Inhibition of polymorphonuclear leukocyte functions by fluorinated nitrobenzenes

Chem.-Biol. Interactions, 52 (1984) 163-172 Elsevier Scientific Publishers Ireland Ltd.

INHIBITION OF POLYMORPHONUCLEAR BY FLUORINATED NITROBENZENES

JAN G.R. ELFERINK

163

LEUKOCYTE

FUNCTIONS

and MARTHA DEIERKAUF

Laboratory of Medical Biochemistry, Sylvius Laboratories, aarseweg 72, 2333 AL Leiden (The Netherlands)

University

of Leiden,

Wassen-

(Received May 25th, 1984) (Revision received August 23rd, 1984) (Accepted August 27th, 1984)

SUMMARY

The bifunctional fluorinated nitrobenzenes, 1,5-difluor&2,4-dinitrobenzene (DFDNB) and 4,4’-difluoro-3,3’-dinitrodiphenyl sulfone (DFDNDPS), and the monofunctional 1-fluoro-2,4-dinitrobenzene (FDNB) inhibit chemotaxis, phagocytosis, exocytosis and the respiratory burst of rabbit polymorphonuclear leukocytes. Inhibition occurs in the micromolar concentration range; the bifunctional compounds are stronger inhibitory than the monofunctional one. The inhibitory effect can be counteracted by sulfhydryl compounds and not with amino-group containing compounds. The results suggest that an interaction with vulnerable sulfhydryl groups, located in a hydrophobic surrounding, is the basis of the inhibitory effect of the fluorinated nitrobenzenes.

Key words: Polymorphonuclear leukocytes - Fluorinated nitrobenzenes Chemotaxis - Phagocytosis - Exocytosis - Respiratory burst

-

INTRODUCTION

The primary task of the polymorphonuclear leukocyte (PMN) is phagocytosis and destruction of invading microorganisms. For the latter purpose the neutrophil is activated during phagocytosis, resulting in degranulation and the ‘respiratory burst’ [l-3]. Degranulation comprises the fusion of lysosomes and other granules with the microorganism containing phagosome Abbreviations: CB, cytochalasin B; DFDNB, 1,5difluoro-2,4dinitrobenzene; DFDNDPS, FMLP, 4,4’-difluoro-3,3’dinitrodiphenyl sulfone; FDNB, l-fluoro-2,4dinitrobenzene; LDH, lactate dehydrogenase; PMA, phorbol formyl-methionyl-leucyl-phenylalanine; myristate acetate; NBT, nitroblue tetraaolium; PMNs, polymorphonuclear leukocytes. 0009-2797/84/$03.00

o 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

164 or with the plasma membrane, allowing digestive enzymes to get access to the extracellular space (exocytosis). In the respiratory burst a membranebound oxidase is activated, leading to the conversion of molecular oxygen into superoxide (0;) ; this superoxide is then subsequently processed into highly reactive oxygen metabolites, such as hydrogen peroxide (H202), hydroxyl radicals (‘OH) and singlet oxygen (‘0,) [ 4,5]. Whereas intracellular degranulation and the intracellular release of oxygen metabolites is of importance for the killing of invaders, exocytosis and the extracellular release of oxygen metabolites can result in inflammation [ 3,4]. It is possible to study the processes of degranulation and respiratory burst apart from phagocytosis, because it is possible to induce degranulation and respiratory burst, in the absence of phagocytosis, with certain soluble stimuli such as ionophore A23187, chemotactic peptide, phorbol myristate acetate and others [ 6-31. A complete physiologic response of the PMN includes chemotaxis, followed by phagocytosis and activation of the cell to respiratory burst and degranulation. The physiology of these functions shares common determinants and interrelated biochemical pathways. For an understanding of the mechanisms of these functions, and to establish their interrelationship, highly reactive inhibitors, acting in low concentrations, may be useful. In this respect we considered the effect of DFDNB and related compounds on chemotaxis, phagocytosis, degranulation and respiratory burst.

DFDNDPS

DFDNB NO2

FDNB METHODS

PMNs Rabbit peritoneal polymorphonuclear leukocytes were obtained as described previously [9]. The medium used consisted of 140 mM NaCl, 5 mM KCl, 10 mM glucose, 1 mM CaCl,, 1 mM MgCl, and 20 mM Hepes; the pH of the medium was 7.3. In the experiments PMNs were preincubated

165 with or without fluorinated volume of 1 ml, containing stimulus was given, followed

nitrobenzenes for 10 min at 37”C, in a total 3 X lo6 PMNs. After this preincubation time a by incubation.

Exocy tosis

Exocytosis was measured as the release of the granule-associated enzymes lysozyme and p-D-glucuronidase, in the absence of a significant release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH). As a stimulus to induce exocytosis the combination formylmethionylleucylphenylalanine (FMLP) (lo-’ M) + cytochalasin B (5 X 10m6 M) was used. After an incubation of 30 min at 37°C the mixture was centrifuged, and enzyme release in the supernatant was determined as described previously [ 91. Respiratory

burst

.

NBT reduction was used as a measure for the respiratory burst [lo]. We found that both NBT reduction and cytochrome c reduction are indicators of the activation of the respiratory burst, though both methods do not cover exactly the same aspect of the metabolic burst [lo]. During the burst reducing molecules (e.g. superoxide) are formed; these reduce the yellowish, soluble nitro-blue tetrazolium chloride into a dark, insoluble formazan. PMNs were incubated in a medium containing 0.04% NBT and 1 mM KCN. The respiratory burst was induced with the combination FMLP (lo-’ M) + cytochalasin B (5 X lob6 M). After incubation for 15 min 5 ml 0.5 N HCl was added to stop the reaction. The mixture was centrifuged, and the residue was dissolved in 2 ml pyridine, by warming in a boiling waterbath for 10 min. After cooling to room-temperature the extinction of * the pyridine-formazan solution was measured at 509 nm. By comparison with the extinction of a pyridine solution of formazan, obtained by chemical reduction of NBT with ascorbic acid, the extinction was converted into nmoles NBT reduced per 3 X lo6 PMNs per 15 min. Phagocy tosis

Uptake of opsonized zymosan was taken as a measure for phagocytosis. Phagocytosis was determined after 30 min incubation at 37°C. The mixture was centrifuged. In the supematant the release of enzymes was determined. To the residue 5 X low3 M EDTA was added to block phagocytosis. Subsequently the zymosan particles taken up were counted using oil immersion microscopy. Cells with two or more zymosan particles were counted as phagocytic. Chemo taxis

Chemotaxis was measured with the Boyden chamber technique, using agent, as previously described [ll] . The 1O-9 M FMLP as a chemotactic PMS’s were preincubated for 10 min with the fluorinated nitrobenzenes in the concentrations indicated, after which the medium was supplemented

166 with 0.5% bovine serum albumin 37°C.

(BSA), followed

by incubation

for 1 h at

Reagents Cytochalasin B (CB) was obtained from Aldrich Co. and the other chemicals were from Sigma. Opsonized zymosan was prepared as described before [9]. DFDNB and DFDNDPS were dissolved in DMSO to give a concentrated stock solution, just before the experiment. Dilutions of these compounds and of FDNB were made in ethanol. Microliter quantities were added to the cell suspension; the final concentration of organic solvent did not exceed 0.3%. This concentration of DMSO did not interfere with PMN functions. RESULTS

Exposure of polymorphonuclear leukocytes to the chemotactic peptide FMLP in the presence of CB results in extensive degranulation, measured as lysozyme release in the absence of LDH release, and in a respiratory burst, measured as NBT reduction. Both degranulation and respiratory burst are inhibited by the fluorinated nitrobenzenes (Figs. 1 and 2). Degranulation refers to exocytosis of specific granules and to exocytosis of azurophilic granules. Lysozyme is present in both types of granules, whereas fl-glucuronidase is confined to the azurophilic granules only. As can be seen in Fig. 1,

Fig. 1. Effect of fluorinated nitrobenzenes on exocytosis by PMNs, induced by FMLP + CB. Exocytosis was measured as the selective release of the granule-associated enzymes lysozyme and p-glucuronidase; the release of the cytoplasmic enzyme LDH was determined as a measure for cell damage, The values given are the mean values of 4 experilysozyme release; ----, @-glucuronidase release; 3, DFDNB; -1, ments. DFDNDPS; 2, ‘FDNB. 3, A, m LDH release for DFDNB, DFDNDPS and FDNB, respectively.

167

M inhibitor Fig. 2. Inhibition of FMLP + CB-induced NBT reduction by fluorinated nitrobenzenes. NBT reduction is expressed as nanomoles NBT reduced per 3 X lo6 PMNs per 15 min. The values given are the mean values of 3 experiments. -v-, DFDNB; --A-, DFDNDPS; --c--, FDNB.

inhibition of lysozyme release and glucuronidase occurs in a parallel fashion, thus degranulation of both types of granules is inhibited alike. Some experiments were performed with ionophore A23187 to induce exocytosis, and with phorbol myristate acetate (PMA) to induce a respiratory burst. Exocytosis and metabolic burst, induced by these reagents, are inhibited at about the same concentration of fluorinated nitrobenzenes (results not shown) as is FMLP-induced exocytosis and respiratory burst. Phagocytosis of zymosan is accompanied by release of granule associated enzymes. Both phagocytosis and concomitant degranulation are inhibited by the fluorinated nitrobenzenes in the micromolar concentration range (Figs. 3a-c). DFDNB and DFDNDPS are a tenfold stronger in inhibiting phagocytosis than is FDNB. The absence of LDH release indicates that the fluorinated nitrobenzenes are not cytotoxic in the concentration range used in our experiments. Slightly higher concentrations are required to inhibit chemotaxis (Fig. 4). This may perhaps be due to the fact, that during incubation bovine serum albumin is present in the chemotaxis experiments, in order to get optimal migration conditions, whereas this product is not required for the other functions tested. Because the results presented in Figs. l-4 are carried out on several days on PMNs from different animals, we have compared the inhibiting effect of the fluorinated nitrobenzenes on cells of one animal on the same day (Table I). This experiment confirms the results presented in the figures: all fluorinated nitrobenzenes investigated inhibit all neutrophil functions in the

168

169

60-

M inhlbitor Fig. 4. The effect of fluorinated nitrobenzenes on FMLP-induced chemotaxis. Chemotaxis is expressed as distance travelled in micrometers. The results given are the mean -^i; DFDNB; ---A---, DFDNDPS; -D-, FDNB. value of 3 experiments.

micromolar concentration range. DFDNB is a slightly stronger inhibitor than DFDNDPS, whereas for FDNB the inhibiting concentration is an order of magnitude higher; this sequence applied for all PMN functions. Phagocytosis is inhibited at somewhat lower concentrations than the other functions. In order to evaluate the relative importance of --SH and -NH, groups as a target during the effect of fluorinated nitrobenzenes, DFDNB was given the opportunity to react with certain sulfhydryl compounds (dithiothreitol, TABLE

I

COMPARISON BETWEEN THE INHIBITING EFFECT NITROBENZENES ON ALL PMN FUNCTIONS TESTED

OF

THE

FLUORINATED

Experiments were carried out on cells of one animal on the same day; the results may thus slightly differ from those given in the figures. Three concentrations of each inhibitor, around the expected concentration, were tested. The data were plotted in a graph and from this graph the concentration of inhibitor, required to give 50% inhibition of a given function, was estimated. Concentration

DFDNB DFDNDPS FDNB

of inhibitor

required

to give 50% inhibition

of

Chemotaxis (rM1

Phagocytosis (PM)

Exocytosis (rM1

NBT reduction (rM1

2.5 4 17

0.8 0.9 6

2 2.5 6

1 1.5 7

170 TABLE

II

EFFECT DFDNB

OF SULFHYDRYL AND AMINOGROUP-CONTAINING rNHIBITION OF EXOCYTOSIS

COMPOUNDS

ON

Medium containing sulfhydryl compounds or amino group compounds, with or without DFDNB were preincubated for exactly 10 min at 37°C. Then the cells were added, followed by the exocytotic stimulus (FMLP + CB), and the mixture was incubated for 30 min at 37°C. Lys, lysozyme; Glut, glucuronidase. The values given are the mean values of 4 experiments t S.D. Enzyme

release (%)

Control

0.5 mM dithiothreitol 0.5 mM glutathione 0.5 mM arginine 0.5 mM lysine 5 mM lysine

5 * 1O-6 M DFDNB

LDH

Lys

2*1 5 + 1 4*1 2to It0 3+1

74 84 86 87 71 82

Glut f f f f f r

7 5 5 3 6 7

58 62 73 60 49 64

t f * * f +

3 1 3 6 6 5

-

LDH

Lys

Glut

0+-o 4fl 3*2 O+O Ok0 lkl

lo+1 84+7 81*4 17?2 16t3 15k3

10 t 2 55 * 3 71 t 5 6+-l 8tl 552

glutathione), or NH,-compounds (lysine, arginine), under comparable conditions. The results are represented in Table II. Under the conditions of our experiments (10 min preincubation in medium at 37°C) the inhibiting effect of DFDNB on exocytosis is annihilated when it is combined with a sylfhydry1 compound. When during preincubation an amino-compound is present, the inhibiting effect of DFDNB remains about the same upon exposure to activated PMNs. DISCUSSION

The results obtained show that the fluorinated nitrobenzenes tested are strong inhibitors of the examined PMN functions. There is little difference in the concentration required of a given compound to inhibit the several functions. Phagocytosis is inhibited at slightly lower concentrations than the other PMN functions, but the difference is not striking. The PMN functions tested are very complicated and consist of a large number of steps, eventually resulting in a given function. A number of these steps they have in common and a number of them is different and specific for one function. The resemblance in inhibitions suggest that the fluorinated nitrobenzenes interfere at a step which is common to all PMN functions tested. The bifunctional reagents DFDNB and DFDNDPS, which have two reactive fluoro atoms in one molecule, are stronger inhibitors than the monofunctional reagent FDNB. A similar difference in reactivity has been found in several systems [12,13]. An explanation of this phenomenon may be that the spatial separation of the reactive groups on the reagent molecule coincides with the distance of two reacting groups, and the formation of

171 cross-links, which may be the result of such a reaction. Another explanation of the difference in reactivity between the bifunctional and monofunctional reagents is based on their difference in polarity: the bifunctional reagents DFDNB and DFDNDPS are less polar than the monofunctional FDNB. This is reflected in the octanol: water partition coefficients which are higher for DFDNB and DFDNDPS than for FDNB [14]. This implies that bifuncticnal reagents can cross more easily the hydrophobic lipid bilayer core of the membrane, and can easily reach reactive groups in an apolar surrounding. The fluorinated nitrobenzenes are known as amino group reagents [15-171. This study was primarily undertaken to evaluate the possible relevance of NH2 groups for neutrophil functions. Whereas however, sometimes the suggestion is made that these compounds are special reagents for NH2 groups [18], they may react with other groups as well, such as SH groups [14]. Fluorinated nitrobenzenes can react with aminogroups on phospholipids, giving rise to cross-linking in the case of bifunctional reagents [ 19-211. However, the concentrations of DFDNB and DFDNDPS (+400 PM [19] ), used in the experiment to demonstrate crosslinking of phospholipids in erythrocyte membranes, are much higher than in our experiments. In order to find out whether NH, groups or SH groups on proteins were involved in the reaction with fluorinated nitrobenzenes, under the conditions of our experiments, DFDNB was given the opportunity to react with NH,or SH-containing aminoacid before addition of PMNs. The inactivation of DFDNB by dithiothreitol and glutathione, and the relative ineffectiveness of lysine and arginine indicates, that under the conditions of our experiments this reagent is much more apt to react with sulfhydryl groups, than with amino groups on proteins. It has been shown previously that neutrophil functions requires a sulfhydryl containing protein, whose function is dramatically dependent on the intactness of sulfhydryl groups [22,23]. We also found, that strongly hydrophobic sulfhydryl reagents, i.e. N-naphthylmaleimide, were much more potent inhibitors of exocytose than weakly hydrophobic reagents [23]. The same was found by Yamashita et al. [24] for inhibition of chemotaxis. It seems therefore likely that the fluorinated nitrobenzenes inhibit neutrophil functions as a consequence of a reaction with vulnerable sulfhydryl groups. These sulfhydryl groups are either located in a hydrophobic surrounding, or can only be reached after passage through a hydrophobic barrier, i.e. the membrane. Because the PMN has a high intracellular concentration of glutathione (4 mM) [25], a likely location of the sulfhydryl groups involved is the inner side of the plasma membrane. REFERENCES 1 T.P. Stossel, Phagocytosis, New Engl. J. 2 M.N.I. Walters and J.M. Papadimitriou, col. (1978) 377. 3 G. Weissmann, J.E. Smolen and H.M. from stimulated neutrophils, New Engl.

Med., 290 (1974) 717. Phagocytosis: a review, CRC Crit. Rev. ToxiKorchak, Release of inflammatory J. Med., 303 (1980) 27.

mediators

172 4 J.C. Fantone and P.A. Ward, Role of oxygenderived free radicals and metabolites in leukocytedependent inflammatory reactions, Am. J. Pathol., 107 (1982) 397. 5 J.A. Badwey and M.L. Karnovsky, Active oxygen species and the function of phagocytic leukocytes, Annu. Rev. Biochem., 49 (1980) 695. 6 I.M. Goldstein and G. Weissmann, Nonphagocytic stimulation of human polymorphonuclear leukocytes: role of the plasma membrane, Sem. Hematol., 16 (1979) 175. I L.R. DeChatelet, Initiation of the respiratory burst in human polymorphonuclear neutrophils: a critical review, J. Reticuloendothel. Sot., 24 (1978) 73. 8 G. Weissmann, KM. Korchak, H.D. Perez, J.E. Smolen, J.M. Goldstein and S.T. Hoffstein, The secretory code of the neutrophil, J. Reticuloendothel. Sot., 26 (1979) 687. 9 J.G.R. Elferink, Chlorpromazine inhibits phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes, Biochem. Pharmacol., 28 (1979) 965. Elferink, Measurement of the metabolic burst in human neutrophils: A com10 J.G.R. parison between cytochrome c and NBT reduction, Res. Commun. Chem. Pathol. Pharmacol., 43 (1984) 339. 11 J.G.R. Elferink and M. Deierkauf, The effect of verapamil and other calcium antagonists on chemotaxis of polymorphonuclear leukocytes, Biochem. Pharmacol., 33 (1984) 35. 12 N. Salem, C.J. Lauter and E.G. Trans, Selective chemical modification of plasma membrane ectoenzymes, Biochim. Biophys. Acta, 641 (1981) 366. of sarcoplasmic reticulum ATPase protein with 1,5-difluoro13 G. Bailin, Crosslinking 2,4dinitrobenzene, Biochim. Biophys. Acta, 624 (1980) 511. 14 W.E. Harris and W.L. Stahl, Organization of thiolgroups of electric-cell electric-organ sodium-plus-potassium ion-stimulated adenosine triphosphatase studied with bifunctional reagents, Biochem. J., 185 (1980) 707. The aminoacid sequence in the phenylalanyl chain of 15 F. Sanger and H. Tuppy, insulin, Biochem. J., 49 (1951) 463. G.V. Marinetti and G.B. Segel, The interaction of l-fluoro-2,4-dinitro16 S.E. Gordesky, benzene with aminophospholipids in membranes of intact erythrocytes, modified erythrocytes and erythrocyte ghosts, J. Membrane Biol., 14 (1973) 229. 17 J. Poensgen and H. Passow, Action of 1-fluoro-2,4-dinitrobenzene on passive ion permeability of the human red blood cell, J. Membrane Biol., 6 (1971) 210. Reactivity of fluorodinitrobenzene 18 S.S. Joski, V.S. Basrur and M.B. Sahasrabudhe, with intact human leukocytes: examination of sites of action and the molecular entities involved in interaction, J. Biosci., 4 (1982) 441. 19 S.P. Marley and K.H. Tsai, Crosslinking of phospholipids in human erythrocyte membrane, Biochem. Biophys. Res. Commun., 65 (1975) 31. and R. Love, Extent of crosslinking of amino-phospholipid neigh20 G.V. Marinetti bors in the erythrocyte membrane as influenced by the concentration of difluorodinitrobenzene, Biochem. Biophys. Res. Commun., 61 (1974) 30. 21.G.V. Marinetti and R. Love, Differential reaction of cell membrane phospholipids and proteins with chemical probes, Chem. Phys. Lipids, 16 (1976) 239. and M.A. Lichtman, The role of sulfhydryl groups in human neutro22 G.F. Gordiano phi1 adhesion, movement and particle ingestion, J. Cell. Physiol., 82 (1973) 387. 23 J.G.R. Elferink and J.C. Riemersma, Effects of sulfhydryl reagents on phagocytosis and exocytosis in rabbit polymorphonuclear leukocytes, Chem.-Biol. Interact., 30 (1980) 139. 24 T. Yamashita, Y. Tanake, T. Horigome and E. Fujikawa, Dependence on the lipophilicity of maleimide derivatives in their inhibitory action upon chemotaxis in neutrophils, Experientia, 35 (1979) 1054. 25 J.M. Oliver, D.F. Albertini and R.D. Berlin, Effects of glutathione-oxidizing agents on microtubule assembly and microtubule-dependent surface properties of human neutrophils, J. Cell Biol., 71 (1976) 921.