Immunology 1992 77 416-421

Calcitonin gene-related peptide (CGRP) activates human neutrophils inhibition by chemotactic peptide antagonist BOC-MLP J. RICHTER, R. ANDERSSON,* L. EDVINSSONt & U. GULLBERG Division of Haematology and tDivision of Angiology, Department of Medicine, University Hospital, Lund and *Department of Cell Biology, University of Linkoping, Link6ping, Sweden Acceptedfor publication 15 June 1992

SUMMARY The effect of the neuropeptides substance P, neurokinin A and a-calcitonin gene-related peptide (CGRP) on human neutrophil granulocytes was investigated. Substance P induced secondary granule secretion at a concentration of 100 yM. CGRP induced a significant secretory response at 10 pM and thus appeared to be about 10 times more potent than substance P. Calcitonin and a fragment of CGRP, CGRP(8-37), had no effect on neutrophil degranulation. The chemotactic peptide antagonist BOC-MLP (100 yM) inhibited lactoferrin secretion mediated both by CGRP and chemotactic peptide FMLP almost completely, while secretion in response to tumour necrosis factor (TNF) was unaffected. Results from receptor binding studies showed that CGRP and N-formylmethionyl-leucyl-phenylalanine (FMLP) do not compete for binding. This indicates that CGRP does not exert its effects by binding to the chemotactic peptide receptor. CGRP induced a rapid increase in the cytosolic-free calcium concentration and this increase was not, unlike that induced by FMLP, abolished by preincubation of the cells with pertussis toxin (1000 ng/ml). Therefore CGRP signal transduction in neutrophils appears to involve rapid changes in the cytosolic-free calcium concentration but not a pertussis toxin-sensitive G-protein. In summary, this is the first report to show that CGRP can directly activate neutrophil granulocytes, and this probably occurs via a cell surface receptor which is distinct from that of FMLP although both the CGRP and FMLP-mediated effects can be blocked by BOC-MLP.

INTRODUCTION The undecapeptide substance P (SP) is widely distributed throughout the central and peripheral nervous system where it probably functions as a neutrotransmitter.' In addition, it can be released from some primary sensory nerves following axon reflexes and also affect non-neuronal tissues.2 A number of studies have suggested that SP is involved in the inflammatory response. SP can induce a vasodilator response, cause tissue oedema and also directly affect inflammatory cells.3 The neuropeptide can for example, stimulate lymphocyte proliferation,4 Ig synthesis,5 mast cell degranulation6 as well as neutrophil and macrophage function.7-'0 To describe this link between the nervous system and the immune response the term neurogenic inflammation has been used. The neutrophil granulocyte plays an important role in the inflammatory process and the effect of SP on neutrophil function has been described in several studies. SP triggers the respiratory burst as well as secretion of both primary (azuro-

philic) and secondary (specific) granule constituents.8 SP is chemotactic for neutrophils, induces aggregation of these cells and can also modulate neutrophil-dependent cytotoxicity against antibody-coated targets." On rabbit neutrophils SP has been shown to bind to the same receptor as the chemotactic peptide FMLP.7 However, several investigators have presented data that indirectly show that this is not the case for human

neutrophils.8",2 Two other neuropeptides also stored in and released from peripheral terminals of primary neurons are a-calcitonin generelated peptide (CGRP) and neurokinin A (NKA). CGRP is a 37 amino acid peptide which is generated from the same gene as calcitonin by alternative splicing of the mRNA transcripts.'3" 4 CGRP is a potent dilator of peripheral blood vessels.'5 NKA is

co-stored and co-released with SP and also believed to bind to the same receptor. 16 While SP, as noted above, has been described to affect directly neutrophil granulocytes, little is known of the effect of CGRP and NKA on these cells. In this work we have investigated the effects of SP, CGRP, NKA on neutrophil granulocytes and also compared the effects to that of the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP).

Correspondence: Dr J. Richter, Research Department 2, E-blocket, University Hospital, S-221 85 Lund, Sweden.

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Activation of human neutrophils by CGRP MATERIALS AND METHODS Special reagents

Anti-lactoferrin and anti-myeloperoxidase (MPO) were obtained by immunization of rabbits with purified human proteins. The antisera were highly specific and did not react with other neutrophil proteins, as judged by experiments with radial immunodiffusion. SP, FMLP, BOC-methionyl-leucyl-phenylalanine (BOC-MLP), human calcitonin and pertussis toxin were from Sigma Chemical Co. (St Louis, MO). Human-a-CGRP was from Bachem Feinchemikalien AG (Budendorf, Switzerland). Human-a-CGRP (8-37) and human NKA were from Peninsula Laboratories (Merseyside, U.K.). Tritiated FMLP (formyl-Met-Leu-[3H]Phe) with a specific activity of 55-8 Ci/ mmol was obtained from New England Nuclear Research Products (Boston, MA). Recombinant human tumour necrosis factor (TNF) was kindly supplied by Dr G.R. Adolf (Ernst Boehringer Institut, Vienna, Austria). Fura-2/AM and fura-2 free acid were from Molecular Probes (Eugene, OR). Isolation ofgranulocytes Blood was obtained from healthy volunteers. One part of 2% Dextran (Pharmacia, Uppsala, Sweden) in 015 M NaCl was added to one part of heparinized blood. After red cell sedimentation the leucocyte containing supernatant was layered on top of Lymfoprep (Nyegaard and Co, Oslo, Norway) and centrifuged, resulting in a pellet containing granulocytes and erythrocytes. After hypotonic lysis of the erythrocytes with sterile water for 30-45 seconds, the granulocytes were washed once in 0-15 M NaCl and once in Hanks' balanced salt solution (HBSS; Flow Laboratories, Irvine, U.K.) with 1% foetal bovine serum (FBS) (Gibco, Paisley, U.K.). A Coulter Counter was used for cell counting. Initially a Burker chamber was used for checking the Coulter Counter readings and granulocyte purity, which was from 96 to 98%. Viability was in excess of 97%, as judged by trypan blue staining. The cells were allowed to equilibrate for at least 30 min in HBSS with 1% FBS before being used in the haemolytic plaque assay.

Coupling of protein A to sheep red blood cells Protein A (Pharmacia) was coupled to sheep red blood cells (SRBC; kindly supplied by the Dept. of Microbiology, University of Lund, Sweden) as previously described.'7 In short, SRBC, stored in Alsevers solution and aged 1-3 weeks, were washed three times in 0-15 M NaCl. One part packed SRBC was mixed with one part protein A (0-5 mg/ml in 0-15 M NaCl) and 10 parts 2-5 x 10-4 M CrCI3 in 0 15 M NaCl. The mixture was incubated at 300 for 1 hr, washed once in 0 15 M NaCl and twice in HBSS, and kept at 4°. The cells were used within 1 week.

Haemolytic plaque assay We have previously described a modification of the original monolayer plaque assay.'8 Plastic dishes, 60 mm in diameter (Falcon Plastics, Oxnard, CA), were treated with poly-L-lysine solution (25 pg/ml 015 M NaCl; Sigma) for 15 min at room temperature. The dishes were rinsed once in 0 15 M NaCl and once in HBSS followed by addition of 1 ml of a 1% suspension of protein A-SRBC in HBSS. After 15 min of incubation at 40, surplus protein A-SRBC were poured off, followed by addition of anti-lactoferrin (1/2560) or anti-MPO (1/300), guinea-pig complement (1/100; Flow Laboratories), granulocytes and a

stimulatory agent dissolved in HBSS. Approximately 330 granulocytes were added to each dish. The dishes were incubated for 2 hr at 370 and then fixed with a 5% solution of glutaraldehyde in phosphate buffer and stored at 4° until the number ofhaemolytic plaques was counted under a microscope. It should be noted that the haemolytic plaque assay is semiquantitative as the number of cells that secrete lactoferrin or MPO is determined, not the amount of protein secreted. Determination of cytosolic-free calcium concentration The medium used for fura-2 loading and fluorometric analysis contained 136 mm NaCl, 4-7 mm KCI, 1 2 mm MgSO4, 11 mM CaCl2, 0.1 mm EGTA, 1.2 mm KH2PO4 5 mm NaHCO3, 55 mM glucose and 20 mm HEPES (pH 7 4). Cells were suspended in the previously described medium at a concentration of 5 x 106 cells/ ml and then incubated with 2 yM fura-2/AM (from 2 mm stock solution in DMSO) for 20 min. Cells were kept on ice until used. In experiments where pertussis toxin was used, batches of cells were incubated with 1000 ng/ml of the toxin for 2 hr at 370 with frequent agitations; fura-2/AM was added to the cell suspension during the last 20 min. Immediately before use I ml aliquots of cell suspension were spun down and resuspended in 2 ml of the above medium in a quartz cuvette. Fluorescence measurements were performed with a Perkin-Elmer fluorimeter (LS 3B). The cuvette holder was thermostated to 370 and equipped with a continuous stirring device. The excitation and emission wavelengths were 340 and 510 nm, respectively. Calibrations of the fluorescence signal were carried out as previously described.'9

Determination of formyl-Met-Leu-[3H]Phe binding to neutrophils. Neutrophils (107 cells) were incubated with 4 5 nM tritiated FMLP in binding buffer [phosphate-buffered saline (PBS) and 1% bovine serum albumin (BSA)] with or without unlabelled CGRP or FMLP in a total volume of 200 Ml in 1 5-ml Eppendorf centrifuge tubes. After incubation by rotation for I hr at 40 the cell suspensions were layered on top of siliconoil and centrifuged for 10 seconds at 8000 g, thus separating the cells from unbound peptide in the supernatants. The pellets were resuspended and transferred to liquid scintillation vials for measurement of cellbound radioactivity. All experiments were performed in triplicate.

RESULTS

Effect of neuropeptides on lactoferrin secretion from adherent neutrophil granulocytes In the haemolytic plaque assay neutrophils were exposed to various concentrations of three different neuropeptides, CGRP (Fig. Ia), NKA and SP (Fig. lb). At concentrations of 10 yM NKA and SP had little effect on lactoferrin secretion from adherent neutrophils (Fig. lb). At 100 gM the effect of SP was more pronounced. CGRP, on the other hand, induced a marked secretory response at a concentration of 10 gM. From the doseresponse curves CGRP thus seems to be 10 times more potent than SP as a neutrophilic secretagogue. A C-terminal fragment of human CGRP consisting of amino acids 8-37, CGRP(8-37), had no effect on lactoferrin secretion (Fig. la). For comparison the effect of the chemotactic peptide FMLP is shown in Fig. Ic. Calcitonin possesses structural characteristics similar to that of

J. Richter et al.

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Figure 1. Effect of CGRP, CGRP(8-37), calcitonin, SP, NKA and FMLP on lactoferrin secretion. In the plaque assay neutrophils were exposed to various concentrations of: (a) CGRP (0), CGRP(8-37) (-), calcitonin (v); (b) SP (0), NKA (0); (c) FMLP (A). The values given are mean + SEM from three to eight separate determinations. SEM values not given fall within the outline of the corresponding symbol.

CGRP. However, we found no effect of calcitonin on lactoferrin secretion (Fig. 1 a). The effect of CGRP on secretion of primary granule content was also assayed, using MPO as a marker for primary graunules. CGRP, in contrast to the forbolester phorbol myristate acetate (PMA), did not induce degranulation of primary granules (data not shown).

100%

V 60%-

Effect of BOC-MLP on lactoferrin secretion induced by CGRP, FMLP and TNF BOC-MLP is a structural analogue to FMLP which binds to the chemotactic peptide receptor and functions as a competitive antagonist.20 Neutrophils were preincubated with 100 yM BOCMLP (in HBSS with 0-1% DMSO) at 370 for 5 min prior to assay of degranulation. The cells were then exposed to CGRP (10 IM), FMLP (100 nM) in the presence of 100 pM BOC-MLP in the plaque assay. Control cells were treated in the same way, but not exposed to the peptide antagonist. BOC-MLP reduced FMLPinduced secretion by 87% and CGRP-induced secretion by 91% (Fig. 2). To exclude an unspecific effect of BOC-MLP on the cells, TNF, which also binds to receptors on the neutrophil surface, was used as a positive control. BOC-MLP did not inhibit TNF-induced lactoferrin secretion. Effect of CGRP

on

formyl-Met-Leu-13HIPhe binding to neutro-

phils

Neutrophils were incubated with 4 5 nM tritiated FMLP with or without unlabelled CGRP or FMLP for 1 hr at 4°. Cell-bound radioactivity was then measured to determine binding of formyl-Met-Leu-[3H]Phe to the cells. While unlabelled FMLP competed with binding of formyl-Met-Leu-[3H]Phe in a dosedependent manner, no effect of CGRP was seen even at a concentration as high as 30 pM (Fig. 3). Essentially the same results were obtained when the experiment was carried out at 20° (data not shown).

A A*40%01 2. E* 20%-

TNF

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Figure 2. Effect of BOC-MLP on lactoferrin secretion induced by CGRP, TNF and FMLP. Neutrophils were preincubated with 100 gm BOC-MLP at 37° for 5 min and then exposed to 10 pm CGRP, 1 nm TNF or 100 nM FMLP in the presence of 100 ,UM BOC-MLP in the plaque assay. Lactoferrin plaques are expressed as percentages of plaques formed by cells incubated without BOC-MLP but otherwise treated in the same way. The values given have been corrected for spontaneous plaque formation and are mean+SEM from three to seven separate determinations.

Effect of CGRP on concentration of cytosolic-free calcium in neutrophils Changes in cytosolic-free calcium were measured with a spectrophotometer as fluorescence of fura-2 loaded cells in suspension. Representative data from several measurements of changes in the cytosolic-free calcium concentrations in response to CGRP and FMLP are shown in Fig. 4. CGRP (10 gM) caused a rapid rise in the cytosolic-free calcium concentration (Fig. 4a). The response to CGRP was, however, not of the same magnitude as when neutrophils were stimulated with 100 nM FMLP (Fig. 4b). The slope of the rapid rise in calcium in response to CGRP was

Activation of human neutrophils by CGRP

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also consistently less steep compared to the FM LP response. No changes in cytosolic-free calcium were seen when the cells were exposed to CGRP at lower concentrations (1 yM and 100 nm, data not shown).

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Effect of pertussis toxin on changes in cytosolic-free calcium induced by CGRP

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Figure 3. Neutrophils were incubated with 4-5 nM tritiated FMLP in binding buffer (PBS and 1% BSA) with or without unlabelled CGRP or FMLP. After incubation by rotation for 1 hr at 4' the cell suspensions were layered on top of siliconoil and centrifuged, thus separating the cells from unbound peptide in the supernatants. The pellets were resuspended and cell-bound radioactivity was measured. The values given are means from two separate determinations. [Ca 2+]

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In order to investigate the possible involvement of a pertussis toxin sensitive G-protein in CGRP-induced activation of neutrophils cells were preincubated with pertussis toxin (1000 ng/ ml) for 2 hr at 37~prior to determination of the cytosolic-free calcium concentration. Pertussis toxin treatment completely abolished the changes in cytosolic-free calcium elicited by FMLP (Fig. 4d). CGRP, on the other hand, induced a rapid rise in cytosolic free calcium also in cells pretreated with pertussis toxin (Fig. 4c). Preincubation of neutrophils with 0-1 mm BOC-MLP partially blocked the increases of cytosolic-free calcium in response to CGRP and FMLP. One millimolar of BOC-MLP totally blocked calcium changes in response to FMLP and reduced the CGRP-induced calcium transient by 78%.

DISCUSSION The neuropeptide CGRP is a potent vasodilator.'5 When injected intradermally it significantly increases microvascular blood flow.'5 In synergy with inflammatory mediators like the bacterial peptide FMLP or complement fragment C5a CGRP can induce oedema formation and neutrophil accumulation.2' [Ca 2+] nf

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Figure 4. Effect of CGRP and FMLP on the cytosolic-free calcium concentration. Neutrophils were loaded with fura-2 and fluorescence was measured as described in Materials and Methods. The cells were exposed to 10 pm CGRP (a,c) or 100 nm FMLP (b,d). Cells in (c) and (d) were preincubated with pertussis toxin (1000 ng/ml) for 2 hr at 37° prior to calcium measurements. Traces shown are from the same batch of cells and representative of at least four separate experiments.

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Thus CGRP like other neuropeptides localized in and released from small unmyelinated sensory C-fibres (Substance P and Neurokinin A and B) has been implicated as acting as a physiological mediator between the peripheral nervous system and the immune system. However, while several studies have shown that SP has direct effects on neutrophil granulocytes,7 9,11,12 no reports exist to our knowledge, which demonstrate that CGRP alone can activate neutrophils. In the present study we have confirmed previous observations showing that SP can induce secretion of secondary granule content from neutrophils.8 In accordance with those findings SP at a concentration of 100 yM was necessary to induce secretion to a significant degree. NKA proved to be only a weak inducer of secretion, thus our findings correlate with those of Brunelleschi et al. who investigated the priming effects of NKA on superoxide anion production.22 CGRP on the other hand induced a marked secretory response at a concentration of 10 LM. The number of plaque-forming cells in response to 10 yM CGRP was about 50% of the number seen in response to 100 nM FMLP. In the degranulation assay CGRP appeared to be approximately 10 times more potent than SP. Also if one compares the concentration of CGRP used in our measurements of intracellular calcium with the concentration of SP necessary to induce a significant change in the cytosolic-free calcium concentration in two other studies, it appears that CGRP is about one order of magnitude more potent than SP as an activator of neutrophil granulocytes.8'23 Even if the concentrations of CGRP used in this study to activate neutrophils are much higher than those required for a vasomotor response it is possible that CGRP at low concentrations can act in concert with other inflammatory mediators to activate neutrophil granulocytes. Thus the present study gives further support to the concept of neurogenic inflammation. CGRP and calcitonin are generated from the same gene by alternative splicing of the mRNA. The two peptides possess similar structural characteristics (similar size, a disulphide bridge at the NH2-terminal portion and an amidated carboxyterminal end) even though they exhibit little sequence homology. Distinct receptors exist for calcitonin and CGRP in various cells, but because of the structural similarities the two peptides have also been shown to cross-react at the receptor level.2426 Calcitonin, however, in contrast to CGRP had no effect on lactoferrin secretion from adherent neutrophils in our degranulation assay. We also tested a fragment of CGRP consisting of amino acid 8-37 and found that it could not induce degranulation. Thus it seems that the ring structure with the disulphide bond at the NH2-terminal is necessary for cell activation.27 The inhibitory effect of BOC-MLP on CGRP-induced secretion suggests that CGRP in neutrophils may also act via a cell surface receptor. However, the results raised two possibilities concerning the nature of this putative receptor. BOC-MLP has been considered to be a specific antagonist of FMLP20 and therefore the results may be interpreted as CGRP binding to FMLP receptor. This, however, seems unlikely in the light of the differences in size and structure between the two peptides. Another possibility is that CGRP acts via a distinct receptor, but that BOC-MLP can block this receptor also. In order to distinguish between these two possibilities we performed binding studies with tritiated FMLP and tried to explore the signaltransduction mechanisms involved in CGRP-mediated acti-

vation of neutrophils. We were unable to show any displacement of tritiated FMLP from the cells when they were exposed to CGRP at a concentration of 30 kLM, while 10 nM FMLP reduced binding of the radioactive ligand. This indicates that FMLP and CGRP do not bind to the same cell surface receptor. In other cell types that have been studied, CGRP activates the adenylate cyclase-cAMP system.25'27 However, as many receptor-operating neutrophil stimulants (like FMLP, ATP, C5a, interleukin-8) use increases in cytoplasmic-free calcium as a second messenger we were interested in determining if this was also the case for CGRP. Indeed we did find that CGRP induced an increase in cytosolic-free calcium, but this response was slower and of lower magnitude than the response to FM LP. The experiments with pertussis toxin show more clearly the differences in signal-transduction mechanisms between CGRP and FMLP. It is well known that pertussis toxin abolishes second messenger generation from chemoattractant receptors, even at concentrations as low as 200 ng/ml.28 However, although we used a fivefold higher concentration of the toxin in the present study, only minor effects were seen on CGRP-induced calcium transients. This indicates that if a G-protein is involved in CGRP signal transduction, it must differ from the G-protein linked to the FMLP receptor. To summarize, this is the first report to show that CGRP can directly activate neutrophil granulocytes. From binding studies and the signal-transduction mechanisms involved this seems to occur via a pathway distinct from that employed by FMLP even though it can also be blocked by BOC-MLP. It is likely that CGRP activates neutrophils via a cell surface receptor but our data cannot fully prove this. Thus the existence of CGRP receptors on neutrophils remains to be established definitely and also whether these putative CGRP receptors are identical to CGRP receptors on other cells. It would also be of interest to determine whether BOC-MLP can block the action of CGRP in other tissues, for example blood vessels.

ACKNOWLEDGMENTS The authors wish to thank K. Vegas for excellent technical assistance. This work was supported by the Swedish Cancer Society, the Alfred Osterlund Foundation, the King Gustav V Memorial Foundation and the Medical Faculty of Lund.

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19. GRYNKIEWICZ G., POENIE M. & TsIEN R.Y. (1985) A new generation of Ca2+-indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440. 20. FREER R.J., DAY A.R., BECKER E.L., SHOWELL H.J., SCHIFFMAN E. & GROSS E. (1979) Structural requirements for synthetic peptide chemoattractants and antagonists. In: Peptides, Structure and Biological Function. Proceedings of the Sixth American Peptide Symposium (eds E. Gross and J. Meienhofer). Pierce Chemical Company, Rockford, IL, p. 749. 21. BUCKLEY T.L., BRAIN S.D., RAMPART M. & WILLIAMS T.J. (1991) Time-dependent synergistic interactions between vasodilator neuropeptide, calcitonin gene-related peptide and mediators of inflammation. Br. J. Pharmacol. 103, 1515. 22. BRUNELLESCHI S., TARLI S., Gio-ri A. & FANTOZZI R. (1991) Priming effects of mammalian tachykinins on human neutrophils. Life Sci. 48, PL-1. 23. IWAMOTO I., YAMAZAKI H., NAKAGAWA N., KIMURA A., TOMIOKA H. & YOSHIDA S. (1990) Differential effects of two C-terminal peptides of substance P on human neutrophils. Neuropeptides, 16, 103. 24. MICHELANGELI V.P., FINDLAY D.M., FLETCHER A. & MARTIN T.J. (1986) Calcitonin gene-related peptide (CGRP) acts independently of calcitonin on cyclic AMP formation in clonal osteogenic sarcoma cells (UMR 106-01). Calcif. Tissue Int. 39,44. 25. WOHLWEND A., MALMSTROM K., HENKE H., MURER H., VASSALLI J.-D. & FISCHER J.A. (1985) Calcitonin and calcitonin gene-related peptide interact with the same receptor in cultured LLC-PKI cells. Biochem. biophys. Res. Commun. 131, 537. 26. YAMAGUCHI A., CHIBA T., OKIMURA Y., YAMATANI T., MORISHITA T., NAKAMURA A., INUI T., NODA T.& FUJITA T. (1988) Receptors for calcitonin gene-related peptide on the rat liver plasma membranes. Biochem. biophys. Res. Commun. 152, 383. 27. CHIBA T., YAMAGUCHI A., YAMATANI T., NAKAMURA A., MORISHITA T., INUI T., FUKASE M., NODA T. & FUJITA T. (1989) Calcitonin gene-related peptide receptor antagonist human CGRP(8-37). Am. J. Physiol. 256, E3 11. 28. KRAUSE K.H., SCHLEGEL W., WOLLHEIM C.B., ANDERSSON T., WALDVOGEL F.A. & LEW P.D. (1985) Chemotactic peptide activation ofhuman neutrophils and HL-60 cells: pertussis toxin reveals correlation between inositol trisphosphate generation, calcium ion transients and cellular activation. J. clin. Invest. 76, 1348.