Vol.m~ Z~2:. numl~r ~. 412~414 I~IEB~ 09"/!~ I~PH l.+~detaltott of l~urop,~alt I|t,~¢heml~a| ~o~ietie~ 0014~l?9)eglt$)J0

M ~ y 1991

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Generation of interleukin-8 by plasmin from AVLPR-interleukin-8, the human fibroblast-derived neutrophil chemotactic factor H i d e o N~kag¢~wa, Shinji H a t a k e y a n u t , A t s u t o s h i Ikesu¢ and Hisato +M i y a i Faruh.r o f Phormar~utica! So/cures. ;Ta).ama Medical and Pharmaceutical Unlver:dry, SuSlltanL 7"oyanta 930+01, Japan Received 5 March 1991: revised version received 13 March 199 I Phtsmin mai,dy cle;tved the ArlI~,$eP bond o1' ArlpVaI.Leu.Pro.Arll.inletleukin.8 (AVLPR-IL-a} produced by human derm.I Itbroblast=t, which r¢=ulted in the conversionorAVLPR.IL.8 to IL.~t and the in,clive pentapeptide,though, minor cleavall~of AVLPR-IL.It by phtsmin at Ly~':,Ght'* bond occurred. Interl~ukin I~; Fibroblasl; Pla~min: Neutrophil chemolaCti¢ Factor

I. iNTRODUCCTION

In recent years it has become clear that a novel neutrophil chemotactic factor, IL-8, is secreted b~ several types of cells including monocytes, fibroblasts and endothelial cells in response to IL.1 and tumor necrosis factor [1-61. The IL.8 is a peptide consisting of 72 amino acid residues, and its amino acid sequence is identical to the deduced amino acid sequence of a fragment o f the 3.10(2 eDNA sequence [7]. Recently it has been demonstrated that human fibroblasts stimulated with cytokines mainly produce AVLPRIL-8 (FDN(2F) which is the NH.,-terminal extended 77-residue variant of IL-8 [8-10], We report here that plasmin, an important proteinase in the regulation of inflammation, mainly cleaves FDN(2F to IL-8 and the reactive AVLPR pentapeptide. 2. MATERIALS AND METHODS 2.1. Purtfication of FDNCF Human dermal fibroblasts (SF-TY cell line) were maintained in Dulbecco's modified Eagle's medium supplemented with 5°7o fetal calf sert~,m, 25 mM Hepes, penicillin (0, I mg/ml) and streptomycin (O. 1 mg/ml) until they reached confluence. The cells were cultured in serum-free culture medium supplemented with tO "m M recombinant human IL-I# and 0.1o70 bovine serum albumin. After culture for 2 days, cell.free conditioned medium was obtained by centrifugation at 1600× g for 20 rain. The chemoattractant in the conditioned medium

Abbreviations;

AVLPR-IL-8, Ala-Val-Leu-Pro-Arg-interleukin-8; FDNCF fibroblast-derived neutrophil chemot~ctic factor; RP. HPLC, reverse-phase high performance liquid chromatography; SDS-PAGE, sodium dodccyl sulfate polyacrylamide gel electrophoresJs.

Correspondence address: H. Nakagawa, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani, Toyarna 930-01, Japan,

412

was purified essentially ns described previously [11]; the sample was

chromatoliraphedsequential/y on CM-Seph~dex, SephadexO-?$ and RP-HPLC, 2.2. Tree/men/of FDNCF =+'ithplasmin FDNCF (40 j+g) in a final volume o f 0,2 ml ¢0f 50 mM Tris.HCI buffer (pH 8.0) was incubated with 0 , g p g of plasmin (purified from human plasma; Sigma, MO. USA) at 37*C for 30 rain, and the rent. lion was stopped by the addition of leupeptin (final 1 raM), The reae. lion mixture was concentrated by a Speed-Vac centrifuge, dissolved in 6 M 8uanidine solution and loaded onto a C.18 reverse-phase col.

umn (0.45 x l$cm; ODS.120T, Tosoh Co, Tokyo, Japan). Chemotaetic factors were ehtted with a linear co~centration gradient of aeetonitrile from 0% to 50.4% in 0.05% trifluoroacetic acid at z~ flow rate of 0.8 ml/min, Neutrophil chemotaxls of plasmin.treated FDNCF was performed in vitro by using rat and human neutrophils as described previously [121, As an index of ehemotaxis, the number of neutrophils migrated into the lower chamber was expressed as percentage (migration rate) of that of neutrophils applied in the upper chamber, $DS-PAGE was carried out on a slab gel of a discontinuous 9,6%/16,507o acrylamide in tricine buffer system [13].

2.3. NH2.terminal atnino acid sequencing analysis NH,-terminal amino acid sequence determination was performed by automated Edman degradation on a gas-plmse protein sequencer (model 470A, Applied Biosystems, CA, USA) e¢~uipped with a PTH analyzer (model 120A HPLC system).

3. RESULTS AND DISCUSSION H um an dermal fibroblasts stimulated with 10 -t° M IL-1 mainly produced FDNCF, which is the NH2-termina] extended 77-residue variant of IL-8 (AVLPR-IL-8). The homogeneity of the purified FDNCF was confirmed by SDS-PAGE, its NH2-terminal amino acid sequence and its COOHterminal amino acid determination. We found that the purified FDNCF attracts human neutrophils, but rat neutrophils are not attracted by the FDNCF at concentrations less than 10 -8 M. On the other hand, I0 -s M

Published by Elsevier Science Publishers B. V.

M~y 1991

FEB$ LI~TTI~RS

Volume 2t!2, numbw 2

I

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Fill. I t A ) RII| neutrophtl cheroot.eric .¢tlvlty of pl~=mln4re=t(~d FDNCF. FDNCF [I .4/~) wa~ Incubated wi~h the indicated ~mcunts of plawmlnat )'PCfor 30 rain. 'rl~e reacdon wa~ ,topped by addition or leupeplin (final concentration I raM}. (B) Time eour~c for rat neutrophtl ~hemotaetl¢ activity or plasmin.treat~d FDNCF, (e} FDNCF (!.4/~11) was incubated with plantain (30 na] at 37'C for the indicated time and the reaction was stopped by addilion leupepdn (final eoncentraUon 1 raM), while as a con|tel (c~) plantain was inactlv~t~¢l wilh I~upcptin anti th©n incubated with FDNCF. neutrophtl chemotaedc activity of the reactio~ mixtures was measured at 3 x 10"~ M lhe plasmin.treated FDNCF. Each r~oint repr~=ent~ che mean 6 determinations. Horiaon[al bars Indicate the

of

Ra~

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SE, IL.8 attracts b o t h rat and h u m a n neutrophils. T h e results indicate that rat neutrcphils are attracted by II.-8, but not by F D N C F at a concentration of 10 "~ M. In the present studies, therefore, we used rat n e u t r o p h i l s at a b o u t 10," M concentration of attractants in c h e m o t a x i s assay to determine the conversion o f F D N C F to IL-8 by plasmin. W h e r F D N C F was incubated with various a m o u n t s o f ptasmin, the reaction mixture dose-dependently increased the chemotactic activity for rat neutrophils (Fig, IA). The c h e m o t a c t i c activity o f the reaction mtxture rapidly increased and reached a m a x i m u m within 40 rain when F D N C F (l.4,ag) was incubated with L

50 (A)

i~.olated front the plasmin.¢reated FDNCF by RP-HPLC (Fig. 29), Th~ amino add rcddu¢~ identified by protein ~¢quencer ar~ marked by arrc~w~ under Ih¢ residtl¢~. Partial sequence; of IL.8 and the FDNCF purified from conditioned medium of SF,'rY cdl cul=ureare shown.

plasmin (30 ng) at 37=C (Fig. I B ) . The results suggest that IL-8 is r a p i d l y formed f r o m F D N C F by plasmin. The reaction products o f plasmin-treated F D N C F

were isolated by R P - H P L C to c o n f i r m the f o r m a t i o n o f IL-8. T h e R P - H P L C revealed one m a j o r peak (peak 2) and two m i n o r peaks (peaks 1 and 3); the area of p e a k 2 was a b o u t 90¢1o o f total peak areas on RPH P L C (Fig. 2 r . . P e a k s 2 and 3 h a d c h e m o t a c t i c activity for rat neutrophils, but chemotactic activity was ot f o u n d in peak 1 (Fig. 2B). T h e p e a k s 1-3 obtained f r o m the R P - H P L C were c o n c e n t r a t e d and loaded o n t o a gas-phase protein sequencer. NH2-terminal a m i n o acid of peaks I - 3 are s u m m a r i z e d in Fig, 3, T h e sequence o f peak ] is A V L P R , aud the subsequential a m i n o acid was not detected (Fig. 3). In addition, a m i n o acid analysis of p e a k 1 showed that the peptide o f peak 1 is constituted b y Ala, Val, Leu, P r o and Arg in equal n u m b e r of m o l e s (data not shown). T h e results indicate that peak 1 is the p e n t a p e p t i d e , A V L P R . N H z - t e r m i n a l a m i n o acid sequence o f peak 2 was identical to that o f IL-8 (Fig. 3), and other sequences

sequences

FDNCF

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Fig. 2. RP-HPLC of the purified FDNCF (A) and the plasmintreated FDNCF (B). Chemotactic activity of each fraction was assayed at 3 × 10-8 M in duplicate. Experimental conditions are described in section 2.

(kDa)

Fig. 4. SDS-PAGE of FDNCF, peak 2 and peak 3. Peaks 2 and 3 were isolated from the plasmin-treated FDNCF (Fig. 2B). Jne disulfide bonds of samples were reduced with 25% (v/v) 2-mercaptoethanol, and subjected to SDS-PAGE. The molecular mass standards (Std) were run simultaneously. 413

Volume 2~t2, number 2

FEB$ LI~TTERS

includin= F D N C F were not found in siInifl~ant amount= by N H r t e r m i n a l sequence ~nalysls of" peak 2. although F D N C F and IL-8 were eluted at the same retention time on R P - H P L C (Fi~, 2A and B). The

resultssuEtest that F D N C F was almost ¢ompletell/convetted to [L.8 by pin=rain under the present conditions. The carboxypeptidase A treatment of peak 2 and F D N C F resulted in the release of the same amino acid, Ser which is identical to carboxy-terminal amino acid o f [L-8 (data not shown). In addition, both the peak 2 and F D N C F revealed a similar molecular mass (about 6.4 kDa) on SDS-PAGE (Fig. 4). "*fhe results indicate that peak 2 is IL-8 with 72 amino acids, On the basis o f NH=-terminal amino acid sequence nf peak 3 (Fig, 3) and the molecular mass of peak 3 which was similar to that o f F D N C F (Fig, 4), it is p r o b a b l e that peak 3 is the NH=-terminal truncated 69-amino acid variant of IL-8, T h e ratio o f peak 2 (IL-8)/peak 3 was about 9: l on the basis o f the area o f each peak on R P - H P L C (Fig, 2B), suggesting that plasmin mainly cleaves the ArgS-Ser ~ b o n d , and t h e partial cleavage o f Lys~-Giu 9 bond may occur as a result of the secondary attack of plasmin, Plasmin did not cleave any other A r g - X and L y s - X bonds of F D N C F under the present experimental conditions, This stability of F D N C F except for NHz-terminal portion may be due to the fact that IL.8 contains a triple stranded anti-parallelP-sheet structure and a long COOH-terminal hcIL,~ [14,15]. It can be assumed that plasmin is generated from plasminogen by plasminogen activator which is secreted by the stimulated inflammatory cells including m o n o c y t e s / m a c r o p h a g e s and endothelial cells in the in. f l a m m a t o r y region [16], In the present study we have shown for t h e first time that plasmin is responsible for the limited proteolysis o f F D N C F ; plasmin can selectively cleave F D N C F to IL-8 and the inactive A V L P R pentapeptide, though a minor secondary reaction occurs.

414

M~ty 1991

A¢lmuwledxememx: We iI~tnk OBuka Phatm~eulk~tl Co. Ltd, tTt~kushima, Japan} and Japan¢~ Canwr ResearchReso~r~o Bank ¢Tokyo. Japan| for ~h¢tri!=nero~ llltt~ or re¢~mblaam human IL. I# and ~F-TY gelh. respegtively. We al~o Ihank Dr K. Walan~tbe for

helpful d|i~ulSlOlB,

REFERENCES

[11 Yoshlmura, T., Malsu~hima, K,, Tanaka, $., Robinson, E,A,, Appalls, E,. Oppenl~elm, J,J, and Leonard, E.J, (|9#'/) Prec, N~tl, Aged. Sol. USA 84, 9233-9237. [21 Walx, A,, Peveri, P,, Aschauer, H. and Balllioiini, M. (19#?} Bier:hem, BIophys, Res. Commun, 149. `/$$-761. [3] $~:hrodcr, J.-M,, Mrowlel=, U., Merits. E. and Christopher=, E. (1987) J. lmmunol, 139, 34'/4=3483, 14] Van Dammc, J,, Van Beeumen,J,, Opdertakker, G. and Biliau, A, (1988) J, Exp. Mad, 167. 1364~1376, [~} Larsen. C,G,, Anderson, A.O, App©lla, E , Oppenh¢im, J.J, and Malsushinla K. (1989} Setence243, 1464-1466. [6} Baaliolini, M,. Walt, A. and Kunkel, S,L, (1989) J. Cltn: Invest 84, 1045-1049, ['/] $¢hmld, J, and Weissmann. C. (198T} J. [mmunol. 139, 2~0-256. [8] Schroder, J,.M.. Sticherling, M., Henneicke, H.H,, Pteissn©r, W.C. and Christopher=, E, (1990) J. lmmunol. 144, 2223-2232. [9] Van D~mm©, J., De¢ock, B., Coning=, R,, Lenacr|s, J,.P,, OlvJenakker. G. and Billiau, A. (1989) Eur, J. lmmunol, 19, 189- 1194.

[10] Golds, E,E,, Mason. P. and Nyirkos, P. 0989) Biochem, J, 259. 585-588 lit} Watanabe, K,, Kinoshita, S, ~nd Nakal~awa, H. (1989) Bio. cllem, Biophys, Res. Commun. t6l, 1093-1099. [12] Watanab¢, K,, Nakagawa, H, and Tsurufuji, S. (1985) Jap, J, Pharmaco]. 39, I02-I04 [13] Schagser, H. and Von Jagow, G. (1987) Anal, Biochem. 166, 268-3'/9. [1~] Clore, G.M,, Appalls0 E., Yamada, M,, Matsushima, K. and Gronenborn, A.M, (1989] J Biol. Chern, 264, 18907-18911. [IS} CIore, G,M.. Appalls, E., Yamada, M,. Matsushima, K, and Gronenborn) A,M, (1990) Biochemistry 29, 1689-1696, [16] Dane, K., Andreasen, A,, Grondahl-Hansen, J,, Kristensen, P., Nielsen. L.S. and Skriver, L, 0985) Adv, Cancer Res. 44, 139-266.

Generation of interleukin-8 by plasmin from AVLPR-interleukin-8, the human fibroblast-derived neutrophil chemotactic factor.

Plasmin mainly cleaved the Arg5-Ser6 bond of Arg-Val-Leu-Pro-Arg-interleukin-8 (AVLPR-IL-8) produced by human dermal fibroblasts, which resulted in th...
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