Volume 300, number 1.21-N Q 1992Fcdcralionof Eumpn
BiochemicalSociks
FEW 10852
QO14579.392/!&00
hliirch 1922
Inhibition of neutrophii superoxide production hy human plasma a,-antitrypsin
Exposure of neutmphik to a variety of stimuli fin&ding conunavalin A plus cytochalasi~ E) activates a membnne-bound NADPHoxidase to catalyst the gnemtion of the superoside anion radical (O;-t
Rbhrr*rti~&s:q-AT. a,-antitrypsinr TPCK. L-l-tosylamide 3phenylahyl chloromethyl ketone: SBTI. soybean tqpsin inhibitor: Con A. connnovalin A: Cyto E. cytochalasin E. Corwsp~rrdc~~~ a&rcs:
P.G. Winyard. Ictlammation
Rewarch
Group. Arthritis and Rheumatism Council Buildmg. Lczdon Hospi-
tal Medical College. Z-29 AshficldStrcct, Londcn Ei 2.4D. Err&ml. Fax {44) (7:) 377 M??.
During the course of eqxrintenrs designed to study the mcvhanisms of a,-AT inactivation by isolared huma‘; neutrophi8 WC noted that rhc amaunt of detecloblc CrT* relcascd by cel!s stimulazed with a vari~ig of agents :wru,s~-&ed qmosan. nhziom ionophow E :cg:c Or SiX’YZXNaliFSA fCkX r’ ’ .;w qK.&Gx? E)) was Iowrcd in the pxencc of q-AT. Eulicr swiils. which wrc largely performed with Con A + Cyto E as the slimulus. showed that L-I-tosylamide 2-phenylcth>l chlommethyl ketone IT&X). soybean trypsia inhibitor (SBTU and other sMinc pmtease inhibitors have the ability to inhibit cX_- production by neutmphils(Wl and references therein) and by other cefl QF j15.161. Therefore. the dose-dependent inhibition by at-AT of Con A + C’)TO E-stimulated neutrophil a- production was cornpar& wi\?rhthe dose responses for TPCK, SBTI and q-AT-A&*_ The latter is a human recombinant X,-AT vntiant in which the reactive centre Met”’ has k;; q+~&ss ;ay an aqinine residue [I 7]_ Thii single amino acid substitution results in a 10.~fold dccrease in anti-CLasroly-tic activity and a 1O.OWfold incww ir? &rnmzhin inhibitory capacity.
Volutnc
300, number
I
MiWh 1992
FEDSLETTERS stiniulntory
i\gClllS only) endonens 11ncgnlivc conwol (Cells + sliinulatory tiycnts + supcroxidc dismuturic). The unwunt of0;’ produced(nmol O,‘U/lOhwlls) wits d~ulutcd using nn cxlinction cdlkdcnt 01’21 I( 10’ M” ‘*cm”’id plottd us II
limainn ol’ thno ul\crrrddhianol’rdmulimtr to obtnin the inhid nltc rcnction. The inhibitory ca~pncity wus onlculu~cd aY the % inhihhion 01’ the inill:\! nW oT the posilivc cannnl.
01’ cxh
3. RESULTS e,=AT, TPCK und SBTt showed vrrrious&grees of inhibition of the ncutrophil Oi* production clicitd by
serum-trcutcd zymosun, culcium ionophorc A 23187 and Con A plus Cyta E (Tnblc I). Whatcvcr the tri ing agent, siynilicunt umounts of inhibition wcrc o tuincd with both a,AT and TPCK ut similrir molnr conentratians (11.3 ~md 10.0yM, rqxdvcly). HOWcvcr, t’orSBTI, the molnr conccntrution rcquipcdto ivc tdmih\r extents cd’inhibition was about ?Wold higher (Tublc I). a,-AT at n cxxtccntrution of 0.6 mg/ml (IL5 pM) ulnmt complctcly inhibit& O human ncuwophih. Values uplp cdcukt~cd form Fig. 2.
.sminepro1ea.w Inhibitors in Con A
Serine protease inhibitor a,-A-la,-AT-Arg”” TPCK SBTI
5.1 (_uhl) 93.6 (UN! 4.7 yrhij ~07.0 (UhS)
---
c%ntact with rite cxlla e.g, wmpwtmentat in &lysis tubing in tlw prclscnce of e.Rtcm& stimukwd neutrophib &Sl)or. the al-AT VW irutctiwtrd by the addition of a supermeant which hvld been conditioned by stimufatcd neutrophils 1t t 25). WC conclude that. in inf!ammatory fluids contzzining rclatiwly high concentrations of q-AT. this serpin might play a physiological role in directly inhibiting ncutrophil 02*- production. Although it might bc argued &at the stiz~ulus mSnly used in this study Con A c Cyu E) is not physiclogiilly F? =nt. other stud&. #SC refc;Tzncx.sciiic : {I SJ]as WCEJS cur twn rcsui~~ obaind with serum-trcatcd zynosan and calcium iw nophon- show that wine protcasc inhibitors inhibit wutrophil cX_ production in response to a variet)’ of stimuli. Ektstase h;ls been shown to print neutrophil
02*- release dited
by dininxtt stimulants [XXI.
Our
system contained no added elastase 10 prime the system prior to stimulant addition. suggesting that a,-AT inhibited neutrophil C&- release by inhibiting a membmne bound wine protease. HOWWW.in
experimental
viva. inhibition of elastase may be an additional mcchanism by which q-AT down-regulates the neutrophil wi;arive wsponse. our ~~ufts also suggest that. in those inflammatory &ids where the q-AT concentration is relatively hi& th:Loxidation of a,-AT by neutrophil-released rcactiw o~~g~u ;+&s is unlikely tc occur. However. other cc;1 typ may mediate the oxidation of a,-AT and it ‘5
REFERENCES [I] Cwcll, R,W, (1986) J. Clin, luvcnl,78, IJll-1431. [S] Wong, P,S, und l’ruvir, J. (IYBO)Uiochwr. Uiophyn.Rcn, Commun, 96, 1449-1454. [.I] Cnrp* H,, Ml&r, F.; Huidal, J.R, nnrl JnncrlGA. (IUS?) Prw, Nud. Arad. Scl. ,USA 79. 2041-204Ss 141Oyurhi. F,, Hubhatd. R.C., Vog~hrcicr.C.. Fdlr. CIA. umdCry+ Mel, K.G. (lY91)J, Clin, Invcrl. H7, IO(iO-IObS. [S] Chidwick.IL, W\, snP((,P.O., Zhlmy, 2.. Fudl. A-J, und Ulukc. D.R. (I9911 Ann. Rhcum. Dir, 50, YIS-YI6, 161StockIcy.R.h, und hll’wd, C. (IYH4) HoppeScylur’r 2. Phyriol. Chcm.365. 503-510. 171Zhnny, Z,, WCy~ird, P.Q., Chidwick,K.. Farrell, A.. l’cmbwton, P,. Cwmll. R, nnd Bhrkc. D.R. (1’80) Biuchan, Sot. Trnna. 1H, 89H”“HYY. IH) Wclux,S,J. [lYH9l N. Egl. J. Mcd. Xl). 365 376. [Ul Huhhard,R.C.,O~ushi, l’s, Follr, CIA,, Cunrin. A.M.. J41lltrt.S.. Courlncy.M. und Crynlul.R.G, (I%71 J. Clia, Invcs~. HO,139 1295. [IOl Lalripr. V.. Rcinkc. H. und Tschc!xhc.N. (IWO) FELIS Lcu. w-357. c‘61., .bS [I II Mlchuclix.J.. Viascrs. M.C.M. strtlWh~~crhourrr.CC, (IYYI1) Uiochau,J, 270, NwNIJ,
[I?] Winyutul, PG.. Zhung. Z.. Chidwick, K,, Blukc. D.R., Currcll, K.W. and Murphy, C. (1991) FEBS Lctl. 279. 91-94. [IJ] Dcwochcrs. P.B.. JclTrcy, J.J. und Weiss, S.J. (1991) J. Ctin. Invcsl. X7, 2358-2265. [14] Mass. A.B.. Enghild. J.J., Nugusc,H.. Suzuki, K., Pitm. S.V. and Sulvcscn,G. (1991) J. Blol. Chcm.266. 15810-lSHl6. II 51 Kitapwn. S. and Tnknku. I:, (1982) Adv. Exp. MIA. Bid. 141. 441-4511 HolIInan, M,, Fclldmun,R. and Pizza, S.V. 11913) Blochlm. Blophyr, Acru 760,421-42X C~urincy, M., Jullul, S.. Tcssicr. L-H., knuvcnlu, A.. Ctyslat, R.O. und Ltwcq. J,P. (19115) Nuwrc 313,149-151. Bicl, J., Spice. B. snd Wcrmulh, CU. (1974) tliochcmm. Med. I I, so-357. Brudt‘ord, M.M. (1976) Awl. tliochcm.72.248-254. [Johisrcin, I.M., ROOH,D., Kuplnn, H.B. und Wcirsnumn, 0. 11975)J. Clin. lnwsl. 56, 1155-I 163. Boyum, A. (1968) !&and. J. Clin. Lab. Invat, ?I, 77-89. Kilptrick, L., Johnson, J.L., Nickbwg. J.L.. Wang. TM.. ClilTord. T,F.. Bnnuch.M., Cvoprmnn, U.S., Dou&w, SD. und Rubln, H. (1991) J. Immunol. I46,2388-2393. Ownnu. P.S.. Tclrl, &I’., Malhaon, N.R., Rcgianl.r;, andWclsa S,J. 119Rb)5, Clin. Invw 77. 193% 1951. Curp, H, 41ndJnnoll’, A. (1979)J. Clin. Invw, 63.793-797. Dcsrwhcn. P,E, und Wcln, S.J. (198X)J, Clln. Inval. 81, If&iIf&). Kurncr. D.J, und King, C.H. (1989)J. Immunol. 143. If+&-1702. Kumcr. D.J.. Anwc, J.N.. Fmncwchi, D.. Surusw MM,. Spagnuoh~. P.J. andKiclg.C.14.( 1991)J. Itid Char, 266, Im 16,371.
BiochemicalSociks
FEW 10852
QO14579.392/!&00
hliirch 1922
Inhibition of neutrophii superoxide production hy human plasma a,-antitrypsin
Exposure of neutmphik to a variety of stimuli fin&ding conunavalin A plus cytochalasi~ E) activates a membnne-bound NADPHoxidase to catalyst the gnemtion of the superoside anion radical (O;-t
Rbhrr*rti~&s:q-AT. a,-antitrypsinr TPCK. L-l-tosylamide 3phenylahyl chloromethyl ketone: SBTI. soybean tqpsin inhibitor: Con A. connnovalin A: Cyto E. cytochalasin E. Corwsp~rrdc~~~ a&rcs:
P.G. Winyard. Ictlammation
Rewarch
Group. Arthritis and Rheumatism Council Buildmg. Lczdon Hospi-
tal Medical College. Z-29 AshficldStrcct, Londcn Ei 2.4D. Err&ml. Fax {44) (7:) 377 M??.
During the course of eqxrintenrs designed to study the mcvhanisms of a,-AT inactivation by isolared huma‘; neutrophi8 WC noted that rhc amaunt of detecloblc CrT* relcascd by cel!s stimulazed with a vari~ig of agents :wru,s~-&ed qmosan. nhziom ionophow E :cg:c Or SiX’YZXNaliFSA fCkX r’ ’ .;w qK.&Gx? E)) was Iowrcd in the pxencc of q-AT. Eulicr swiils. which wrc largely performed with Con A + Cyto E as the slimulus. showed that L-I-tosylamide 2-phenylcth>l chlommethyl ketone IT&X). soybean trypsia inhibitor (SBTU and other sMinc pmtease inhibitors have the ability to inhibit cX_- production by neutmphils(Wl and references therein) and by other cefl QF j15.161. Therefore. the dose-dependent inhibition by at-AT of Con A + C’)TO E-stimulated neutrophil a- production was cornpar& wi\?rhthe dose responses for TPCK, SBTI and q-AT-A&*_ The latter is a human recombinant X,-AT vntiant in which the reactive centre Met”’ has k;; q+~&ss ;ay an aqinine residue [I 7]_ Thii single amino acid substitution results in a 10.~fold dccrease in anti-CLasroly-tic activity and a 1O.OWfold incww ir? &rnmzhin inhibitory capacity.
Volutnc
300, number
I
MiWh 1992
FEDSLETTERS stiniulntory
i\gClllS only) endonens 11ncgnlivc conwol (Cells + sliinulatory tiycnts + supcroxidc dismuturic). The unwunt of0;’ produced(nmol O,‘U/lOhwlls) wits d~ulutcd using nn cxlinction cdlkdcnt 01’21 I( 10’ M” ‘*cm”’id plottd us II
limainn ol’ thno ul\crrrddhianol’rdmulimtr to obtnin the inhid nltc rcnction. The inhibitory ca~pncity wus onlculu~cd aY the % inhihhion 01’ the inill:\! nW oT the posilivc cannnl.
01’ cxh
3. RESULTS e,=AT, TPCK und SBTt showed vrrrious&grees of inhibition of the ncutrophil Oi* production clicitd by
serum-trcutcd zymosun, culcium ionophorc A 23187 and Con A plus Cyta E (Tnblc I). Whatcvcr the tri ing agent, siynilicunt umounts of inhibition wcrc o tuincd with both a,AT and TPCK ut similrir molnr conentratians (11.3 ~md 10.0yM, rqxdvcly). HOWcvcr, t’orSBTI, the molnr conccntrution rcquipcdto ivc tdmih\r extents cd’inhibition was about ?Wold higher (Tublc I). a,-AT at n cxxtccntrution of 0.6 mg/ml (IL5 pM) ulnmt complctcly inhibit& O human ncuwophih. Values uplp cdcukt~cd form Fig. 2.
.sminepro1ea.w Inhibitors in Con A
Serine protease inhibitor a,-A-la,-AT-Arg”” TPCK SBTI
5.1 (_uhl) 93.6 (UN! 4.7 yrhij ~07.0 (UhS)
---
c%ntact with rite cxlla e.g, wmpwtmentat in &lysis tubing in tlw prclscnce of e.Rtcm& stimukwd neutrophib &Sl)or. the al-AT VW irutctiwtrd by the addition of a supermeant which hvld been conditioned by stimufatcd neutrophils 1t t 25). WC conclude that. in inf!ammatory fluids contzzining rclatiwly high concentrations of q-AT. this serpin might play a physiological role in directly inhibiting ncutrophil 02*- production. Although it might bc argued &at the stiz~ulus mSnly used in this study Con A c Cyu E) is not physiclogiilly F? =nt. other stud&. #SC refc;Tzncx.sciiic : {I SJ]as WCEJS cur twn rcsui~~ obaind with serum-trcatcd zynosan and calcium iw nophon- show that wine protcasc inhibitors inhibit wutrophil cX_ production in response to a variet)’ of stimuli. Ektstase h;ls been shown to print neutrophil
02*- release dited
by dininxtt stimulants [XXI.
Our
system contained no added elastase 10 prime the system prior to stimulant addition. suggesting that a,-AT inhibited neutrophil C&- release by inhibiting a membmne bound wine protease. HOWWW.in
experimental
viva. inhibition of elastase may be an additional mcchanism by which q-AT down-regulates the neutrophil wi;arive wsponse. our ~~ufts also suggest that. in those inflammatory &ids where the q-AT concentration is relatively hi& th:Loxidation of a,-AT by neutrophil-released rcactiw o~~g~u ;+&s is unlikely tc occur. However. other cc;1 typ may mediate the oxidation of a,-AT and it ‘5
REFERENCES [I] Cwcll, R,W, (1986) J. Clin, luvcnl,78, IJll-1431. [S] Wong, P,S, und l’ruvir, J. (IYBO)Uiochwr. Uiophyn.Rcn, Commun, 96, 1449-1454. [.I] Cnrp* H,, Ml&r, F.; Huidal, J.R, nnrl JnncrlGA. (IUS?) Prw, Nud. Arad. Scl. ,USA 79. 2041-204Ss 141Oyurhi. F,, Hubhatd. R.C., Vog~hrcicr.C.. Fdlr. CIA. umdCry+ Mel, K.G. (lY91)J, Clin, Invcrl. H7, IO(iO-IObS. [S] Chidwick.IL, W\, snP((,P.O., Zhlmy, 2.. Fudl. A-J, und Ulukc. D.R. (I9911 Ann. Rhcum. Dir, 50, YIS-YI6, 161StockIcy.R.h, und hll’wd, C. (IYH4) HoppeScylur’r 2. Phyriol. Chcm.365. 503-510. 171Zhnny, Z,, WCy~ird, P.Q., Chidwick,K.. Farrell, A.. l’cmbwton, P,. Cwmll. R, nnd Bhrkc. D.R. (1’80) Biuchan, Sot. Trnna. 1H, 89H”“HYY. IH) Wclux,S,J. [lYH9l N. Egl. J. Mcd. Xl). 365 376. [Ul Huhhard,R.C.,O~ushi, l’s, Follr, CIA,, Cunrin. A.M.. J41lltrt.S.. Courlncy.M. und Crynlul.R.G, (I%71 J. Clia, Invcs~. HO,139 1295. [IOl Lalripr. V.. Rcinkc. H. und Tschc!xhc.N. (IWO) FELIS Lcu. w-357. c‘61., .bS [I II Mlchuclix.J.. Viascrs. M.C.M. strtlWh~~crhourrr.CC, (IYYI1) Uiochau,J, 270, NwNIJ,
[I?] Winyutul, PG.. Zhung. Z.. Chidwick, K,, Blukc. D.R., Currcll, K.W. and Murphy, C. (1991) FEBS Lctl. 279. 91-94. [IJ] Dcwochcrs. P.B.. JclTrcy, J.J. und Weiss, S.J. (1991) J. Ctin. Invcsl. X7, 2358-2265. [14] Mass. A.B.. Enghild. J.J., Nugusc,H.. Suzuki, K., Pitm. S.V. and Sulvcscn,G. (1991) J. Blol. Chcm.266. 15810-lSHl6. II 51 Kitapwn. S. and Tnknku. I:, (1982) Adv. Exp. MIA. Bid. 141. 441-4511 HolIInan, M,, Fclldmun,R. and Pizza, S.V. 11913) Blochlm. Blophyr, Acru 760,421-42X C~urincy, M., Jullul, S.. Tcssicr. L-H., knuvcnlu, A.. Ctyslat, R.O. und Ltwcq. J,P. (19115) Nuwrc 313,149-151. Bicl, J., Spice. B. snd Wcrmulh, CU. (1974) tliochcmm. Med. I I, so-357. Brudt‘ord, M.M. (1976) Awl. tliochcm.72.248-254. [Johisrcin, I.M., ROOH,D., Kuplnn, H.B. und Wcirsnumn, 0. 11975)J. Clin. lnwsl. 56, 1155-I 163. Boyum, A. (1968) !&and. J. Clin. Lab. Invat, ?I, 77-89. Kilptrick, L., Johnson, J.L., Nickbwg. J.L.. Wang. TM.. ClilTord. T,F.. Bnnuch.M., Cvoprmnn, U.S., Dou&w, SD. und Rubln, H. (1991) J. Immunol. I46,2388-2393. Ownnu. P.S.. Tclrl, &I’., Malhaon, N.R., Rcgianl.r;, andWclsa S,J. 119Rb)5, Clin. Invw 77. 193% 1951. Curp, H, 41ndJnnoll’, A. (1979)J. Clin. Invw, 63.793-797. Dcsrwhcn. P,E, und Wcln, S.J. (198X)J, Clln. Inval. 81, If&iIf&). Kurncr. D.J, und King, C.H. (1989)J. Immunol. 143. If+&-1702. Kumcr. D.J.. Anwc, J.N.. Fmncwchi, D.. Surusw MM,. Spagnuoh~. P.J. andKiclg.C.14.( 1991)J. Itid Char, 266, Im 16,371.
