Biochimica et Biophysica Acta, 1055(1990) 193-196

193

Elsevier BBAMCR10262

BBA Report

Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils Errol Lobo, Fred Elfman, Edward Kelly and H. Daniel Perez Rosalind Russell Arthritis Research Laboratories, University of California, San Francisco, and The Medical Service, San Francisco General Hospital, San Francisco, CA (U.S.A.)

(Received8 March 1990) (Revisedmanuscript received30 July 1990)

Key words: Neutrophil; N-Formylpeptide receptor; Phorbolmyristate acetate; Receptorrecycling; Receptorregulation; (Human) We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.

Exposure of human neutrophils to the synthetic peptide N-formyl-Met-Leu-Phe stimulates these cells to migrate in a directed fashion (i.e., respond chemotactically) [1]. This process is initiated by the binding of fMet-Leu-Phe to specific receptors present on the neutrophil membrane [2]. Previously, we reported that fMet-Leu-Phe-induced neutrophll chemotaxis may require the re-expression (or recycling) of a population of formyl peptide receptors (FPR) [3,4]. Recently, we provided evidence indicating that fMet-Leu-Phe-induced neutrophil chemotaxis did not require either marked changes in cytosolic free calcium or specific granule discharge, but could be inhibited by preventing FPR re-expression [5]. These results provided indirect evidence suggesting that human neutrophils are able to re-express FPR (independently from mobilization of an intracellular pool) in a process that required minimal changes in cytosolic free calcium. To examine this possibility further, we have affinity labeled the neutrophll FPR. By stimulating affinity labeled neutrophils with the calcium-independent protein kinase C activator, phorbol myristate acetate (PMA) under low calcium conditions, we have been able to provide the first direct

Abbreviations: FP, N-formyl-Nle-Leu-Phe-Tyr;FPR, formyl peptide receptor; EGS, ethylene glycolbis(succinimidylsuccinate). Correspondence: H.D. Perez, Building 30, Room 3300, San Francisco General Hospital, 1001 Potrero Avenue, San Francisco, CA 94110, U.S.A.

evidence indicating that human neutrophils process FPR in a phenomenon compatible with either receptor recycling or quantal internalization. Highly purified (> 98%), platelet-poor neutrophil suspensions were prepared as described [3]. N-FormylNle-Leu-Phe-Tyr (FP) (Sigma Chemical Corp., St Louis, MO) was radioiodinated with carrier-free Na125I (Amersham, Arlington Heights, IL) by the chloramine T method, as described [4,6]. Specific activity of 125I-FP was 1000 Ci/mmol. Equilibrium binding (4 ° C, 15 min) of 125I-FP (0.1-30 nM) to neutrophils (2.0.106 cells) was assessed as described previously [3]. A collection method involving centrifugation through silicon oil was used to separate bound from free peptide [3]. Nonspecific binding (i.e., binding in the presence of 100-fold excess unlabeled FP) did not account for more than 5-8% of total binding. Affinity labeling of neutrophil FPR was performed as follows. Neutrophils (10 6 cells/ml) suspended in phosphate (10 mM)-buffered 140 mM NaC1, pH 7.4 (PBS) containing 5.0 mM EDTA, were incubated with 1.0 pmol aESI-FP/106 cells for 15 min at 4 ° C to achieve equilibrium binding [3]. After incubation, ethylene glycol bis (succinimidyl succinate) (EGS) (Pierce Chemical Co., Rockford, IL) (0.2 mg/ml) was added, and mixtures incubated for an additional 60 rain at 4 ° C under continuous mixing conditions. At the end of incubation, neutrophils were pelleted by centrifugation ( 1 5 0 × g for 8 min, 4°C) and washed three times with cold buffer. Neutrophils were resuspended in PBS containing 5.0 mM EDTA. Croslinked neutrophils were as capable as control (i.e., non-crosslinked) cells in

0167-4889/90/$03.50 © 1990 Elsevier SciencePublishers B.V. (BiomedicalDivision)

194

A.

B. 600

100

1600 -

2~'~/~1~

X

FP

800-

300

~"'",, FMLP ",,

50 o

0 o

1

T

12 ~251-FP (nM)

T

q

- 2;o Bound (pM)

I

"--fl I

24

I

I

I

I

10-~010-9 10-8 10-7 10-6 10-5

Unlabeled peptide (M)

Fig. 1. A. Binding of 12SI-FP to neutrophils. Cells (2.106/ml) were incubated with increasing concentrations of 12SI-FP at 4 ° C for 15 min in the presence and absence of 100-fold excess unlabeled FP. Reactions were terminated by centrifugation through silicon oil. lnset: Scatchard plot analysis of 125I-FP binding to neutrophils. B. Competition of binding of 125I-FP (5.0 nM) to neutrophils by increasing concentrations of either unlabeled FP (Q O) or FMLP (z~. . . . . . zx). Reactions were carried out as described for panel A.

their ability to generate superoxide anion radicals when challenged with either suboptimal or optimal concentrations of PMA (not shown). In some instances, washed cells were solubilized using 0.5 ml PBS containing 5.0 mM EDTA, 5.0 mM diisopropylfluorophosphate (DFP, Sigma) and 2.0% (w/v) octyl glucoside (Calbiochem, San Diego, CA). For the papain digestion studies, affinity-labeled neutrophils (equal cell numbers in all reactions) were incubated at 4 ° C for 15 min with 5.0 U / m l papain (Sigma). Reactions were stopped by the addition of 1.0 mM (final concentration) cystatin (Sigma). Tubes were placed on ice for 10 min, after which neutrophils were solubilized and samples subjected to SDS-PAGE. Samples were run on SDS-PAGE (reducing conditions, 7-17% gradient slab gels) [3] and dried gels analyzed by autoradiography, as described previously [3]. PMA was from Sigma. fMet-Leu-Phe was from Peninsula Laboratories, San Carlos, CA. Initially, we determined the ability of 125I-FP to 125 interact with the neutrophil FPR. I-FP bound to neutrophils in a specific and saturable fashion (Fig. 1A) exhibiting an EDs0 of approx. 3.0 nM. Scatchard analysis of binding (Fig. 1A inset) was consistent with the presence of high (30600 sites, dissociation constant [KD] 0.14 nM) and low (146000 sites, K D 2.0 nM) affinity binding sites [7]. Binding of 125I-FP to neutrophils could be competed by the presence of either unlabeled FP (EDs0 8.0 nM) or unlabeled fMet-Leu-Phe (EDs0 600 nM) (Fig. 1B). Furthermore, the EDs0 of inhibition exhibited by unlabeled FP and fMet-Leu-Phe were similar to the EDs0 of these peptides required to induce lysosomal enzyme release from cytochalasin Btreated neutrophils (not shown). Thus, FP and fMetLeu-Phe interact with the same receptor on the neutrophil membrane. Neutrophil FPR was affinity labeled using t25I-FP and EGS. After labeling, cells were solubilized and

aliquots subjected to SDS-PAGE, followed by autoradiography. Autoradiograms of affinity labeled neutrophils revealed the presence of a single broad band exhibiting a M r of 50000-65000 (Fig. 2A, lane 1). Labeling was specific since it could be competed by the presence of either 50-fold excess unlabeled FP (Fig. 2A,

B.

AI

MW(xl0-3)

92-69-46-ii~!i!~i!i~i~i/ii~i!'~?i'i/

i!!ii i!iii!ili i !!!ii !i Lane

1 2 3

1

2

Fig. 2. A. Autoradiogram of affinity labeled neutrophil FPR. Lane 1: affinity labeled neutrophil FPR. Lane 2: identical reactions performed in the presence of 50-fold unlabeled FP. Lane 3: identical reactions performed in the presence of 500-fold excess unlabeled FMLP. B. Autoradiogram demonstrating the ability of papain to digest neutrophil 12SI-FPR. Lane 1: neutrophil 1251-FPR, no papain. Lane 2: neutrophil ~2SI-FPR incubated with 5.0 U of papain for 15 min at

4°C.

195 lane 2) or 500-fold excess unlabeled fMet-Leu-Phe (Fig. 2A, lane 3). Neutrophil 125I-FPR can be digested with papain (by limited digestion) to a species exhibiting a M r of approx. 32000-34000 that retains the crosslinked labeled peptide (Fig. 2B) [8]. Consequently, we used papain digestion of extraceUular 125I-FPR to assess distribution of FPR. Intracellular 125I-FPR would be protected from extracellular papain and exhibit its usual M r of 50 00065000 by autoradiography. Extracellular (? re-expressed) 125I-FPR would become papain sensitive and be cleaved to a species of M r 32000-34000. Affinity labeled neutrophils (107 cells/ml) were incubated in PBS containing 5.0 mM EDTA at 37 °C for varying periods of time (5-40 min), in the presence and absence of PMA (10 ng per 3 • 106 cells). At each time point, aliquots (0.5 ml) were removed, placed on ice and 0.5 ml cold buffer containing papain was added. Digestion was allowed to proceed for 15 rain, after which cystatin was added. After digestion, neutrophils were pelleted by centrifugation, washed, solubilized with octyl glucoside and samples subjected to SDS-PAGE followed by autoradiography. In the absence of PMA (Fig. 3, right) no undigested (i.e., papain resistant) 125I-FPR was detected over the 40 min incubation. Degraded

material (some of which probably represents internalized and degraded 125I-FPR) was present after 10 min in the absence or presence of PMA (Fig. 3). When neutrophils were incubated in the absence of PMA (and not subjected to papain digestion) FPR appeared as a species of M r 50000-65000 during all time points examined. Degraded material was present to the same extent as shown in Fig. 3, fight. In the presence of PMA (Fig. 3, left) undigested (i.e., M r 50000-65000, papain-resistant) 125I-FPR was present after 5 min and again after 15, 30, 35 and 40 min. The amount of undigested t25I-FPR at 35 min was less than that observed at 30 and 40 min, suggestive of partial re-expression of internalized 125I-FPR. Two possible explanations exist for the findings reported here. First, is possible that PMA induces quantal internalization of 125I-FPR. This explanation would require fairly constant 'waves' of receptor internalization by stimulated neutrophils. The second possibility (which we favor) [5,9] is that neutrophils recycle 125I-FPR upon stimulation with PMA. PMA induced neutrophils to internalize 125I-FPR after 5 min (i.e., papain-resistant) of incubation. Internalized lzSI-FPR was re-expressed after 10 min (papain-sensitive), reinternalized after 15 min (papain-resistant) and so on. The fact that

MW (xl 0 -3)

92-69-46--

30 m

Incubation time (min)

0

5 10 15 20 25 30 35 40 5 10 1 5 2 0 2 5 11

I

PMA-treated

30 35 40 I

No PMA

Fig. 3. Processing of 125I-FPR by neutrophils. Affinity labeled neutrophils (107 cells in 1.0 ml PBS, 5.0 mM EDTA) were incubated at 37 ° C for varying periods of time in the presence and absence of PMA (10 ng per 3.106 ceUs). At each time point an aliquot was removed, placed on ice and papain added. After 15 min of incubation at 4 ° C, reactions were stopped by the addition of 1.0 mM final cystatin. Tubes were kept at 4 ° C for 10 min, after which neutrophils were washed, solubilized and samples subjected to SDS-PAGE and autoradiography. One of four experiments with essentially identical results.

196 processing of 125I-FPR occurred only u p o n stimulation of neutrophils with P M A suggests a role for protein kinase C in the process. Because F P R was crosslinked, experiments using F P as stimulus are not feasible. H o w ever, processing of 125I-FPR did not occur when neutrophils were stimulated with optimal concentrations of the complement-derived peptide C5a (not shown). It should be noted that in Fig. 3, only a population of F P R is processed b y stimulated neutrophils. This is consistent with our previous report [3]. It is likely that a population of F P R m a y recycle while another population of F P R gets internalized and degraded. Finally, these results are similar to recycling experiments reported using photoaffinity labeled insulin receptors [10] and indicate that dissociation of ligand-receptor complexes is not an absolute requirement for F P R processing by h u m a n neutrophils. This research was supported by U S P H S grants A M 28566, DE-08138 and AI-28290.

References 1 Showell, H.J., Freer, R.J., Zigmond, S.H., Schiffman, E., Aswanikumar, S., Corcoran, B. and Becker, E.L. (1976) J. Exp. Med. 143, 1154-1169. 2 Williams, L.T., Snyderman, R., Pike, M.C. and Lefkowitz, R.J. (1977) Proc. Natl. Acad. Sci. USA 74, 1204-1208. 3 Perez, H.D., Elfman, F., Lobo, E., SEar, L., Chenoweth, D. and Hooper, C. (1986) J. Immunol. 136, 1803-1812. 4 Perez, H.D., Elfman, F. and Lobo, E. (1987) J. Immunol. 139, 1978-1984. 5 Perez, H.D., Elfman, F., Marder, S., Lobo, E. and Ives, H. (1989) J. Clin. Invest. 83, 1963-1970. 6 Niedel, J.S., Wilkinson, S. and Cuatrecasas, P. (1979) J. Biol. Chem. 254, 10700-10706. 7 Koo, C., Lefkowitz, R.J. and Snyderman, R. (1982) Biochem. Biophys. Res. Commun. 106, 442-449. 8 Dolmatch, B. and Niedel, J. (1983) J. Biol. Chem. 258, 7570-7577. 9 Perez, H.D., Marder, S., Elfman, F. and Ives, H. (1987) Biochem. Biophys. Res. Commun. 145, 976-981. 10 Huecksteadt, T., Olefsky, J.M., Brandenberg, D. and Heidenreich, K.A. (1986) J. Biol. Chem. 261, 8655-8659.