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The Journal of Immunology, 2000, 164: 3783-3789.
Copyright © 2000 by The American Association of Immunologists

Characterization of Synthetic C3a Analog Peptides on Human Eosinophils in Comparison to the Native Complement Component C3a1

Holger Petering2,*, Jörg Köhl{dagger}, Ansgar Weyergraf2,*, Yasmin Dulkys*, Daniela Kimmig*, Regina Smolarski*, Alexander Kapp* and Jörn Elsner3,*

* Department of Dermatology and Allergology and {dagger} Institute of Medical Microbiology, Hannover Medical University, Hannover, Germany


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


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The anaphylatoxin C3a is a 77-aa peptide derived from C3 by proteolytic cleavage of the {alpha}-chain after activation of the alternative or classic pathway of the complement system (1, 2). As a proinflammatory mediator, C3a evokes a variety of in vivo and in vitro effects via a specific G protein-coupled seven-transmembrane receptor that shows a broad expression in different tissues and organs (3, 4, 5). Moreover, cells of the peripheral blood express the C3a receptor emphasizing its role as a pleiotropic cell activator. It was demonstrated that the anaphylatoxin C3a triggers specific cellular responses in human mast cells (6, 7, 8), monocytes (9), neutrophils (9, 10), IL-3-treated basophils (11), and also leukemia-derived basophils (12).

Previous studies using short synthetic C3a analogue peptides indicated that the C-terminal domain of the anaphylatoxin C3a is the binding and effector site in the guinea pig system (13, 14). The pentapeptide LGLAR (C3a 73–77) was found to be the minimal sequence with C3a-specific activity (15). This pentapeptide is highly conserved in human, bovine, rat, mouse, and guinea pig C3a (16). N-terminal elongation of synthetic C3a-related peptides with the 9-fluorenylmethoxycarbonyl (F-moc)4 and the 6-aminohexanoyl (Ahx) group revealed that even the LGLAR sequence can be further shortened to the minimal sequence LAR (17).

Within the last few years, a number of factors have been described to activate human eosinophil granulocytes such as platelet-activating factor (18, 19), 5-oxo-eicosanoids (20, 21), and the CC family of chemotactic cytokines represented e.g. by RANTES, monocyte chemoattractant protein-3 and -4, and eotaxin (22, 23, 24). Besides C5a (25), the anaphylatoxin C3a could be identified as the most potent proinflammatory mediator derived from the complement system inducing biologic effects of human eosinophils like chemotaxis (26) and the release of reactive oxygen species (ROS) (19). These findings indicate the physiologic importance of C3a for diseases typically associated with a peripheral blood or tissue eosinophilia like lymphoma, atopic dermatitis, and allergic asthma. In the case of chronic allergic airway inflammation, eosinophils have been reported to be the primary cell responsible for the induction of bronchial mucosal injury by the respiratory burst. Furthermore, eosinophils are thought to contribute to bronchial obstruction associated with the asthmatic response by the release of granular matrix proteins like major basic protein and eosinophilic cationic protein (27).

In this study, we report the effect of synthetic C3a analogue peptides based on homologies to the C terminus of C3a on binding and effector functions of human eosinophils. In the first set of experiments the binding pattern of C3a-related peptides was investigated to find out which amino acid sequence confers human eosinophil binding. Furthermore, we investigated whether the leading sequence responsible for C3a receptor binding is also necessary for relevant eosinophil effector functions. For this purpose, desensitization experiments detecting transient changes of intracellular Ca2+ concentration ([Ca2+]i) were performed as an indirect method for measuring the receptor specificity of different synthetic C3a analogue peptides in comparison to native C3a. In addition, the in vivo relevance of C3a-related sequences on the respiratory burst as a measure of the microbicidal and tissue destructive power of eosinophils was evaluated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of human eosinophils, neutrophils, and mononuclear cells

Human granulocytes were isolated from heparin-anticoagulated venous blood from normal nonatopic healthy donors using Ficoll (Pharmacia, Piscataway, NJ) density gradient centrifugation as described (9, 19). For further purification, granulocytes were resuspended in HEPES-buffered HBSS (Life Technologies, Grand Island, NY), pH 7.4, containing 1 mg/ml BSA. Eosinophils were purified by negative selection with anti-CD16 Ab (clone 3G8; Immunotech, Hamburg, Germany) coated Dynabeads M-450 (Dynal, Hamburg, Germany) as described (19). The resulting eosinophil purity was >99.5%, as determined by Kimura staining and flow cytometrical analysis (FACScan; Becton Dickinson, Heidelberg, Germany) using PE-conjugated anti-CD16 Ab (clone 3G8; Immunotech). Viability of eosinophils after purification was >95% as assessed by propidium iodide (Molecular Probes, Eugene, Oregon) staining in flow cytometry in a second experiment.

Peptide synthesis

The peptides were prepared by solid-phase synthesis and analyzed by TLC, HPLC, and amino acid analysis as described earlier (17, 28). The amino acid sequences and m.w. of synthesized C3a analogue peptides are summarized in Table IGo.


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  		Table I. Amino acid sequence and m.w. of C3a analog peptides and human C3a

 
Monoclonal Abs and cytokines

The murine anti-human C3a receptor single-chain Fv fragment (scFv) 3G7 was used as described (29). Human recombinant C3a was obtained from Advanced Research Technologies (San Diego, CA), and C5a was obtained from Sigma (Deisenhofen, Germany).

C3a receptor mRNA expression in human eosinophils

Total RNA was isolated from eosinophils, lymphocytes/monocytes (mononuclear cells), and neutrophils using TRIzol (Life Technologies) according to the manufacturer’s instructions based on the guanidine isothiocyanate method. First-strand cDNA synthesis was performed as described (23). Primers for the amplification of C3a receptor (sense, 5'-GGCTGCTGGGGACTGTCTAT-3'; and antisense, 5'-TGACGTGCCAAGAACTC CTT-3'), CD16 (sense, 5'-GGCCTCGAGCTACTTCATTG-3'; and antisense, 5'-GGAGCCG CTATCTTTGAGTG-3'), CD2 (sense, 5'-TCCAAAGGTGCAGTCTC-3'; and antisense, 5'-TTGTGTGTGATGACCCT-3'), and CD52 (sense, 5'-GCCACGAAGATCCTACCAAA-3'; and antisense, 5'-GCTTGGCCCCTACATCATTA-3') were designed according to the published sequences (accession nos.: C3a receptor, U62027; CD16, M24854; CD2, M16445; CD52, A23013). PCR was performed as described earlier (18). PCR samples were run on a 1.8% agarose gel stained with 0.2 µg/ml ethidium bromide, and the PCR products were visualized with UV light and photographed (23).

Rapid-cycle PCR with the Light Cycler system

LightCycler PCR was carried out by rapid-cycling in glass capillaries with a reaction volume of 20 µl using the LightCycler Instrument (Roche Molecular Biochemicals, Mannheim, Germany). PCR was performed with LightCycler-DNA Master SYBR Green I as a ready-to-use reaction mix (Roche Molecular Biochemicals). This buffer contains nucleotides, Taq DNA-polymerase, reaction buffer, SYBR Green I dye, and 10 mM Mg2+. Template DNA, primers (2.0 µM), sterile H2O, and additional MgCl2 (the final Mg2+ concentration was adjusted to 3 mM) had to be added. SYBR Green is a double-strand DNA-specific dye, which binds to the amplified PCR products, and the amplicon can be detected by its fluorescence. The following experimental protocol was adapted to use CD2, CD16, and CD52. After an initial denaturation step at 95°C for 30 s, 40 cycles of amplification were performed. Beginning with a denaturation step at 95°C, followed by primer-specific annealing, the elongation was carried out at 72°C with an amplicon-depending incubation time (t = (bp ÷ 25) seconds). SYBR Green I fluorescence was detected at the end of each elongation cycle to monitor the amount of PCR product formed during that cycle. After the end of amplification, a final melting curve was recorded by cooling the sample to 65°C at a rate of 20°C/s, maintaining 65°C for 15 s, and then heating slowly at 0.2°C/s up to 95°C with continuous measurement of fluorescence. At the end of each run, a melting curve analysis on the amplification reaction was performed to increase the specificity and sensitivity of SYBR Green I detection. The fluorescence signal (F) was plotted against temperature (T) to produce melting curves for each sample (F vs T). Melting curves were then converted to melting peaks by plotting the negative derivative of the fluorescence with respect to temperature against temperature (–dF/dT vs T). Thus, each specific PCR product produce a product-specific signal that results in a product-specific melting peak.

Immunofluorescence of eosinophils with the anti-human C3a receptor scFv 3G7

Immunofluorescence of eosinophils was performed using standard techniques (18). In brief, eosinophils were adjusted to a density of 1 x 107 cells/ml. Aliquots (20 µl) containing 2 x 105 cells were incubated at 4°C for 30 min with the C3aR scFv 3G7. Thereafter, cells were washed twice with cold PBS. The scFv 3G7 comprise a c-myc tag that is recognized by mAb 9E10. This mAb was then detected using FITC-conjugated anti-mouse antiserum. Thereafter, cells were analyzed by flow cytometry (FACScan) and stained with the anti-human C3a receptor scFv 3G7 derived from combinatorial phage Ab libraries (29). The sample was excited at 488 nm, and emission was measured at 530 nm (FITC-labeled Abs, Fluorescence 1, green fluorescence) and at 585 nm (anti-CD16 PE, Fluorescence 2, red fluorescence). The C3a receptor expression was analyzed after preincubation of human eosinophils for 30 min at 37°C with different synthetic C3a analogue peptides or medium alone. The mean channel fluorescence of five experiments was determined. Results were expressed as the median fluorescence intensity after peptide preincubation for 30 min vs median fluorescence intensity after incubation with medium. Incubation of cells for longer than 30 min did not change the results.

Measurement of [Ca2+]i in spectrofluorometry

Eosinophils were loaded with 2 µM fura-2 (Molecular Probes), and [Ca2+]i was measured at 37°C in an Aminco Bowman Series 2 spectrofluorometer (SLM-Aminco, Urbana, IL) as described (30, 31). Autofluorescence represents ~10% of the total fluorescence of cells loaded with fura-2. In addition, autofluorescence is higher at 340 nm than at 380 nm. To minimize these effects, the fluorescence of unloaded cells was subtracted from an equivalent density of cells loaded with fura-2 to obtain fluorescence signals that were solely representative of intracellular fura-2. The autofluorescence of cells was virtually unchanged upon addition of stimulus. Calibration was conducted after addition of the stimuli. Thus, in all experiments, the fluorescence of untreated and unloaded cells was used for subtraction of autofluorescence. Dual excitation spectra were collected at 340 and 380 nm; emission was determined at 510 nm (31). Absolute [Ca2+]i was calculated automatically by AB2 Series 2 software (SLM-Aminco) according to the equation described by Grynkiewicz (32).

Lucigenin-dependent chemiluminescence

Lucigenin-dependent chemiluminescence represents a sensitive method for the measurement of ROS and was performed as described (10, 19, 22). In brief, eosinophils were suspended at a density of 5 x 104 cells/ml in HBSS plus BSA containing 200 µM lucigenin (Sigma). Aliquots (100 µl) containing 5 x 103 eosinophils were placed into flat-bottom white microtiter plates (Microfluor; Dynatech, Denkendorf, Germany) and stimulated with the indicated stimuli or medium. Measurements were performed in triplicates at 37°C. Triplicates were measured and indicated as intensity integral counts or ratio between stimulus-induced integral intensity counts and medium-induced integral intensity counts.

Statistical analysis

Unless otherwise stated, the data in the text and figures are expressed as the mean ± SEM as determined by SigmaStat analysis. Values of p < 0.05 were accepted as significant using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anaphylatoxin receptor C3a is expressed on human eosinophils

First, RT-PCR analysis was performed to demonstrate the expression of the human C3a receptor (C3aR) on highly purified human eosinophils from healthy nonatopic donors. Using the indicated primer pairs, DNA fragments with an expected size of 655 bp specific for the human C3a receptor could be detected in human eosinophils (Fig. 1GoA). As expected, highly purified human neutrophils and a mononuclear cell preparation did express constitutively C3aR mRNA as indicated by specific RT-PCR products and were used as positive controls. To demonstrate that all mRNA preparations were highly purified, RT-PCR was performed simultaneously using primers for CD52 (384 bp), CD2 (479 bp), and CD16 (297 bp). In addition, rapid-cycle PCR with the Light Cycler system was performed for the detection of mRNA specific for different cell-surface molecules. Derivative melting curve plots of the amplified RT-PCR products specific for CD52 (eosinophils and mononuclear cells), CD16 (neutrophils and mononuclear cells), and CD2 (mononuclear cells; a mixture of monocytes and lymphocytes) are shown and demonstrate the purity of the cell preparations (Fig. 1GoB). Our results indicate that the anaphylatoxin C3a receptor is expressed at mRNA level in highly purified human eosinophils.



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  		FIGURE 1. A, Anaphylatoxin receptor C3a is expressed on human eosinophils. Human eosinophil, neutrophil, and mononuclear cell (containing lymphocytes and monocytes) mRNA was isolated, and first-strand cDNA synthesis was performed. PCR was conducted with primer pairs specific for C3aR (expected size, 655 bp), CD52 (expected size, 384 bp), CD 16 (expected size, 297 bp), and CD 2 (expected size, 479 bp) for each cell type. The amplicons were separated by electrophoresis in 1.8% agarose gel and stained by ethidium bromide. The different cell-surface molecules were loaded for each cell type in the following manner: lane 1, C3aR; lane 2, CD52; lane 3, CD16; and lane 4, CD2. The lanes marked with M show a 50-bp DNA size marker. One representative experiment of four is shown. B, Rapid-cycle PCR with the Light Cycler system demonstrates the purity of cell preparations. Rapid-cycle PCR was performed for the detection of cell-surface molecules CD52, CD16, and CD2 with cDNA from eosinophils (Eos), neutrophils (Neutr), and mononuclear cells (MNC) with Cybergreen as an unspecific DNA-marker. Following amplification, a melting analysis was immediately received. The melting curve plot of fluorescence signal (F1) vs temperature (T) was transformed into a derivative melting curve plot with -dF1/dT vs T for each PCR product. PCR amplicons specific for CD52 (eosinophils and mononuclear cells), CD16 (neutrophils and mononuclear cells), and CD2 (mononuclear cells) could be detected demonstrating the purity of the cell preparations. The bold line represents CD52, the dotted line CD16, and the broken line symbolizes CD2.

 
Flow cytometry revealed that the C3a receptor is expressed on human eosinophils and that synthetic C3a analogue peptides led to C3aR internalization

In the next set of experiments, highly purified human eosinophils were stained with a site-directed anti-human C3a receptor scFv 3G7 derived from combinatorial phage Ab libraries (29). ScFv 3G7 does not affect C3aR binding as has been demonstrated (29). Histogram analysis showed homogeneous binding of the C3aR scFv and revealed that the C3a receptor is constitutively expressed on the surface of highly purified human eosinophils (Fig. 2Go, A and B). Then, human eosinophils were incubated for 30 min with synthetic peptides (A1-C10) at a concentration of 10-6 M, C3a (10-8 M), or medium. Kola et al. performed a comparison study with an ATP release assay using guinea pig platelets and a guinea pig ileum contraction assay measuring the biologic activities of C3a and synthetic C3a analogue peptides. They observed a significant activity of synthetic peptides at a concentration of 10-6 M (33). As expected, preincubation of human eosinophils with native C3a led to a significant down-regulation of C3aR on the cell surface, indicating that the native anaphylatoxin C3a was able to bind specifically to the C3aR (Fig. 2GoB). Next, the pentapeptide LGLAR (A1), found to be the minimal sequence for C3a-specific activity in the guinea pig system (16), was used for preincubation with human eosinophils. A1 did not down-regulate the C3aR at a concentration of 10-6 M, indicating that peptide A1 is not able to bind to the C3aR at this concentration (Fig. 2GoB).



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  		FIGURE 2. Flow cytometry revealed that the C3a receptor is expressed on human eosinophils and that synthetic C3a analogue peptides led to C3aR internalization. A, Overlay histograms for the expression of C3aR on human eosinophils. As indicated, cells were preincubated for 30 min with human C3a (10-8 M), medium, or different synthetic C3a analogue peptides (10-6 M). Eosinophils were stained for C3aR with the scFv 3G7. The thin lines represent medium-incubated eosinophils with high expression of C3aR as a positive control. The dotted lines indicate purified eosinophils preincubated with C3a showing a down-regulation of the C3aR and, therefore, represent a negative control. The bold lines represent eosinophils preincubated with different synthetic C3a analogue peptides. Data from one typical experiment of six performed is given. B, The effect of human C3a and the indicated synthetic C3a analogue peptides on C3aR expression on human eosinophils. Results are expressed as the mean ± SEM of the fluorescence intensity of six experiments. **, p < 0.001 compared with medium incubated cells (Student’s t test). *, p < 0.01 compared with medium incubated cells (Student’s t test).

 
Then, tri- and tetrapeptides F-moc-Ahx-LAR (A2) and F-moc-Ahx-GLAR (A3) were used for preincubation experiments. Peptide A2 containing only the C-terminal tripeptide connected with F-moc-Ahx did not down-regulate the C3a receptor (Fig. 2GoB). In contrast to A2, the tetrapeptide F-moc-Ahx-GLAR (A3) slightly decreased the C3aR-mAb binding on human eosinophils (Fig. 2Go, A and B).

To determine whether the replacement of the aminohexanoyl groups of A2 and A3 by repetitive alanine residues did influence the biologic potency, peptide B9 (F-moc-YAAALGLAR) was used. In contrast to the pentapeptide A1 and the F-moc-Ahx-conjugated tripeptide A2, peptide B9 did significantly down-regulate the C3aR on human eosinophils (Fig. 2Go, A and B). Peptide B9 was more effective than A3 and nearly as effective as native C3a at a concentration of 10-6 M (Fig. 2Go, A and B). Therefore, the amino-terminal elongation of the peptide backbone of B9 seems to be important for the binding to the C3aR on human eosinophils. A glycine/alanine replacement at position 74 and variations of the repetitive alanine elements resulted in peptide C10, which was nearly as effective as B9 (Fig. 2Go, A and B).

Surprisingly, B4 representing a pentapeptide with an amino-terminal alanine backbone missing the hydrophobic F-moc-anchor did not induce a C3aR down-regulation on human eosinophils (Fig. 2Go, A and B). In addition, preincubation of highly purified human eosinophils for 30 min with peptides B5–B8 did not lead to C3aR internalization on eosinophils (Fig. 2GoB).

Effects of synthetic C3a analogue peptides on [Ca2+]i in human eosinophils

As an indirect method for studying the receptor binding and desensitization capacity of different synthetic C3a analogue peptides, transient cytosolic changes of [Ca2+]i were measured. In the first set of experiments, the pentapeptide LGLAR (A1) was used for stimulation of human eosinophils. Neither changes of [Ca2+]i nor desensitization effects to native C3a could be observed after stimulation with A1 (10-6 M) (Fig. 3Go).



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  		FIGURE 3. Effects of synthetic C3a analogue peptides on [Ca2+]i in human eosinophils. Spectrofluorometric measurements of [Ca2+]i in fura-2-loaded highly purified human eosinophils were performed. Cells were prestimulated with C3a (10-8 M) (A) or the indicated synthetic C3a analogue peptides (10-6 M) (B–F) and restimulated with C3a (10-8 M). The graph shown for peptide B6 is representative for the results obtained with peptides B5, B7, and B8. One representative experiment of six is shown.

 
In contrast with A1, the tri- and tetrapeptides F-moc-Ahx-LAR (A2) (data not shown) and F-moc-Ahx-GLAR (A3) (Fig. 3Go) were able to induce an increase of [Ca2+]i in eosinophils at a concentration of 10-6 M. In addition, prestimulation of eosinophils with peptide A3 induced complete desensitization to restimulation with C3a (10-8 M) (Fig. 3Go). These results indicate that the increase of [Ca2+]i is due to a C3a-specific activity and that the amino-terminal elongation of peptide A3 by a single glycine residue is of marginal importance in terms of biologic potency.

Next, peptide B9 (F-moc-YAAALGLAR) was investigated for its biologic activities on human eosinophils. In contrast with A2 and A3, peptide B9 does not contain an aminohexanoyl group. In B9, the peptide backbone was elongated by repetitive alanine residues connected to the hydrophobic F-moc anchor. In contrast to A2 (data not shown), peptide B9 did induce changes in [Ca2+]i transients in eosinophils and did show desensitization to C3a (Fig. 3Go). Therefore, the amino-terminal elongation of the peptide backbone of B9 seems to be responsible for biological effects on human eosinophils comparable to peptide A3, which correlates to the binding experiments of B9 to the C3a receptor. The addition of the aminohexanoyl group and the replacement of glycine vs alanine at position 74 (peptide C10) did not alter the biologic potency in this assay at a concentration of 10-6 M (data not shown).

To further evaluate the effect of carboxyl-terminal amino acid replacements on the increase of [Ca2+]i in human eosinophils, peptides B4–B8 were used: B4 represents the physiologic C terminus (C3a 73–77) containing an N-terminal spacer consisting of the tripeptide AAA. In contrast to A1, the peptide B4 induced an increase of [Ca2+]i in human eosinophils and showed desensitization to human C3a at a concentration of 10-6 M (Fig. 3Go). These results emphasize the biologic importance of amino-terminal alanine elongations. Single amino acid replacements at positions 73–76 (B5–B9) resulted in a loss of activity of these peptides and did not show desensitization to C3a at a concentration of 10-6 M (Fig. 3Go). These results emphasizes the biologic importance of the conserved C-terminal pentapeptide LGLAR for C3a effector functions.

Synthetic C3a analogue peptides are potent activators of the respiratory burst

Human C3a is a potent inducer of the respiratory burst in neutrophil (10) and eosinophil granulocytes (19). Here, the efficacy of synthetic C3a analogue peptides as eosinophil agonists was investigated. Stimulation of highly purified human eosinophils with native C3a induced the respiratory burst. A release of ROS was observed at concentrations of 10-5 M to 10-7 M (Fig. 4Go). Stimulation of eosinophils with synthetic peptides B9, C10, and also A3 induced the production of ROS indicating that the F-moc moiety is necessary for functional activity (Fig. 4GoA). A much lower production of ROS was observed after stimulation with B4 (YAAALGLAR) or the tripeptide A2 (F-moc-LAR) (Fig. 4Go). Peptides A1 and B5-B8 exhibit only a marginal signaling activity (Fig. 4Go). In summary, synthetic C3a analogue peptides were able to induce the respiratory burst. The biologic potency depends on their carboxyl-terminal alterations, which is in accordance to the binding and [Ca2+]i experiments described above.



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  		FIGURE 4. Effect of different synthetic C3a analogue peptides as potent activators of the respiratory burst of human eosinophils. Human eosinophils were stimulated with the indicated synthetic C3a analogue peptides at different concentrations (10-7 M to 10-5 M). Data are expressed as relative ROS release, i.e., the ratio of peptide-stimulated vs medium-stimulated cells. A, Stimulation of human eosinophils with peptides A1-A3, B9, C10, and C3a. B, Stimulation of human eosinophils with peptides B4–B8 and C3a.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The presence of peripheral blood eosinophilia and activated eosinophils in the inflammatory tissue are characteristic features of allergic diseases such as allergic asthma and atopic dermatitis (34, 35, 36). The mechanisms that control the tissue recruitment and activation of human eosinophils are of fundamental importance and could explain the propagation of inflammation (37, 38). Derived from the complement system, the anaphylatoxin C3a has been described as one of the most potent proinflammatory mediators inducing a variety of biologic effects on human eosinophils (9, 19, 26, 39). Human C3a binds to a specific cellular receptor and subsequently triggers cellular responses (1, 2, 9, 29, 40). However, a detailed analysis of the C3a C-terminal effector domain with respect to binding and functional responses on the human C3aR has not yet been performed. In this study, synthetic C3a analogue peptides optimized for binding to the guinea pig-C3aR were tested for binding and functional responses on human eosinophils.

Expression of the C3aR on human eosinophils has recently been published using a polyclonal rabbit anti-C3aR serum for flow cytometrical analysis (9). Martin et al. differentiated between leukocyte populations using CD16 as a specific marker (9). In this study, the expression of C3aR was demonstrated with the help of a monoclonal anti-human C3aR scFv. Functional receptor studies were performed using a human eosinophil preparation of >=99.5% purity. At first, the expression of the C3a receptor at mRNA level as well as on the cell surface of human eosinophils was investigated before starting the functional analysis of synthetic C3a analogue peptides. RT-PCR analysis revealed a baseline transcription of the C3a receptor mRNA in human eosinophils derived from healthy nonatopic donors. In addition, flow cytometrical scFv staining showed that C3aR is expressed constitutively on the cell surface of unstimulated highly purified human eosinophils and, therefore, C3aR mRNA trancripts were translated into specific membrane receptor proteins. These data are in accordance with recently published results indicating that human eosinophils can be activated by the C3a anaphylatoxin (19, 26).

In earlier studies, it was shown that the biologic activity of C3a resides mostly in the C terminus of the molecule. Caporale et al. described the C-terminal pentapeptide LGLAR (A1) as the minimal sequence required for a C3a-specific effect (15). In addition, this sequence could be further reduced to the tripeptide LAR when the peptide was coupled to the aromatic F-moc and the aliphatic Ahx sequence (17). As published recently by Settmacher et al., C3aR internalization could be demonstrated using a C3aR-specific scFv 3G7 (41). C3aR expression analysis using the C3aR-specific scFv 3G7 revealed that preincubation of human eosinophils with A1 did not alter the C3aR expression, suggesting that the peptide LGLAR did not induce C3aR down-regulation at this concentration. However, we cannot exclude that higher concentrations of peptide LGLAR might induce such a response. Furthermore, no changes of [Ca2+]i in human eosinophils have been detected as an indirect method for studying receptor-ligand interactions. In addition, only a marginal production of ROS occurred after stimulation with A1. These data are in accordance with functional analysis of LGLAR in the guinea pig system indicating a functional activity lower than 0.01% compared with native C3a (17).

As described earlier, the biological potency of short synthetic C3a analogue peptides can be substantially enhanced by the N-terminal addition of hydrophobic groups such as the aromatic F-moc connected to the aliphatic Ahx moiety (42, 43). Federwisch et al. demonstrated that these effects depend on hydrophobic, unspecific interactions of the F-moc group with the lipid bilayer of the cell membrane leading to a local enrichment of the C3a analogue peptides (44). The direct interaction between the membrane-bound ligand and its receptor is supposed to induce a C3aR down-regulation and, therefore, a process of receptor internalization (17). Indeed, C3aR-specific binding could be demonstrated for peptides B9 and C10 by flow cytometrical analysis. In fact, these synthetic C3a analogue peptides bound specifically to the C3a receptor expressed on human eosinophils and were nearly as effective as the native anaphylatoxin C3a in inducing C3aR down-regulation. As demonstrated for the guinea pig system, the tripeptide F-moc-Ahx-LAR showed C3a-specific activity on human eosinophils indicating that the mechanisms by which the human C3aR recognizes the C3a ligand must be very similar (17). In contrast with our observations, Ember et al. postulated that the attachment of amino-terminal hydrophobic groups to C3a analogues markedly enhances the potency of synthetic C3a peptides by a specific interaction of the hydrophobic unit with a second binding site on the C3a receptor (45). They presumed the presence of both a primary (effector) and a secondary (hydrophobic) binding site on their linear synthetic C3a analogue peptides, which can interact cooperatively with the C3a receptor (45). Our data do not exclude one of theses mechanisms. Both mechanisms, which are either unspecific interactions of the F-moc group with the lipid bilayer of the cell membrane or specific interactions of the hydrophobic unit with a second receptor binding site, may contribute to the observed effects.

To evaluate the effect of amino-terminal elongations on C3aR binding, synthetic C3a analogue peptides B4–B8 were tested containing an N-terminal spacer of three alanine residues and single amino acid replacements at position 73–77. C3aR staining by C3aR scFv 3G7 was not altered using peptides B4–B8, suggesting that replacements, in particular within the LAR sequence, decrease functional potency. In contrast, transient changes of [Ca2+]i were detected showing that peptide B4, containing the pentapeptide LGLAR connected with an alanine spacer, did induce changes in [Ca2+]i. In addition, the production of ROS was increased after stimulation of eosinophils with B4 (10-5 M). Therefore, the alanine elongation of the peptide backbone of B4 increased biologic potency on human eosinophils. These data are comparable to functional studies of C3aR expressed on guinea pig platelets showing that amino acid spacers potentate C3a-specific activity as measured by the ATP release assay (17, 33). The fact that B4 induced changes in [Ca2+]i without affecting C3aR down-regulation in flow cytometry studies indicates that the measurement of changes in [Ca2+]i in leukocytes represents the more sensitive method as compared with receptor internalization studies. The threshold for receptor internalization is above the measurable changes in [Ca2+]i mobilization. Preincubation of human eosinophils with B5–B8 did not decrease C3aR scFv 3G7 staining, suggesting that these synthetic C3a analogue peptides do not induce a process of receptor internalization and, therefore, may not function as a ligand for the C3aR. Amino acid replacements in peptides B5–B8 resulted in a loss of biologic activity without inducing changes in [Ca2+]i in human eosinophils. Measurement of ROS revealed that these peptides do not possess intrinsic activity at concentrations up to 10-5 M.

In summary, the analysis of synthetic C3a analogue peptides demonstrates that conserved amino acids essential for binding and functional activity in the guinea pig system are also crucial for the interaction of C3a with the human receptor. Our study revealed that the C-terminal pentapeptide LGLAR and in particular the C-terminal tripeptide LAR are of fundamental importance for C3aR binding on human eosinophils. Moreover, the putative binding motif of the peptide C3a correlates well with the minimal amino acid sequence required for the induction of biologic effects in human eosinophils such as changes of [Ca2+]i and the release of ROS. Therefore, amino acid sequences being important for the C3aR binding seem to be also important for the functional activity of C3a in the guinea pig and the human system.


   Footnotes
 
1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft (EL 160/6-1; to J.E.), a grant from the SFB 244 (B8; to J.K.), and by the Hochschulinterne Leistungsförderung (to H.P.). Back

2 H.P. and A.W. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Jörn Elsner, Department of Dermatology and Allergology, Hannover Medical University, Ricklinger Strasse 5; D-30449 Hannover; Germany. E-mail address: Back

4 Abbreviations used in this paper: F-moc, 9-fluorenylmethoxycarbonyl; Ahx, 6-aminohexanoyl; ROS, reactive oxygen species; [Ca2+]i, intercellular Ca2+ concentration; scFv, single chain Fv fragment. Back

Received for publication June 7, 1999. Accepted for publication January 31, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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