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Institute of Immunology and Allergology, Inselspital, Bern, Switzerland
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Basophils are an important source of IL-4 and IL-13 and are the only cell type capable of expressing these immunoregulatory cytokines in an Ag-independent manner by the combined action of C5a and IL-3 (10). IL-4 and IL-13 have many overlapping and some distinct bioactivities and play a central role in immune responses skewed to a Th-2 phenotype and in other important aspects of allergic inflammation (e.g., VCAM-1 up-regulation and induction of eotaxin expression) (11, 12, 13). Basophils also synthesize large amounts of LTC4 known to play a major role in chronic allergic inflammation, such as asthma (14). Thus, elucidation of the mechanisms leading to C5a-induced cytokine and LTC4 production are important for developing strategies to interfere with the synthesis of these mediators.
C5a acts via a seven-transmembrane G protein-coupled receptor belonging to the subfamily of the chemotactic receptors (1). It has two major regions important for ligand binding (15). The first, interacting with the core of C5a, is in the extracellular N-terminal domain, and the second, interacting with the C terminus of C5a, is poorly characterized and lies within the transmembrane helices (possibly helix V) of the receptor. Several studies have indicated that ligand interaction with the putative transmembrane binding site but not the N terminus of the receptor is responsible for the induction of cellular responses, like chemotaxis and enzyme release from neutrophils (15, 16, 17, 18). Subsequently, a number of C5a C-terminal analogs interacting with the second, "effector" site of the receptor and possessing different agonistic properties were generated (19, 20, 21). Using such peptide analogs (19) and Abs raised against the N terminus of the C5aR, we have investigated the requirements for C5aR-mediated IL-4 and IL-13 production and continuous LTC4 generation in human basophils. Our data demonstrate that interaction of an agonistic ligand with the transmembrane activation site of the receptor is necessary and sufficient to induce these prolonged responses in basophils. Ongoing cytokine and LTC4 production requires persistent activation of the C5aR. Furthermore, the signaling pathways involved can be blocked and mediator release terminated hours after exposure to C5a. Additionally, we show that depending on the nature of the ligand, the C5aR mediates qualitatively and quantitatively different responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HEPES was obtained from Calbiochem (La Jolla, CA); EDTA from Fluka AG (Buchs, Switzerland); Percoll from Pharmacia (Uppsala, Sweden); and fatty acid-free BSA from Boehringer Mannheim (Mannheim, Germany). All other reagents were of the highest purity available. HA buffer contained 20 mM HEPES, 125 mM NaCl, 5 mM KCl, 0.5 mM glucose (pH 7.4), and 0.25 mg/ml BSA. Culture medium was RPMI 1640 supplemented with 10% heat-inactivated FCS, 25 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM nonessential amino acids, and 2 mM L-glutamine (Life Technologies, Paisley, U.K.).
Stimuli and other reagents
The complement products C5a and the C5a cleavage product lacking
the C-terminal arginine (C5adesarg) were purified
from yeast-activated human serum with or without carboxypeptidase N
inhibitor as described (22, 23) and were found to be
homogenous as determined by amino acid analysis, SDS-PAGE,
reverse-phase HPLC, and microzone paper electrophoresis at pH 8.6. All
C5a C-terminal peptides CO28 (N-methyl phenylalanine
(NMePhe)-Lys-Pro-D-cyclohexylalanine
(Cha)-Cha-dArg-COOH; IC50 for the C5aR = 25
nM), CO26 (NMePhe-Lys-Pro-dCha-Phe-dArg-COOH;
IC50 for the C5aR = 50 nM), and CO61
(NMePhe-Lys-Pro-dCha-1-naphthyl-dArg-COOH; IC50
for the C5aR = 80 nM) were kind gifts from Dr. Springer (Merck
Research Laboratories, Rahway, NJ). Purified anti-Fc
RI mAb 29C6,
directed against the non-IgE-binding epitope of the high-affinity IgE
receptor 
-chain (24), was a generous gift from Drs.
Hakimi and Chizzonite (Hoffman-La Roche, Nutley, NJ). Recombinant human
IL-3 was provided by Sandoz (Basel, Switzerland). Murine anti-CD88
mAb S5/1 specific for an N-terminal epitope of the C5aR
(25) was purchased from Serotec (Oxford, U.K.). Pertussis
toxin was purchased from Sigma (St. Louis, MO). All compounds were
added to cells at a 1:100 v/v ratio.
Preparation of basophils
Basophil-enriched leukocyte fractions were prepared as previously described (26). Briefly, blood of unselected healthy volunteers was anticoagulated with 10 mM EDTA and mixed with 0.25 volumes of 6% dextran in 0.9% NaCl, and erythrocytes were allowed to sediment at room temperature. Leukocytes were pelleted by centrifugation (150 x g, 20 min, room temperature) and separated over a three-step discontinuous Percoll gradient (1.0791/1.0695/1.065 g/ml isotonic Percoll solution, respectively) at 400 x g for 30 min at room temperature. Basophil-enriched interphases between densities 1.0791 and 1.0695 g/ml were harvested and washed twice in HA buffer. The purity of basophils ranged from 10 to 30%, with contaminating cells consisting mainly of lymphocytes and variable proportions of neutrophils. Previous studies using basophils purified to near homogeneity have shown that the response of basophils to C5a is not affected by the contaminating leukocytes and that the LTC4 and all the IL-4 and IL-13 is derived exclusively from basophils (9, 10, 27, 28). For the experiments shown in this work, partially purified basophils were used because of a higher recovery from blood allowing the comparison of a larger number of experimental conditions within the same experiment (e.g., in dose-response studies).
Basophils were also purified to near homogeneity (>95%) using a combination of Percoll gradients and negative selection with the "Basophil Isolation Kit" according to the manufacturer’s recommendations (Miltenyi Biotec, Bergisch Gladbach, Germany). Using highly purified basophils from three different donors, we found that the purity of basophils did not influence the results under the different experimental conditions used in this study, except for dose-response and kinetic studies, which could not be repeated with pure basophils due to the necessity of larger numbers of this rare cell type.
Mediator release from human basophils
The assay was performed on basophil preparations as previously reported (29). Cells were resuspended in HACM buffer (HA buffer supplemented with 1 mM CaCl2 and 1 mM MgCl2) at a cell density adjusted to obtain 1 x 106 basophils/ml. Cells were preincubated in a shaking water bath for 15 min at 37°C before adding the first stimulus as indicated or buffer control. After an additional 10 min, the second agent or buffer control was added, and incubation continued for a further 20 min (a time sufficient for maximal mediator release under all experimental conditions). The reaction was stopped by cooling the cell suspension on ice. In kinetic experiments, mediator release was stopped by the rapid addition to the cells of 1 volume of ice-cold buffer at the time points indicated. Histamine and LTC4 were measured in the supernatants using an automated fluorometric method (30) and RIA (31, 32), respectively.
Culture conditions
Basophil preparations were resuspended at a cell density of 1 x 106 basophils/ml in culture medium and incubated in sterile round-bottom 96-well microtiter plates using 200 µl/well (Becton Dickinson, Lincoln Park, NJ) at 37°C in a humidified atmosphere with 5% CO2. Different agents were added to the cells at 37°C on a thermostated heater within 15-min intervals, except for the experiments in which a different time interval is specified. After 18 h of cell culture, cell-free supernatants were harvested and stored at -20°C until the measurements of cytokine production by ELISA and LTC4 synthesis by RIA (31, 32). IL-4 was measured using the "Eli-pair" kit supplied by Diaclone (Besançon, France), according to manufacturer’s protocols. IL-13 ELISA was performed as described (10) with anti-IL-13 mAb pairs from PharMingen (San Diego, CA) and IL-13 standards from Peprotech EC (London, U.K.). All the data were derived within the linear dynamic range of the assays by diluting the samples when needed.
Presentation of data
All experiments were performed in duplicate or triplicate with variations of <10% of the mean values. All experiments were repeated at least three times, demonstrating that the pattern of results was always reproducible. However, the amounts of mediators released differed among experiments due to the well-known variability of mediator release and cytokine expression among basophils from different donors (9, 10). Thus, for clarity of presentation, the mean values of replicates of representative single experiments are shown for dose-response curves and kinetic experiments. The relatively large SE values obtained from the combined data of independent experiments from different donors are due to the donor variability rather than variations in the pattern of results. Histamine release was expressed as the percentage of the total cellular histamine content determined by cell lysis and cytokines or LTC4 as weight per million basophils.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C5a induces exocytosis in basophils and, after a short
preincubation with IL-3, also a rapid and transient burst of LTC4
generation. Three C5a C-terminal hexapeptide analogs, CO28, CO26, and
CO61, which differ only at amino acid position 5, display agonistic and
partial agonistic properties on neutrophil functions despite similar
binding affinities to the C5aR in the nanomolar range
(19). These peptides and C5a were tested for their
activity profile on basophils with and without priming with IL-3. Fig. 1
shows that CO28 is a full agonist for
degranulation and induces LTC4 formation in IL-3-primed cells with
similar efficacy as C5a, but with an 
3 log lower potency, consistent
with the correspondingly lower binding affinity for the C5aR. The
partial neutrophil agonists CO26 and CO61 (19) did not
induce mediator release in unprimed cells, and only CO26 promoted some
exocytosis and low levels of LTC4 release after preincubation of the
cells with IL-3 with similar potency as CO28. The kinetics of the
release responses to CO28 and the dependence on the order of IL-3 and
peptide addition is shown in Fig. 2. Thus, the bioactivities of CO28 on
these rapid basophil responses are identical with those induced by C5a
and C5adesarg (23, 27, 33).
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Recent studies have shown that culturing basophils with IL-3 and
C5a leads to the expression of considerable amounts of the
immunoregulatory cytokines IL-4 and IL-13 in parallel with a
continuous-phase LTC4 production (9, 10). The cytokines
and LTC4 are formed steadily during several hours and reach maximal
levels after 16–20 h of culture. In contrast to the rapid release
responses, these late cellular functions do not depend on the order of
addition of the stimuli to the cells (9). To study whether
signaling through the putative transmembrane activation site is
sufficient for inducing cytokine release and the late phase of LTC4
production, basophils were cultured with different concentrations of
C5a or the three hexapeptides for 18 h. To prevent the rapid burst
of LTC4 formation, all of the agonists were added before IL-3 in these
experiments. Fig. 3 shows that CO28 was
an effective agonist for cytokine release and continuous LTC4
formation, with an 1000 times lower potency on a molar basis
compared with C5a. However, CO26 and CO61 were completely inactive
under these conditions, because no LTC4 was detectable and IL-4 and
IL-13 levels did not exceed that induced by IL-3 alone. Thus, CO26
behaves like many other basophil agonists, such as IL-8,
platelet-activating factor, and eotaxin, which interact with other
chemoattractant seven-transmembrane receptors and are only able to
induce rapid release responses and/or chemotaxis (3).
Interestingly, CO28 was a superagonist for the induction of a late
phase of LTC4 production, but not for IL-4 and IL-13 synthesis and
release as shown in Fig. 3. This difference of lipid mediator formation
was statistically significant when the response to maximally effective
concentrations of C5a and of CO28 was compared in 10 independent
experiments with cells from different donors (87.2 ± 8.2 SEM vs
132.8 ± 14.4 SEM ng of LTC4 per million basophils;
p < 0.05 paired t test; for C5a + IL-3 vs
CO28 + IL-3, respectively).
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A mAb against a N-terminal C5aR epitope inhibited the response to
C5a but not to the agonistic hexapeptide (Fig. 4). In fact, there was even a trend for
an enhancement of peptide-induced IL-4 production in the presence of
the Ab. The anti-C5aR Ab did not affect the anti-IgE receptor
Ab-induced mediator release used as a control.
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shows that both IL-4 and LTC4
release by basophils cultured with an optimal concentration of C5a and
IL-3 could be inhibited dose-dependently by the C-terminal analog CO61.
Thus, in this experimental system, CO61 acts as a full antagonist.
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To determine whether continuous signaling through the C5aR is
still required hours after the addition of C5a, we added the
antagonistic C-terminal analog, CO61, at different time points after
stimulating the basophils with C5a and IL-3. Fig. 6 shows that indeed both cytokine as well
as LTC4 release could be inhibited even after 8 h of stimulation.
Moreover, the amounts of IL-4 and LTC4 produced in the presence of CO61
peptide added at different time points corresponded to the amounts of
both products generated at the corresponding time points (as shown by
the kinetics of the release after C5a + IL-3 addition reported earlier
(9) and illustrated in the insets). This inhibition is
specific as CO61 did not influence basophil responses to anti-IgE
receptor Ab.
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C5adesarg has a different bioactivity profile on late continuous responses of basophils
In contrast to the anaphylatoxin C3a, C5a retains bioactivity
after cleavage of the C-terminal arginine by serum carboxypeptidase N.
However, C5adesarg is generally less potent and
has a different pattern of activity than intact C5a dependent on the
cell type and response under investigation (23, 34, 35).
In human basophils, C5adesarg induces exocytosis
and, in IL-3 primed cells, also a burst of LTC4 formation with similar
efficacy but 1 log lower potency compared with C5a and is thus a full
C5aR agonist for the rapid responses of basophils (23).
However, when cells were stimulated with
C5adesarg followed by IL-3 and cultured for
18 h, LTC4 production was minimal up to a concentration of 100 nM
C5adesarg (Figs. 7 and 8). Thus, in contrast to the rapid
transient burst of LTC4 release, the late continuous phase of LTC4
generation is inefficiently induced by C5adesarg.
Nevertheless, C5adesarg efficiently promoted
cytokine production and release with about a 1 log lower potency as
C5a, showing that the C-terminal arginine is not required for this
long-lasting response of basophils. Somewhat unexpectedly, IL-13
production in response to C5adesarg was almost
twice as high as that induced by its precursor C5a (70% mean increase
of IL-13, as determined in seven independent experiments;
p < 0.01).
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). Furthermore, pertussis toxin
added 3 h after cell stimulation with
C5adesarg blocked these ongoing responses to the
same extent as CO61. (Percent inhibition of product release by the
antagonistic peptide and pertussis toxin, respectively, are as follows:
IL-4, 94% ± 2.4 SEM and 98% ± 0.5 SEM; IL-13, 95% ± 2.8 SEM and
100% ± 3.3 SEM; n = 3). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The requirements for the ligand receptor interaction to induce the continuous release of cytokines and LTC4 were studied using peptides and receptor Abs. Our experiments show that the activation site of the C5aR is clearly sufficient to induce all cellular responses. However, only the most active analog, CO28, was able to induce IL-4/IL-13 and LTC4 release under these conditions, and even the most active partial agonist for neutrophils, CO26, which differs from CO28 only by the presence of a phenylalanine instead of a cyclohexylalanine at position five, was completely inactive. It thus appears that late cellular responses are more dependent on an optimal ligand receptor interaction and signaling strength than are other cellular functions. Furthermore, the weak partial C5aR agonist for neutrophils CO61 acted as a full antagonist, further demonstrating that the putative transmembrane activation site is both sufficient and necessary for these late basophil responses induced by C5a. With regard to the whole C5a molecule, an interaction of the core of C5a with the negatively charged N-terminal C5aR domain is also involved in binding but not in receptor activation, as shown by inhibition with the mAb S5/1 known to bind to the negatively charged motif DLTDKDD (25).
Several conclusions can be drawn from experiments in which the antagonistic peptide was added at different time points after stimulating cells with C5a and IL-3. If activation of the C5aR during a short time period would lead to an irreversible signaling cascade, then the antagonist should only inhibit the response if added before but not shortly after C5a. Obviously, this was not the case. Even when the peptide was added 1 and 4 h after C5a, the inhibitory effect persisted, indicating that any signaling event or gene expression having occurred within this time frame does not lead to cytokine and LTC4 production in an irreversible manner. This is particularly evident for lipid mediator formation starting with a lag time of 2–4 h after addition of stimuli (9). Finally, significant inhibition is still observed by antagonizing the C5aR with the hexapeptide CO61 8 h after cell activation when cytokines and LTC4 are released at a maximal rate, indicating that even during the period of product release there is a continuous ligand-receptor interaction needed for an ongoing response. The conclusion that continuous signaling through the C5aR is required for the late responses is further supported by the fact that pertussis toxin added 3 h after C5a blocks IL-4, IL-13, and LTC4 release to the same extent as the antagonistic peptide. In addition, this experiment showed that not only the well characterized rapid responses but also late cellular events are all mediated through pertussis toxin-sensitive G proteins.
Particularly through the introduction of leukotriene antagonists as efficient antiasthmatic drugs, it has become evident that LTC4 is a major mediator of chronic allergic inflammation and of allergic late-phase reactions. In vivo, LTC4 is produced continuously and in the absence of an acute exposure to allergen, indicating that effector cells produce lipid mediators in response to IgE-receptor-independent activation for prolonged periods. The combined stimulation of basophils with C5a and IL-3 results in lipid mediator formation for many hours, in contrast to all other cell types and modes of activation for which only a transient burst of lipid mediator release is observed (e.g., IgE-receptor cross-linking or activation of primed cells by chemotactic agonists). Thus, the experimental model investigated in this work is likely to reflect more closely the in vivo situation than other in vitro models. This late phase of LTC4 production appears to be most susceptible to the structure of the C5a C terminus and the way in which it interacts with the putative activation site of the C5aR. The agonistic peptide analog was a superagonist for continuous LTC4 formation but not for cytokine production. In contrast, C5a lacking the C-terminal arginine only weakly induced lipid mediator formation even at concentrations sufficient to maximally induce cytokine release. In fact, the efficacy of C5adesarg to induce the synthesis of IL-4 was identical with C5a and was even higher for IL-13 (almost twice of that of C5a), another difference in bioactivity of the desarg form of C5a. The bioactivity of C5adesarg is also clearly distinct from that of the most active partial agonist CO26 (see above). Thus, C5adesarg is not just a weaker or partial agonist, but rather promotes a distinct profile of the late cellular responses. In vivo, C5a is rapidly degraded to C5adesarg, indicating that persistent LTC4 production may only occur if C5a is continuously generated. However, the immunoregulatory function of basophils expressing IL-4 and IL-13 seems less dependent on the timing and kinetic of complement activation, because C5adesarg is very stable in vivo and cleared only by receptor-mediated endocytosis (1).
The molecular nature of interaction between the C5a C terminus and the putative activation site of the receptor is still ill-defined. For small agonistic peptides, an interaction of the C-terminal arginine with Arg206 in the fifth transmembrane helix of the C5aR has been postulated (36). Also unknown is the molecular basis of the distinct bioactivity of C5adesarg. The fact that the release of IL-4 and IL-13 in response to C5adesarg can be blocked by the anti-N-terminal C5aR mAb as well as by the C-terminal analog CO61 demonstrates that C5adesarg does not act through another unknown receptor. Furthermore, C5a and C5adesarg must use similar or at least very proximal interaction sites on the C5aR and both signal through pertussis toxin-sensitive G proteins. A recent elegant study provided evidence for an interaction between Glu199 of the C5aR with Lys68 of C5adesarg (37). This interaction was necessary for activating the cells by C5adesarg but not by C5a. However, in the bioassay used there were no qualitative differences of the response of the human C5aR-transfected cell-line (RBL-2H3) upon activation by C5a or C5adesarg, and therefore it is unclear whether this interaction is also important for the differences in signaling by these ligands. The fact that the activity of an agonistic peptide was also dependent on Glu199 of the C5aR (the study of Crass et al. (37)) and that the agonistic peptide CO28 acted like C5a rather than like C5adesarg (this study) argues against the hypothesis that this Glu-Lys interaction is responsible for the distinct bioactivity of C5adesarg. It would be important to find C5a analogs that mimic the effect of C5adesarg rather than that of C5a, allowing a better characterization of the molecular basis of the differences in ligand-receptor interaction responsible for distinct activities of C5a and C5adesarg. In other cells, it is not quite clear whether peptides with full or partial agonistic activity act as C5a or rather as C5adesarg mimics. For example in neutrophils, the most preserved function of partial peptide agonists is chemotaxis (19), similar to C5adesarg, which also primarily induces chemotaxis rather than activation of other effector functions (35). The late-phase response of basophils described here thus provides a unique model to further study these questions, because cytokine expression allows the distinction of partial C5a agonists from C5adesarg while LTC4 formation allows the discrimination of C5a-like from C5adesarg-like effects.
In summary, our study shows that, similar to the rapid responses of neutrophils and basophils, the putative transmembrane binding site of the C5aR is also responsible for activating the signaling pathways leading to continuous LTC4 generation and IL-4 and IL-13 production in basophils. The G protein-coupled C5aR can be constantly activated for many hours to promote certain cellular functions. However, these late cellular responses seem to be more strongly influenced by the type of interaction at the putative activation site. The distinct bioactivities of C5a, C5a C-terminal mimics, and C5adesarg suggest that this part of the receptor is also important for differential stimulation of the cellular functions by minor alterations of ligand-receptor interactions. This study also shows that C5adesarg can induce cellular responses that are qualitatively different from C5a. The efficient induction of the immunoregulatory cytokines IL-4 and IL-13 by C5adesarg in combination with IL-3 in basophils indicates a role for complement in the regulation of the Th1/Th2 balance, which is not controlled by inactivation of the anaphylatoxin through carboxypeptidases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Clemens A. Dahinden, Institute of Immunology and Allergology, Inselspital, CH-3010 Bern, Switzerland.
3 Abbreviations used in this paper: C5a, anaphylatoxin derived from the fifth complement component; C5adesarg, C5a cleavage product lacking the C-terminal arginine; C5aR, C5a receptor (CD88); LTC4, leukotriene C4; NMePhe, N-methyl phenylalanine; Cha, cyclohexylalanine; Na1, 1-naphthyl.
Received for publication March 13, 2000. Accepted for publication June 6, 2000.
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References |
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RI): analysis of functional domains of the -subunit with monoclonal antibodies. J. Biol. Chem. 266:11245.This article has been cited by other articles:
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) or before (
) the cytokine.
Histamine release (left) and LTC4 generation
(right) are shown. Arrows indicate the time of addition
of the stimuli. A representative experiment is shown.
) or
absence (
