, 7165–7169[Abstract/Free Full Text]
  19. Okinaga, S., Slattery, D., Humbles, A., Zsengeller, Z., Morteau, O., Kinrade, M. B., Brodbeck, R. M., Krause, J. E., Choe, H. R., Gerard, N. P., and Gerard, C. (2003) Biochemistry 42, 9406–9415[CrossRef][Medline] [Order article via Infotrieve]
  20. Konteatis, Z. D., Siciliano, S. J., Van Riper, G., Molineaux, C. J., Pandya, S., Fischer, P., Rosen, H., Mumford, R. A., and Springer, M. S. (1994) J. Immunol. 153, 4200–4205[Abstract]
  21. Wong, A. K., Finch, A. M., Pierens, G. K., Craik, D. J., Taylor, S. M., and Fairlie, D. P. (1998) J. Med. Chem. 41, 3417–3425[CrossRef][Medline] [Order article via Infotrieve]
  22. Paczkowski, N. J., Finch, A. M., Whitmore, J. B., Short, A. J., Wong, A. K., Monk, P. N., Cain, S. A., Fairlie, D. P., and Taylor, S. M. (1999) Br. J. Pharmacol. 128, 1461–1466[CrossRef][Medline] [Order article via Infotrieve]
  23. Sumichika, H., Sakata, K., Sato, N., Takeshita, S., Ishibuchi, S., Nakamura, M., Kamahori, T., Ehara, S., Itoh, K., Ohtsuka, T., Ohbora, T., Mishina, T., Komatsu, H., and Naka, Y. (2002) J. Biol. Chem. 277, 49403–49407[Abstract/Free Full Text]
  24. Pellas, T. C., Boyar, W., van Oostrum, J., Wasvary, J., Fryer, L. R., Pastor, G., Sills, M., Braunwalder, A., Yarwood, D. R., Kramer, R., Kimble, E., Hadala, J., Haston, W., Moreira-Ludewig, R., Uziel-Fusi, S., Peters, P., Bill, K., and Wennogle, L. P. (1998) J. Immunol. 160, 5616–5621[Abstract/Free Full Text]
  25. Heller, T., Hennecke, M., Baumann, U., Gessner, J. E., zu Vilsendorf, A. M., Baensch, M., Boulay, F., Kola, A., Klos, A., Bautsch, W., and Köhl, J. (1999) J. Immunol. 163, 985–994[Abstract/Free Full Text]
  26. Hennecke, M., Otto, A., Baensch, M., Kola, A., Bautsch, W., Klos, A., and Köhl, J. (1998) Eur. J. Biochem. 252, 36–44[Medline] [Order article via Infotrieve]
  27. Oppermann, M., Raedt, U., Hebell, T., Schmidt, B., Zimmermann, B., and Götze, O. (1993) J. Immunol. 151, 3785–3794[Abstract]
  28. Morgan, E. L., Ember, J. A., Sanderson, S. D., Scholz, W., Buchner, R., Ye, R. D., and Hugli, T. E. (1993) J. Immunol. 151, 377–388[Abstract]
  29. Strachan, A. J., Woodruff, T. M., Haaima, G., Fairlie, D. P., and Taylor, S. M. (2000) J. Immunol. 164, 6560–6565[Abstract/Free Full Text]
  30. Baumann, U., Köhl, J., Tschernig, T., Schwerter-Strumpf, K., Verbeek, J. S., Schmidt, R. E., and Gessner, J. E. (2000) J. Immunol. 164, 1065–1070[Abstract/Free Full Text]
  31. Arumugam, T. V., Shiels, I. A., Strachan, A. J., Abbenante, G., Fairlie, D. P., and Taylor, S. M. (2003) Kidney Int. 63, 134–142[Medline] [Order article via Infotrieve]
  32. De Vries, B., Köhl, J., Leclercq, W. K., Wolfs, T. G., van Bijnen, A. A., Heeringa, P., and Buurman, W. A. (2003) J. Immunol. 170, 3883–3889[Abstract/Free Full Text]
  33. Karp, C. L., Grupe, A., Schadt, E., Ewart, S. L., Keane-Moore, M., Cuomo, P. J., Köhl, J., Wahl, L., Kuperman, D., Germer, S., Aud, D., Peltz, G., and Wills-Karp, M. (2000) Nat. Immunol. 1, 221–226[CrossRef][Medline] [Order article via Infotrieve]
  34. Baumann, U., Chouchakova, N., Gewecke, B., Köhl, J., Carroll, M. C., Schmidt, R. E., and Gessner, J. E. (2001) J. Immunol. 167, 1022–1027[Abstract/Free Full Text]
  35. Soruri, A., Kiafard, Z., Dettmer, C., Riggert, J., Köhl, J., and Zwirner, J. (2003) J. Immunol. 170, 3306–3314[Abstract/Free Full Text]
  36. Klos, A., Ihrig, V., Messner, M., Grabbe, J., and Bitter-Suermann, D. (1988) J. Immunol. Methods 111, 241–252[CrossRef][Medline] [Order article via Infotrieve]
  37. Bock, D., Martin, U., Gartner, S., Rheinheimer, C., Raffetseder, U., Arseniev, L., Barker, M. D., Monk, P. N., Bautsch, W., Köhl, J., and Klos, A. (1997) Eur. J. Immunol. 27, 1522–1529[Medline] [Order article via Infotrieve]
  38. Hennecke, M., Kola, A., Baensch, M., Wrede, A., Klos, A., Bautsch, W., and Köhl, J. (1997) Gene (Amst.) 184, 263–272[CrossRef][Medline] [Order article via Infotrieve]
  39. Suckau, D., Köhl, J., Karwath, G., Schneider, K., Casaretto, M., Bitter-Suermann, D., and Przybylski, M. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9848–9852[Abstract/Free Full Text]
  40. Karp, C. L., Wysocka, M., Wahl, L. M., Ahearn, J. M., Cuomo, P. J., Sherry, B., Trinchieri, G., and Griffin, D. E. (1996) Science 273, 228–231[Abstract]
  41. Kalant, D., Cain, S. A., Maslowska, M., Sniderman, A. D., Cianflone, K., and Monk, P. N. (2003) J. Biol. Chem. 278, 11123–11129[Abstract/Free Full Text]
  42. Wittamer, V., Franssen, J. D., Vulcano, M., Mirjolet, J. F., Le Poul, E., Migeotte, I., Brezillon, S., Tyldesley, R., Blanpain, C., Detheux, M., Mantovani, A., Sozzani, S., Vassart, G., Parmentier, M., and Communi, D. (2003) J. Exp. Med. 198, 977–985[Abstract/Free Full Text]
  43. Or, Y. S., Clark, R. F., Lane, B., Mollison, K. W., Carter, G. W., and Luly, J. R. (1992) J. Med. Chem. 35, 402–406[CrossRef][Medline] [Order article via Infotrieve]
  44. Mollison, K. W., Mandecki, W., Zuiderweg, E. R., Fayer, L., Fey, T. A., Krause, R. A., Conway, R. G., Miller, L., Edalji, R. P., Shallcross, M. A., Lane, B., Fox, J. L., Greer, J., and Carter, G. W. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 292–296[Abstract/Free Full Text]
  45. Köhl, J., Lubbers, B., Klos, A., Bautsch, W., and Casaretto, M. (1993) Eur. J. Immunol. 23, 646–652[Medline] [Order article via Infotrieve]
  46. Ember, J. A., Sanderson, S. D., Taylor, S. M., Kawahara, M., and Hugli, T. E. (1992) J. Immunol. 148, 3165–3173[Abstract]
  47. Klco, J. M., Lassere, T. B., and Baranski, T. J. (2003) J. Biol. Chem. 278, 35345–35353[Abstract/Free Full Text]
  48. Lanza, T. J., Durette, P. L., Rollins, T., Siciliano, S., Cianciarulo, D. N., Kobayashi, S. V., Caldwell, C. G., Springer, M. S., and Hagmann, W. K. (1992) J. Med. Chem. 35, 252–258[CrossRef][Medline] [Order article via Infotrieve]
  49. Pellas, T. C., and Wennogle, L. P. (1999) Curr. Pharm. Des. 5, 737–755[Medline] [Order article via Infotrieve]
  50. Toth, M. J., Huwyler, L., Boyar, W. C., Braunwalder, A. F., Yarwood, D., Hadala, J., Haston, W. O., Sills, M. A., Seligmann, B., and Galakatos, N. (1994) Protein Sci. 3, 1159–1168[Medline] [Order article via Infotrieve]
  51. Bubeck, P., Grötzinger, J., Winkler, M., Köhl, J., Wollmer, A., Klos, A., and Bautsch, W. (1994) Eur. J. Biochem. 219, 897–904[Medline] [Order article via Infotrieve]
  52. Raffetseder, U., Roper, D., Mery, L., Gietz, C., Klos, A., Grotzinger, J., Wollmer, A., Boulay, F., Köhl, J., and Bautsch, W. (1996) Eur. J. Biochem. 235, 82–90[Medline] [Order article via Infotrieve]
  53. Crass, T., Ames, R. S., Sarau, H. M., Tornetta, M. A., Foley, J. J., Köhl, J., Klos, A., and Bautsch, W. (1999) J. Biol. Chem. 274, 8367–8370[Abstract/Free Full Text]
  54. Crass, T., Bautsch, W., Cain, S. A., Pease, J. E., and Monk, P. N. (1999) Biochemistry 38, 9712–9717[CrossRef][Medline] [Order article via Infotrieve]
  55. Cain, S. A., Coughlan, T., and Monk, P. N. (2001) Biochemistry 40, 14047–14052[CrossRef][Medline] [Order article via Infotrieve]
  56. Mellado, M., Rodriguez-Frade, J. M., Manes, S., and Martinez, A. (2001) Annu. Rev. Immunol. 19, 397–421[CrossRef][Medline] [Order article via Infotrieve]
  57. Perianayagam, M. C., Balakrishnan, V. S., King, A. J., Pereira, B. J., and Jaber, B. L. (2002) Kidney Int. 61, 456–463[CrossRef][Medline] [Order article via Infotrieve]
  58. Mukherjee, P., and Pasinetti, G. M. (2001) J. Neurochem. 77, 43–49[CrossRef][Medline] [Order article via Infotrieve]
  59. Mastellos, D., Papadimitriou, J. C., Franchini, S., Tsonis, P. A., and Lambris, J. D. (2001) J. Immunol. 166, 2479–2486[Abstract/Free Full Text]
  60. Campbell, W. D., Lazoura, E., Okada, N., and Okada, H. (2002) Microbiol. Immunol. 46, 131–134[Medline] [Order article via Infotrieve]
  61. Füreder, W., Agis, H., Willheim, M., Bankl, H. C., Maier, U., Kishi, K., Muller, M. R., Czerwenka, K., Radaszkiewicz, T., and Butterfield, J. H. (1995) J. Immunol. 155, 3152–3160[Abstract]
  62. Kiener, H. P., Baghestanian, M., Dominkus, M., Walchshofer, S., Ghannadan, M., Willheim, M., Sillaber, C., Graninger, W. B., Smolen, J. S., and Valent, P. (1998) Arthritis Rheum. 41, 233–245[CrossRef][Medline] [Order article via Infotrieve]
  63. Werfel, T., Oppermann, M., Butterfield, J. H., Begemann, G., Elsner, J., Götze, O., and Zwirner, J. (1996) Scand. J. Immunol. 44, 30–36[CrossRef][Medline] [Order article via Infotrieve]

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