Biochimica et Biophysica Acta, 1091 (1991) 257-260 © 1991 Elsevier Science Publirhers B.V. 0167-4889/'91/$03.50 ADONIS 016748899100090L
257
BBAMCR 12840
The role of adhesive interactions and extracellular matrix fibronectin from human polymorphonuclear leukocytes in the respiratory burst G . F . S u d ' i n a 1, A.V. T a t a r i n t s e v 1, A.A. K o s h k i n 1, S.V. Zaitsev ~, N . A . F e d o r o v 2 a n d S.D. V a r f o l o m e e v 1 I Moscow State University, Moscow 119~899GSP (U.S.S.R.) and " blstitute of the Hygiene o] Work and Occ~Fat~.o::olDiseases, Academy of the Medical Sciences (U.S.S.R.)
(Received 22 December 1989) (Revised manuscript received 2 August 1990)
Key words: Arg-Gly-Asp tripeptide: Ajoene: Fibronectin: Respiratory burst: (Human neutrophil)
The Arg-Giy-Asp (RGD) tripeptide and ajoene were used for studying the role of adhesive receptors in the respiratory burst. Activation of the respiratory burst was examined by using luminol-dependent and lucigenin-dependent chemiluminescence. Recently, it was shown that ajoene, (E, Z )-4,5,9-trithiadodeca-l,6,1|-trien-9-oxide, a substance isolated from garlic extract, inhibits the binding of fibrinogen to activated platelets by direct interaction with fibrinogen receptor (Apitz-Castro, R., Lederma, E., Escalante, J. and ,lain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150). Taking into consideration the structural and functional similarity of integrins, it would be reasonable to assume that ajoene as well as RGD can inhibit adhesive interactions of human neutrophils. We have shown that the effect of various activators on the respiratory burst was abolished by ajoene or RGD treatment. The inhibitory effect of RGD and ajoene was dose-dependent. The treatment of neutrophils with antiserum against human plasma fibronectin inhibited the respiratory burst in response to formyi-methionyl-leucylphenylalanine (fMLP) and phorbol 12-myristate |3-acetate (PMA). This effect is dose-dependent and reversible with the addition of fibronectin. These data indicate that the respiratory burst in human neutrophils is mediated by the integrin family of receptors and that interactions between the extraceilular matrix fibronectin and cells are necessary for the respiratory burst. Introduction
The superoxide anion and other active forms of oxygen are essential for the cytotoxic activity of neutrophils [1]. A cascade of enzymatic reactions of the respiratory burst is initiated by different substances which either bind directly to receptors on the cell surface or penetrate the cell and activate the electron-transport NADPH-oxidase system. The latter transfers the reducing equivalent from NADPH to oxygen producing the superoxide anion Of [2,3]. Metabolic transformations of the superoxide anion under the action of superoxide
Abbreviations: RGD, arginine-glycine-aspartate; fMLP, formylmethionylleucylphenylalanine; PMA, phorbol 12-myristate 13-acetate; Con A, concanavalin A; AA, arachidonic acid; Fn, fibronectin. Correspondence: G.F. Sud'ina, A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, USSR.
dismutase, peroxidase and myeloperoxidase produce hydrogen peroxide, hydroxyl radical, HOC! and singlet oxygen. There is scant information on the role of the cell surface and the state of different receptors, especially adhesive ones, for respiratory burst activation. The experimental facts support the important role of adhesive interactions for respiratory burst activation in neutrophils [4,5]. A respiratory burst in a neutrophil suspension in response to fMLP is short-term, and the O~- production is considerably lower compared with the cells bound to the surface [6]. Suspended neutrophils become insensitive to the repeated action of a stimulus, and conversely, interaction with the surface prevents desensitization of the cells [6]. A good correlation is observed also between the generation of O2 and aggregation of neutrophils in suspension. Disaggregation of neutrophils arrests 02- production [6]. Intercellular adhesive interactions as well as these between the cells and the surfaces, are regulated by adhesive receptors called integrins. Integrins or c~toado hesins form a class of membrane glycoproteins which
258 mediate cell adhesion to the extracellular matrix, basal membrane, other cells and plasma proteins [7,8]. The cytoplasmatic domains of adhesive receptors interact with cytoskeleton components [9]. Many adhesive proteins of the extracellular matrix and plasma, such as fibronectin, vitronectin, collagen, yon Willebrand factor, fibrinogen, thrombin and others, contain the tripeptide Arg-Gly-Asp in the site responsible for binding to cell integrins [10]. Associates of these adhesive glycoproteins with cells are destroyed by synthetic peptides with the RGD sequence [11]. The RGD tripeptide inhibits interaction of ligands with corresponding receptors and blocks adhesive interactions. The latter are also suppressed by ajoene, a substance isolated from garlic extract and possessing physiological activity [12], Ajoe,e also inbJbit.s a~gregation of platelets induced by all known activators [12]. In this work, we have studied the action of adhesive interaction inhibitors, RGD and adjoene, as well as fibronectin antibodies and exogenous fibronectin (Fn) on the respiratory burst in neutrophils initiated by different stimuli. Our aim was to find a relationship between the adhesive interactions and the reactions producing active forms of oxygen. Materials and Methods
fMLP, PMA, calcium ionophore A23187, RGD, AA, luminol (5-amino-2,3-dihydro-l,4-phthlazinedione) and lucigenin (bis-N-methyl acridium nitrate) were from Sigma; Con A, Hepes and human serum albumin were from Fluka, Dextran T-500 was obtained from Pharmacia; Fn from human blood and antiserum to Fn were from Boehringer-Mannheim. Ajoene, the (E, Z)-4,5,9trithiadodeca-l,6,11-trien-9-oxide, was synthesized by Dr. A.A. Schegolev from the Chemistry Department of Moscow State University. Solutions. PBS: KHaPO,,, 0.144 g/l, NaCI, 9 g/l, NaaHPO 4, 0.795 g/l. HBSS (pH 7.4), with the addition of I0 mM Hepes, was prepared from the concentrated stock solution of HBSS (Sigma) without Phenol red and bicarbonate. Isolation of neutrophils. Neutrophils were purified from freshly drawn blood of healthy adult donors. Blood with the citrate anticoagulant was mixed in an equal volume of 3% (w/v) Dextran T-500 in 0,85% NaCI. After sedimentation of erythrocytes at room temperature within 20-30 rain, plasma was laminated on a gradient of Ficoll-Paque, 1.077 [13], and centrifuged at 1500 rpm for 30 rain. The remaining erythrocytes were lysed twice by 0.2% (w/v) NaCI for 25 s. The fraction of polymorphonuclear leucocytes was suspended in HBSS with I0 mM Hepes and kept on ice. laoninescent studies. Luminol up to 1/tM, or lucigenin up to 25/tM were added to (2-10). 105 cells in polystyrene tubes thermostatted at 37°C in luminometer
100
"-E
o:2 o:4 o:8 ols
,:o ~
RGD(mM) Fig. 1. Effect of RGD on the luminol-enhanced chemiluminescence by fMLP-stimulated human neutrophils. Cells (106/ml) were incubated for 10 s at indicated concentrations of RGD; chemiluminescence was induced by 10 pM fMLP. The values of chemiluminescence maxima (in mV) are given in to the value registered in the absence of RGD. Duplicate determinations; S.D. < 10%.
LKB 1251, and then spontaneous luminescence was registered for 3-4 min. A stimulus was finally added and the chemiluminescence response was recorded in mV. Luminol-enhanced chemiluminescence was shown to be primary dependent on a myeloperoxidase-mediated reaction and is inhibited by sodium azide [14]. Lucigenin-amplified chemiluminescence is selective for 02- and is established as assay system for the NADPH-oxidase [15-18]. Results and Discussion
The effect of RGD on the respiratory burst was studied after stimulation of neutrophils by the chemotactic peptide fMLP. In the presence of RGD the fMLP-induced luminol-dependent chemiluminescence was inhibited. The inhibiting effect was enhanced with an increase in the RGD concentration (Fig. 1). After the addition of ajoene to the incubation mixture, the chemiluminescence of neutrophiis, induced by fMLP, AA, calcium ionophore A23187 and Con A, was inhibited (Table I). T h e production of active oxygen forms was arrested as well, if ajoene was added after stimulation (Fig. 2). Ajoene is known to interact with TABLE !
Action of ajoene on iuminol.enhanced chemiluminescence of neutrophiis induced by various stimuli Cell concentration, 5.10S/ml. Peak chemiluminescence values are expressed percentage vise to those obtained in the absence of ajoene. Duplicate determinations; S.D. < 10%. Stimulus
Concn.
Control (%) [Ajoene] (/tM): 0 0.17 1.7
10.5
fMLP Con A A23187 AA
10/tM 100 ttg/ml 1/tM 100/tM
100 100 100 100
0 0 0 19
90 77 100 92
30 5 35 30
259
o]oene
1
20
"~ 16 ~12 10
5
4
23.4
3 i
5
10
15
g ~
20 Vine (rain)
Fig. 2. Kinetics of the luminol-enhanced chemiluminescence by PMA-stimulated human neutrophils. Cells (5.105/ml) were incubated for 10 s in the absence (1) or in the presence (2-1.7/~M; 3-17/~M) of ajoene; chemiluminescence was induced by 4 ng/ml PMA. In experiment (1) ajoene was added at the indicated time at 1.7/tM.
th~ purified g!ycoprotein l l b / l i i a [19]. Considering the structural and functional similarity of integrins, it ,_'s reasonable to assume that ajoene interacts with many other adhesive receptors too. The inhibition of chemiluminescence by R G D and ajoene shows that the state of adhesive receptors in neutrophils is essential for the mechanism of respiratory burst initiation. After washing off the plasma proteins, Fn bound to the extraceilular matrix acts as adhesive protein for cellular receptors in a suspension of neutrophils [20-22]. Adhesive receptors on the cell surface are known to possess higher affinity for the matrix Fn than for the plasma one [23-25]. The action of Fn antibodies on the rate of formation of active oxygen metabolites was detected by assaying lucigenin-dependent chemiluminescence. Figs. 3 and 4 show the effect of Fn antibodies on the chemiluminescence of neutrophils stimulated by fMLP and PMA. An
100
",J
t
t
t
t
t
2 4 6 8 10 1'2 time (rn~-~n) Fig. 4. Kinetics of lucigenin-enhanced chemiluminescence by PMA (4 ng/mi)-induced neutrophils in the absence (1) or in the presence (2-5) of 40/,I antibodies (protein concentration, 5-10 mg/ml) in a cell suspension (]06/ml). In three experiments Fn antibodies were added together with either (3) - human serum albumin (10/~! of 5 mg/ml solution), or (4) - fibrinogen (10 ILl of 5 mg/ml solution), or (5) - Fn (10/,! of 1 mg/mi solution).
increase in the concentration of Fn antibodies in the incubation medium inhibits the respiratory burst. It is known [26] that the matrix and plasma Fn bind the same antibodies. The inhibitory action of antibodies is eliminated by the addition of exogenous Fn, which adsorbs the antibodies (Figs. 3, 4). Conversely, the addition of fibrinogen and albumin to a medium containing Fn antibodies does not eliminate their inhibitory action (Fig. 4). The results of our study suggest the direct involvement of the integrins and extracellular matrix proteins in the activation and regulation of the respiratory burst in neutrophils. The role of integrins in the activation of the oxidative burst is also confirmed by the data of Cabanas et al. [27], who showed that monoclonal antibodies to receptors p150,95 inhibit the Con A induced
100
Q
50
,,,d
10
20
30 40 50 ont i - h - fibronectin, pI
Fibronectin ( pg/ml)
Fig. 3. (A) Effect of Fn antibodies on iucigenin.dependent chemiluminescence of neutrophils. Cells (10~/ml) were incubated for 20 s with the indicated quantities of antibodies (protein concentration, 5-10 mg/ml) and chemiluminescence was induced by 10/~M fMLE (B) Effect of Fn antibodies on iucigenin-dependent chemiluminescence of neutrophils in the presence of exogenous Fn. Cells (106/ml) were incubated for 20 s with 25/~1 antibodies and at indicated concentrations of Fn; chemiluminescence was induced by 10 /LM fMLP. The values of chemiluminescence maxima (in mV) are expressed as mean + S.D. of the duplicate determina6ons and given in % to the value registered in the absence of antibodies.
260 formation of 02- in the monocytoid line U937. It is probable that activation of the respiratory burst enzymes needs definite structural rearrangements of the membrane, which occur as a result of activation of adhesive receptors. Acknowledgments We are indebted Dr. L.B. Margolis and Dr. S.I. Galkina for their collaboration and making available to us useful data. Thanks are also due to Mr. I.S. Kochubey for translation editing. References 1 Beczkowski, J,Z, and Gessner, T. (1988) Int. J. Biochem. 20, 569-580. 2 Makino, R,, Tanaka, T,, lizuka, T., lshimura, Y. and Kanegasaki, S. (1986) J, Biol, Chem. 261, 11444-11447. 3 Clark, R,A,, Leidal, K.G., Pearson, D.W. and Nausef, W.N. (1987) J. Biol. Chem. 262, 4065-4074. 4 Hafeman, D.G., Parce, J.W. and McConnel, H.M. (1979) Biochem. Biophys. Res. Commun. 86, 522-528. 5 Hafeman, D,G., Lewis, J.T. and McConnel, H.M. (1980) Biochemistry 19, 5387-5394. 6 Dahinden, C.D., Fenu, .l. and Hugli, T.E. (1983) J. Clin. Invest. 72, 113-121. 7 Hynes, R.O. (1987) Cell 48, 549-554. 8 Ruoslanti, E. and Piershbacher, M.D. (1987) Science 238, 491-497.
9 Horwitz, A., Diggar, K., Buck, C., Beckerle, M.C. and Burridge, K. (1986) Nature 320, 531-533. I0 Kornblint, A.R. and Gutman, A. (1988) Biol. Rev. 63, 465-507. II, Yamada, K. and Kennedy, D.W. (1987) J. Cell Physiol. 130, 21-28. 12 .lain, M.K. and Apitz-Castro, R. (1987) Trends Biol. Sci. 12, 252-254. 13 Boyum, A. (1976) Scand. J. Immunol. Suppl. 5, 5, 9-15. 14 Suematsu, M., Oshio, C., Miura, S., Suzuki, M., Houzawa, S. and Tsuchiya, M. (1988) Biochim. Biophys. Res. Commun. 155, 106III. 15 Markert, M., Vaglio, M. and Fri, .l. (1988) J. Lab. Clin. MedAl, 577-583. 16 Gyllenhammar, H. (1987) J. Immunol. Methods 97, 209-213. 17 Minkenberg, J. and Ferber, E. (1984) J. Immunol. Methods 71, 61-67. 18 Storch, J. and Ferber, E. (1988) Anal. b[ochem. 169, 262-267. 19 Apitz-Castro, R., Lederma, E., Escalante, J. and Jain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150. 20 Hynes, R.O. and Yamada, K.M. (1981) .l. Cell Biol. 95, 369-377. 21 McKeown-Longo, P.J. and Mosher, D.F. (1983) J. Cell Biol. 97, 466-472. 22 Beppu, M., Masa, H. and Kugawa, K.K. (1989) FEBS Lett. 242, 378-382. 23 Johansson, S. (1985) J. Biol. Chem. 260, No. 3, 1557-1561. 24 Matyas, G.R., Evers, D.C., Radinsky, R. and Morre, DJ. (1986) Exp. Cell Res. 162, 296-318. 25 Patel, V.P. and Lodish, H.F. (1988) J. Cell Biol. 102, No. 2, 449-456. 26 Johansson, S. and Hook, M. (1984) .l. Ceii Biol. 98, 8!0-817. 27 Cabanas, C., Lakal, P., Mollinedo, F., Lopez-Rivas, A., SanchezMadrid, F. and Beernaber, C. (1989) Immunol. Lett. 20, 193-198.
257
BBAMCR 12840
The role of adhesive interactions and extracellular matrix fibronectin from human polymorphonuclear leukocytes in the respiratory burst G . F . S u d ' i n a 1, A.V. T a t a r i n t s e v 1, A.A. K o s h k i n 1, S.V. Zaitsev ~, N . A . F e d o r o v 2 a n d S.D. V a r f o l o m e e v 1 I Moscow State University, Moscow 119~899GSP (U.S.S.R.) and " blstitute of the Hygiene o] Work and Occ~Fat~.o::olDiseases, Academy of the Medical Sciences (U.S.S.R.)
(Received 22 December 1989) (Revised manuscript received 2 August 1990)
Key words: Arg-Gly-Asp tripeptide: Ajoene: Fibronectin: Respiratory burst: (Human neutrophil)
The Arg-Giy-Asp (RGD) tripeptide and ajoene were used for studying the role of adhesive receptors in the respiratory burst. Activation of the respiratory burst was examined by using luminol-dependent and lucigenin-dependent chemiluminescence. Recently, it was shown that ajoene, (E, Z )-4,5,9-trithiadodeca-l,6,1|-trien-9-oxide, a substance isolated from garlic extract, inhibits the binding of fibrinogen to activated platelets by direct interaction with fibrinogen receptor (Apitz-Castro, R., Lederma, E., Escalante, J. and ,lain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150). Taking into consideration the structural and functional similarity of integrins, it would be reasonable to assume that ajoene as well as RGD can inhibit adhesive interactions of human neutrophils. We have shown that the effect of various activators on the respiratory burst was abolished by ajoene or RGD treatment. The inhibitory effect of RGD and ajoene was dose-dependent. The treatment of neutrophils with antiserum against human plasma fibronectin inhibited the respiratory burst in response to formyi-methionyl-leucylphenylalanine (fMLP) and phorbol 12-myristate |3-acetate (PMA). This effect is dose-dependent and reversible with the addition of fibronectin. These data indicate that the respiratory burst in human neutrophils is mediated by the integrin family of receptors and that interactions between the extraceilular matrix fibronectin and cells are necessary for the respiratory burst. Introduction
The superoxide anion and other active forms of oxygen are essential for the cytotoxic activity of neutrophils [1]. A cascade of enzymatic reactions of the respiratory burst is initiated by different substances which either bind directly to receptors on the cell surface or penetrate the cell and activate the electron-transport NADPH-oxidase system. The latter transfers the reducing equivalent from NADPH to oxygen producing the superoxide anion Of [2,3]. Metabolic transformations of the superoxide anion under the action of superoxide
Abbreviations: RGD, arginine-glycine-aspartate; fMLP, formylmethionylleucylphenylalanine; PMA, phorbol 12-myristate 13-acetate; Con A, concanavalin A; AA, arachidonic acid; Fn, fibronectin. Correspondence: G.F. Sud'ina, A.N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow 119899, USSR.
dismutase, peroxidase and myeloperoxidase produce hydrogen peroxide, hydroxyl radical, HOC! and singlet oxygen. There is scant information on the role of the cell surface and the state of different receptors, especially adhesive ones, for respiratory burst activation. The experimental facts support the important role of adhesive interactions for respiratory burst activation in neutrophils [4,5]. A respiratory burst in a neutrophil suspension in response to fMLP is short-term, and the O~- production is considerably lower compared with the cells bound to the surface [6]. Suspended neutrophils become insensitive to the repeated action of a stimulus, and conversely, interaction with the surface prevents desensitization of the cells [6]. A good correlation is observed also between the generation of O2 and aggregation of neutrophils in suspension. Disaggregation of neutrophils arrests 02- production [6]. Intercellular adhesive interactions as well as these between the cells and the surfaces, are regulated by adhesive receptors called integrins. Integrins or c~toado hesins form a class of membrane glycoproteins which
258 mediate cell adhesion to the extracellular matrix, basal membrane, other cells and plasma proteins [7,8]. The cytoplasmatic domains of adhesive receptors interact with cytoskeleton components [9]. Many adhesive proteins of the extracellular matrix and plasma, such as fibronectin, vitronectin, collagen, yon Willebrand factor, fibrinogen, thrombin and others, contain the tripeptide Arg-Gly-Asp in the site responsible for binding to cell integrins [10]. Associates of these adhesive glycoproteins with cells are destroyed by synthetic peptides with the RGD sequence [11]. The RGD tripeptide inhibits interaction of ligands with corresponding receptors and blocks adhesive interactions. The latter are also suppressed by ajoene, a substance isolated from garlic extract and possessing physiological activity [12], Ajoe,e also inbJbit.s a~gregation of platelets induced by all known activators [12]. In this work, we have studied the action of adhesive interaction inhibitors, RGD and adjoene, as well as fibronectin antibodies and exogenous fibronectin (Fn) on the respiratory burst in neutrophils initiated by different stimuli. Our aim was to find a relationship between the adhesive interactions and the reactions producing active forms of oxygen. Materials and Methods
fMLP, PMA, calcium ionophore A23187, RGD, AA, luminol (5-amino-2,3-dihydro-l,4-phthlazinedione) and lucigenin (bis-N-methyl acridium nitrate) were from Sigma; Con A, Hepes and human serum albumin were from Fluka, Dextran T-500 was obtained from Pharmacia; Fn from human blood and antiserum to Fn were from Boehringer-Mannheim. Ajoene, the (E, Z)-4,5,9trithiadodeca-l,6,11-trien-9-oxide, was synthesized by Dr. A.A. Schegolev from the Chemistry Department of Moscow State University. Solutions. PBS: KHaPO,,, 0.144 g/l, NaCI, 9 g/l, NaaHPO 4, 0.795 g/l. HBSS (pH 7.4), with the addition of I0 mM Hepes, was prepared from the concentrated stock solution of HBSS (Sigma) without Phenol red and bicarbonate. Isolation of neutrophils. Neutrophils were purified from freshly drawn blood of healthy adult donors. Blood with the citrate anticoagulant was mixed in an equal volume of 3% (w/v) Dextran T-500 in 0,85% NaCI. After sedimentation of erythrocytes at room temperature within 20-30 rain, plasma was laminated on a gradient of Ficoll-Paque, 1.077 [13], and centrifuged at 1500 rpm for 30 rain. The remaining erythrocytes were lysed twice by 0.2% (w/v) NaCI for 25 s. The fraction of polymorphonuclear leucocytes was suspended in HBSS with I0 mM Hepes and kept on ice. laoninescent studies. Luminol up to 1/tM, or lucigenin up to 25/tM were added to (2-10). 105 cells in polystyrene tubes thermostatted at 37°C in luminometer
100
"-E
o:2 o:4 o:8 ols
,:o ~
RGD(mM) Fig. 1. Effect of RGD on the luminol-enhanced chemiluminescence by fMLP-stimulated human neutrophils. Cells (106/ml) were incubated for 10 s at indicated concentrations of RGD; chemiluminescence was induced by 10 pM fMLP. The values of chemiluminescence maxima (in mV) are given in to the value registered in the absence of RGD. Duplicate determinations; S.D. < 10%.
LKB 1251, and then spontaneous luminescence was registered for 3-4 min. A stimulus was finally added and the chemiluminescence response was recorded in mV. Luminol-enhanced chemiluminescence was shown to be primary dependent on a myeloperoxidase-mediated reaction and is inhibited by sodium azide [14]. Lucigenin-amplified chemiluminescence is selective for 02- and is established as assay system for the NADPH-oxidase [15-18]. Results and Discussion
The effect of RGD on the respiratory burst was studied after stimulation of neutrophils by the chemotactic peptide fMLP. In the presence of RGD the fMLP-induced luminol-dependent chemiluminescence was inhibited. The inhibiting effect was enhanced with an increase in the RGD concentration (Fig. 1). After the addition of ajoene to the incubation mixture, the chemiluminescence of neutrophiis, induced by fMLP, AA, calcium ionophore A23187 and Con A, was inhibited (Table I). T h e production of active oxygen forms was arrested as well, if ajoene was added after stimulation (Fig. 2). Ajoene is known to interact with TABLE !
Action of ajoene on iuminol.enhanced chemiluminescence of neutrophiis induced by various stimuli Cell concentration, 5.10S/ml. Peak chemiluminescence values are expressed percentage vise to those obtained in the absence of ajoene. Duplicate determinations; S.D. < 10%. Stimulus
Concn.
Control (%) [Ajoene] (/tM): 0 0.17 1.7
10.5
fMLP Con A A23187 AA
10/tM 100 ttg/ml 1/tM 100/tM
100 100 100 100
0 0 0 19
90 77 100 92
30 5 35 30
259
o]oene
1
20
"~ 16 ~12 10
5
4
23.4
3 i
5
10
15
g ~
20 Vine (rain)
Fig. 2. Kinetics of the luminol-enhanced chemiluminescence by PMA-stimulated human neutrophils. Cells (5.105/ml) were incubated for 10 s in the absence (1) or in the presence (2-1.7/~M; 3-17/~M) of ajoene; chemiluminescence was induced by 4 ng/ml PMA. In experiment (1) ajoene was added at the indicated time at 1.7/tM.
th~ purified g!ycoprotein l l b / l i i a [19]. Considering the structural and functional similarity of integrins, it ,_'s reasonable to assume that ajoene interacts with many other adhesive receptors too. The inhibition of chemiluminescence by R G D and ajoene shows that the state of adhesive receptors in neutrophils is essential for the mechanism of respiratory burst initiation. After washing off the plasma proteins, Fn bound to the extraceilular matrix acts as adhesive protein for cellular receptors in a suspension of neutrophils [20-22]. Adhesive receptors on the cell surface are known to possess higher affinity for the matrix Fn than for the plasma one [23-25]. The action of Fn antibodies on the rate of formation of active oxygen metabolites was detected by assaying lucigenin-dependent chemiluminescence. Figs. 3 and 4 show the effect of Fn antibodies on the chemiluminescence of neutrophils stimulated by fMLP and PMA. An
100
",J
t
t
t
t
t
2 4 6 8 10 1'2 time (rn~-~n) Fig. 4. Kinetics of lucigenin-enhanced chemiluminescence by PMA (4 ng/mi)-induced neutrophils in the absence (1) or in the presence (2-5) of 40/,I antibodies (protein concentration, 5-10 mg/ml) in a cell suspension (]06/ml). In three experiments Fn antibodies were added together with either (3) - human serum albumin (10/~! of 5 mg/ml solution), or (4) - fibrinogen (10 ILl of 5 mg/ml solution), or (5) - Fn (10/,! of 1 mg/mi solution).
increase in the concentration of Fn antibodies in the incubation medium inhibits the respiratory burst. It is known [26] that the matrix and plasma Fn bind the same antibodies. The inhibitory action of antibodies is eliminated by the addition of exogenous Fn, which adsorbs the antibodies (Figs. 3, 4). Conversely, the addition of fibrinogen and albumin to a medium containing Fn antibodies does not eliminate their inhibitory action (Fig. 4). The results of our study suggest the direct involvement of the integrins and extracellular matrix proteins in the activation and regulation of the respiratory burst in neutrophils. The role of integrins in the activation of the oxidative burst is also confirmed by the data of Cabanas et al. [27], who showed that monoclonal antibodies to receptors p150,95 inhibit the Con A induced
100
Q
50
,,,d
10
20
30 40 50 ont i - h - fibronectin, pI
Fibronectin ( pg/ml)
Fig. 3. (A) Effect of Fn antibodies on iucigenin.dependent chemiluminescence of neutrophils. Cells (10~/ml) were incubated for 20 s with the indicated quantities of antibodies (protein concentration, 5-10 mg/ml) and chemiluminescence was induced by 10/~M fMLE (B) Effect of Fn antibodies on iucigenin-dependent chemiluminescence of neutrophils in the presence of exogenous Fn. Cells (106/ml) were incubated for 20 s with 25/~1 antibodies and at indicated concentrations of Fn; chemiluminescence was induced by 10 /LM fMLP. The values of chemiluminescence maxima (in mV) are expressed as mean + S.D. of the duplicate determina6ons and given in % to the value registered in the absence of antibodies.
260 formation of 02- in the monocytoid line U937. It is probable that activation of the respiratory burst enzymes needs definite structural rearrangements of the membrane, which occur as a result of activation of adhesive receptors. Acknowledgments We are indebted Dr. L.B. Margolis and Dr. S.I. Galkina for their collaboration and making available to us useful data. Thanks are also due to Mr. I.S. Kochubey for translation editing. References 1 Beczkowski, J,Z, and Gessner, T. (1988) Int. J. Biochem. 20, 569-580. 2 Makino, R,, Tanaka, T,, lizuka, T., lshimura, Y. and Kanegasaki, S. (1986) J, Biol, Chem. 261, 11444-11447. 3 Clark, R,A,, Leidal, K.G., Pearson, D.W. and Nausef, W.N. (1987) J. Biol. Chem. 262, 4065-4074. 4 Hafeman, D.G., Parce, J.W. and McConnel, H.M. (1979) Biochem. Biophys. Res. Commun. 86, 522-528. 5 Hafeman, D,G., Lewis, J.T. and McConnel, H.M. (1980) Biochemistry 19, 5387-5394. 6 Dahinden, C.D., Fenu, .l. and Hugli, T.E. (1983) J. Clin. Invest. 72, 113-121. 7 Hynes, R.O. (1987) Cell 48, 549-554. 8 Ruoslanti, E. and Piershbacher, M.D. (1987) Science 238, 491-497.
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