Immunology 1978 35 373
Organophosphorus inhibition of lysosomal enzyme secretion from polymorphonuclear leucocytes EVIDENCE OF A LACK OF A REQUIREMENT FOR ESTERASE ACTIVATION
E. L. BECKER, E. P. KOZA & M. SIG MAN Department of Pathology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
Received 17 November 1977; accepted for publication 21 December 1977
Summary. Previously, di-isopropylphosphorofloridate (DFP) was shown to inhibit the release of lysosomal enzymes induced by chemotactic factors. It was suggested that the inhibition was due to the phosphorylation of a cell bound serine esterase activated by the chemotactic factor. However, as shown here, di-isopropyl methyl phosphate, a nonphosphorylating analogue of DFP, inhibits just as well as DFP, the release of lysozyme and 8 glucuronidase from rabbit polymorphonuclear leucocytes induced by chemotactic factor in the presence of cytochalasin B. Similarly, the poorly phosphorylating phenyl ethyl pentylphosphonate and phenyl ethyl phenylpropylphosphonate inhibit enzyme release, induced under the same conditions, as well as or better than the corresponding good phosphorylators, p-nitrophenyl ethyl pentylphosphonate and pnitrophenyl ethyl phenylpropylphosphonate. Phenyl ethyl pentylphosphonate inhibits at least as well or probably better than p-nitrophenyl ethyl pentyl phosphonate, the chemotactic factor induced release of lysozyme from polymorphonuclear leucocytes
spread on Millipore filters in the absence of cytochalasin B. We conclude that, under the circumstances tested, there is no evidence that the release of lysosomal enzymes from rabbit peritoneal polymorphonuclear leucocytes induced by chemotactic factor involves the activation of an esterase. INTRODUCTION Chemotactic factors in the presence of cytochalasin B induce lysosomal enzyme release from human (Goldstein, Hoffstein, Gallin & Weissmann, 1973) or rabbit (Becker, Showell, Henson & Hsu, 1974) polymorphonuclear leucocytes in suspension or, in the absence of cytochalasin B, when rabbit peritoneal polymorphonuclear leucocytes are spread on filters. Di-isopropyl phosphorofluoridate (DFP) inhibits the lysosomal enzyme secretion from rabbit peritoneal leucocytes induced by chemotactic factors in the presence of cytochalasin B (Becker & Showell, 1974). The inhibition only occurs if DFP is present during the time the chemotactic factor is reacting with the cells. If DFP is used to pretreat the leucocytes and the DFP removed before the addition of cytochalasin B and chemotactic factor, no
Correspondence: Dr E. L. Becker, Department of Pathology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
0019-2805/78/0800-0373So2.00 ©) 1978 Blackwell Scientific Publications
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inhibition results. These results suggested the tentative conclusion that a cell bound esterase was activated by the interaction of chemotactic factor and cell and this esterase was involved in the secretory process (Becker & Showell, 1974). This conclusion was necessarily tentative at best, because it depended on the assumption that under the experimental conditions used, DFP acted only as an irreversible inhibitor of serine esterases. Although DFP is a relatively specific, irreversible inhibitor of this class of enzymes, it is known to have other actions (Woodin & Harris, 1973; Kuba, Albuquerque & Harris, 1973). The irreversible inhibition of serine esterases by organophosphorus inhibitors like DFP occurs through the phosphorylation of the active site of these enzymes (Aldridge & Reiner, 1972). We have tested the possibility that the inhibition of lysosomal enzyme secretion by DFP is not due to the phosphorylation of the active centre of the putative neutrophil esterase by comparing several nonphosphorylating or poorly phosphorylating organophosphorus inhibitors to their structurally similar phosphorylating analogues for their ability to inhibit lysosomal enzyme secretion. In what follows, we shall show that di-isopropyl methyl phosphate, a nonphosphorylating analogue of DFP is inhibitory under the same circumstances and to the same or greater extent than the good phosphorylator, DFP. Similarly, we shall report that several phenyl ethyl phosphonates that phosphorylate very poorly, inhibit chemotactic factor-induced secretion as well as or better than the corresponding p-nitrophenyl ethyl phosphonates. These results will be contrasted with the dissimilar effects of the same agents on two other functions of the neutrophil, chemotaxis (Becker, 1971) and immune dependent erythrophagocytosis (Musson & Becker, 1977).
MATERIALS AND METHODS All the experiments reported here were performed on rabbit peritoneal polymorphonuclear leucocytes. They were obtained 12-14 h after the intraperitoneal injection of 01 % glycogen, as described (Becker & Showell, 1973). The cells were washed in Hanks's balanced salt solution containing 0 01 M tris(hydroxymethyl-amino-methane), pH 7-2 1 mg/ml of glucose and 1 mg/ml crystalline bovine serum albumin. The source of the tris (hydroxymethyl
amino-methane) and crystalline bovine serum albumin was Sigma Chemical Co., St. Louis, MO. The cytochalasin B, obtained from Aldrich Chemical Co., Milwaukee, WI, was diluted just before use from a stock solution containing 4 mg/ml dimethyl sulphoxide. The final concentration of dimethyl sulphoxide was shown to have no effect on the release. The chemotactic factor used to induce release in the presence of cytochalasin B was the pronase-sensitive fraction of an Escherichia coli culture filtrate. Its preparation has been described previously (Schiffmann, Showell, Corcoran, Ward, Smith & Becker, 1975). The chemotactic factor employed in all tests of release induced on filters in the absence of cytochalasin B was the synthetic, chemotactic oligopeptide, formyl-methionyl leucyl phenylalanine, F-Met-Leu-Phe. Its preparation and use have been previously described (Showell, Freer, Zigmond, Schiffmann, Aswanikumar, Corcoran & Becker, 1976). Di-isopropylphosphorofluoridate (DFP) was obtained from Aldrich Chemical Co., Milwaukee, WI. The di-isopropyl methyl phosphate, p-nitrophenyl ethyl phosphonates and phenyl ethyl phosphonates were synthesized at Walter Reed Army Institute of Research, Washington, D.C. and have been described previously (Becker, 1971; Becker, Fukuto, Boone, Canham & Boger, 1963; Henson, Gould & Becker, 1976; Ferluga, Asherson & Becker, 1972). The degree of hydrolysis of the p-nitrophenyl ethyl phosphonates was tested by assaying their content of free pnitrophenol spectrophotometrically at 410 pm (Becker et al., 1963). The degree of hydrolysis of the phenyl ethyl phosphonates was measured by determining their content of free phenol by the technique of Lowry, Rosebrough, Farr & Randall (1965). To test the secretory ability of leucocytes suspended in the presence of cytochalasin B, the washed peritoneal cells were resuspended at a concentration of 1 x 10'/ml in Hanks's buffer containing 2 mg/ml of crystalline bovine serum albumin. In duplicate, 0 25 ml of 20 ug/ml cytochalasin was added to 0-5 ml of cells in 12 x 75 mm test tubes and allowed to stand in the cold for 10 min. Then appropriate dilutions of inhibitor, solvent or buffer and then chemotactic factor were added to bring the final volume to 1 ml. The tubes were immediately placed at 370 for 5 min (the release at this temperature is completed within 1 min) and then centrifuged at 1400 g in the cold. The supernatants were removed
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Inhibition of lysosomal enzyme secretion and aliquots taken for measurement of lactic dehydrogenase (LDH), lysozyme and f glucuronidase, as described (Showell et al., 1976). None of the inhibitors used affected the assay of any of the enzymes. The total concentration of enzyme was measured by lysing the cells with 01% Triton X-100 (Showell et al., 1976). The units of enzyme activity were calculated as described (Showell et al., 1976). In no instance was the release of the cytoplasmic marker enzyme, lactic dehydrogenase, above control levels. The secretion induced by F-Met-Leu-Phe from peritoneal polymorphonuclear leucocytes on filters (25 mm diameter of 0-8 pm average pore size, Millipore Corp., Bedford, MA) was carried out essentially as described (Becker & Showell, 1974) with only minor modifications. Briefly, the washed cells were resuspended to a concentration of 1-5 x 107/ml in Hanks's buffer containing 3 mg/ml of crystalline bovine serum albumin. In quadruplicate, 0 5 ml cells were added to the filters in 15 ml plastic beakers (VWR Scientific Co., Boston, MA) and appropriate concentrations of inhibitor, solvent or buffer and then chemotactic peptide were added. The final concentration of cells was 5 x 106/ml, of peptide, 1 -67 x 10-8 M, and of dimethyl sulphoxide, 0 25% or 0 5%. The beakers were incubated at 370 for 45 min. At the end of that time the beaker contents were poured into 12 x 75 mm test tubes and centrifuged at 1400 g for 5 min in the cold. They were assayed for lysozyme /1 glucuronidase and lactic dehydrogenase. The release of i glucuronidase was so low (Becker & Showell, 1974) that only the lysozyme results were used to calculate the degree of inhibition. The concentration of F-Met-Leu-Phe employed was chosen because it gave 60-80 % of the maximum enzyme released. Dimethyl sulphoxide was employed as the solvent for the inhibitors in place of acetone because with the 45 min incubation period, 0-25 % or 0 5 % dimethyl sulphoxide gave no inhibition of lysosomal enzyme secretion nor induced leakage of lactic dehydrogenase, whereas the same concentrations of acetone did both. RESULTS Inhibition by DFP and di-isopropyl methyl phosphate Figure
compares the
ability of varying
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tions of DFP and the non-phosphorylating disopropyl methyl phosphate to inhibit the secretion of
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mm Inhibitor Figure 1. Inhibition by (DFP) di-isopropyl phosphorofluoridate (0) and (DMP) di-isopropyl methyl phosphate (A) of the secretion of ft glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 11-9 ± 0-18; lower panel, total = 539 ± 7.
P8 glucuronidase (upper panel) and lysozyme (lower panel). The release was induced by a 1 : 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. As is evident, the curves of inhibition given by the two organophosphorus compounds are indistinguishable despite the profound difference in their ability to phosphorylate serine esterases. A second experiment gave the same results. Inhibition by p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate Figure 2 portrays the inhibition given by varying concentrations of p-nitrophenyl ethyl pentylphosphonate and the phenyl ethyl pentylphosphonate on the release of al glucuronidase (upper panel) and lysozyme (lower panel) induced under the same
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E. L. Becker, E. P. Koza & M. Sigman
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difference in the hydrolysis products. The phenyl compound was less hydrolysed than the p-nitrophenyl compound (1 % compared to 7%) and thus had less of the phosphonate salt, sodium ethyl phenylpropylphosphonate, common to both compounds. Phenol was found to be less inhibitory than p-nitrophenol giving no inhibition at 1 6 x 10-5 M, whereas p-nitrophenol even as low as 9 X 10-6 M gave 20% inhibition of lysozyme release. In this connection, in the experiment of Fig. 3, 2 x 10-6 M p-nitrophenol, by itself, inhibited the release of both enzymes by an insignificant 5%. Moreover, complete hydrolysis of the phenyl ethyl phenylpropylphosphonate reduced very greatly its inhibitory capacity (data not shown) even though the amount of hydrolysis products was much greater than in the experiment of Fig. 3.
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Figure 2. Inhibition by p-nitrophenyl ethyl pentylphosphonate (0) and phenyl ethyl pentylphosphonate (A) of the secretion of 6 glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 11 2 ± 0-62; lower panel, total = 700 ± 36.
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circumstance as the experiment of Fig. 1. No differin the curves of inhibition given by the two compounds is evident despite the phenyl derivative being a poor phosphorylator of serine esterases and the p-nitrophenyl compound a good one.
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Inhibition by p-nitrophenyl ethyl phenylpropylphosphonate and phenyl ethyl phenyipropylphosphonate
Figure 3 shows the inhibitory effect of p-nitrophenyl ethyl phenylpropylphosphonate and the poorly phosphorylating phenyl ethyl phenylpropylphosphonate on the release of, glucuronidase (upper panel) and lysozyme (lower panel). Unexpectedly and paradoxically, phenyl ethyl phenylpropylphosphonate inhibited the release of both enzymes much better than p-nitrophenyl ethyl phenylpropylphosphonate. The same result was seen in a second experiment carried out the same way. No explanation can be offered for the greater effectiveness of the phenyl derivative compared to the p-nitrophenyl compound. It could not be due to a
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Inhibitor 3. Inhibition Figure by p-nitrophenyl ethyl phenylpropylphosphonate (0) and phenyl ethyl phenylpropylphosphonate (A) of the secretion of glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 10-6 ± 0 16; lower panel, total = 630 ± 27. mm
Inhibition of lysosomal enzyme secretion In work not shown here, the p-nitrophenyl ethyl phenylethylphosphonate was compared to the corresponding phenyl compound. As in Fig. 3, the phenyl compound inhibited release more effectively than the p-nitrophenyl compound, although the difference between the two was distinctly less than that seen in Fig. 3.
Effect of organophosphorus compounds on release induced on filters All of the work just described was done with cells in suspension in the presence of cytochalasin B. Differences exist between the release induced in this manner compared to the release induced on filters in the absence of cytochalasin B, for example, the release on filters is much slower and shows a greater dependence on Ca2+ than the release from cells in suspension (Goldstein et al., 1973; Becker & Showell, 1974; Goldstein, Hoffstein & Weissmann, 1975; Showell, Naccache, Sha'afi & Becker, 1977). Because of these differences we compared the effects of phosphorylating and poorly phosphorylating inhibitors on release induced on filters by the chemotactic peptide, F-Met-Leu-Phe, as described in Materials and Methods. The first experiments were performed with DFP and di-isopropyl methylphosphate. These were abandoned when it was found that 1 mM-3 mm DFP, in the presence or absence of chemotactic factor, frequently caused leakage of LDH from the cells that was time and concentration dependent. As in the preceding experiments, this was not evident at 5 min. Di-isopropyl methyl phosphate was a less
377
frequent offender. In addition, DFP at the higher concentrations, occasionally gave release that was greater than that given by the controls with no inhibitor. Thus, although the inhibition by diisopropyl methyl phosphate was clearly as great as, and frequently greater than that obtained with DFP, because of the difficulties mentioned, we could not evaluate the significance of the results. We, therefore, turned to a comparison of the effects of p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate. Table 1 shows the average results of three independent experiments comparing the effects of 0 5 and 1 0 mm concentrations of the two compounds. In these experiments, the effect of 0-25% and 05 % dimethyl sulphoxide on the release was also tested. These concentrations had no effect on release and the results obtained were averaged with the controls containing neither inhibitor or solvent. The inhibitory trend is evident from Table 1, even though only the inhibition given by 1 mm phenyl ethyl pentylphosphonate is statistically significant. In any event, it is clear that the p-nitrophenyl ethyl pentylphosphonate is no more and probably less inhibitory than the phenyl ethyl pentylphosphonate. DISCUSSION In the preceding sections, we have shown that the non-phosphorylating, di-isopropyl methyl phosphate inhibits lysosomal enzyme secretion induced by chemotactic factors just as well as the very active phosphorylator, DFP (Fig. 1). Di-isopropyl methyl
Table 1. Effect of p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate on lysozyme release induced by F-Met-Leu-Phe from polymorphonuclear leucocytes on filters*
Inhibitor
p-nitrophenylt
phenylt None
Concentration (mM)
Activity§ (o%)
ActivityT (O%)
05 1.0 05 1-0
17-1 ±2-0 13 2 ±2-4 13-3 ±2-6
105 ±3-3 80 ±9-0 80 11
123±16 16-3 ±-6
74±4 100
* The results reported are the average of three experiments ± s.e.m.
t p-nitrophenyl ethyl pentylphosphonate. t Phenyl ethyl pentylphosphonate.
§ Activity as percent of total lysozyme activity; total activity = 1 120 ± 47 units. ¶ Activity as percent of control with no inhibitor.
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phosphate does not inhibit serine esterases. Similarly, the poorly phosphorylating phenyl ethyl pentylphosphonate (Fig. 2, Table 1) or phenyl ethyl phenylpropylphosphonate (Fig. 3) inhibits secretion as well, or better than the structurally very similar, active phosphorylators, p-nitrophenyl ethyl pentylphosphonate or p-nitrophenyl phenylpropylphosphonate. The phenyl derivatives are 0 04-0 07 % as active as serine esterase inhibitors as the corresponding p-nitrophenyl compounds (Becker, 1971). The inhibitory activity of the non- or poorly phosphorylating organophosphorus compounds is evident whether the release is induced by chemotactic factors from rabbit peritoneal polymorphonuclear leucocytes in suspension in the presence of cytochalasin B or in the absence of cytochalasin B from cells on filters. One possible explanation for the ability of the diisopropyl methyl phosphate or the phenyl ethyl phosphonates to inhibit chemotactic factor induced secretion is that they contain small amounts of a very active phosphorylating impurity. This is very unlikely for several reasons: the purification of these compounds involved washing in strong alkali; this had the purpose of destroying the more active phosphorylating impurities. In addition, one would have to postulate that the putative impurity(s) is highly active against the supposed esterase involved in lysosomal enzyme release but poorly active, if at all, against known serine esterases like acetylcholinesterase and chymotrypsin. Moreover, this hypothesis would not explain why some compounds are inactive in other responses of the neutrophil and other cells where DFP and the phosphorylating phosphonates inhibit (see below). Rather, the results imply that inhibition by DFP or the p-nitrophenyl ethyl phosphonates is occurring through a mechanism other than the phosphorylation of an esterase. What the mechanism of the inhibition is, is unclear but from the results we must conclude that at present we have no evidence that esterase activation is involved in lysosomal enzyme release induced by chemotactic factors. The inhibition by organophosphorus compounds of chemotactic factor-induced lysosomal enzyme release from neutrophils on surfaces seems to occur through the same non-phosphorylating mechanism as that occurring from cells suspended with cytochalasin B (Table 1). It is unknown why the phenyl ethyl pentylphosphonate and the p-nitrophenyl analogue gave much less inhibition of the release
induced from leucocytes on filters (0-25% inhibition, Table 1) than when the cells were in suspension in the presence of cytochalasin B (80% inhibition, Fig. 2). It is unlikely to be due to the use of different chemotactic factors for the two reactions. We have shown that the pronase-sensitive bacterial chemotactic factor reacts with the same receptor on the peritoneal neutrophil as the synthetic, chemotactic peptides like F-Met-Leu-Phe (Aswanikumar, Corcoran, Schiffmann, Day, Free, Showell, Becker & Pert, 1977). The chemotactic factor-induced secretion of cells in suspension and presumably also of cells on surfaces is related to an influx of Ca2+ and possibly of Na+ (Naccache, Showell, Becker & Sha'afi, 1977). On this basis, we speculate that the susceptibility of the stimulated transport of Ca2+ and possibly Na+ to the inhibitory action of the organophosphorus compounds might be greater when the cells are in suspension with cytochalasin B than when they are on surfaces in its absence.* We have not tested the ability of poorly phosphorylating organophosphorus compounds to inhibit the release induced by the CSa chemotactic factor. In view of the essential similarity of the release process induced by CSa and the simple peptides or the bacterial chemotactic factor it is unlikely that a difference will be found in this respect. The non- or poorly phosphorylating organophosphorus compounds are not able to inhibit all neutrophil functions inhibited by the good phosphorylators. Phenyl ethyl butylphosphonate has no effect on the locomotion of rabbit peritoneal polymorphonuclear leucocytes stimulated by chemotactic factors even though, under the same conditions, p-nitrophenyl ethyl butylphosphonate is distinctly inhibitory (Becker, 1971). Similarly, di-isopropyl methyl phosphate is without effect on immune dependent erythrophagocytosis, whereas DFP inhibits the same reaction very significantly (Musson & Becker, 1977). Thus, there seems to be a clear-cut divergence with respect to esterase activation in the mechanism of these latter two neutrophil responses compared to chemotactic factor induced lysosomal enzyme secretion. The basis for this difference is unknown. This and other work (Woodin & Wieneke, 1970; * It is to be noted that in this sentence we have selfconsciously refrained from using the cliche 'It is tempting to speculate . . .'. This noble self denial arises from the realization that in this instance, if in no other, many that do use it follow Oscar Wilde in being able to resist everything but temptation.
Inhibition of lysosomal enzyme secretion Woodin & Harris, 1973; Kuba, Albuquerque & Harris, 1973) underlines in stark strokes the necessity for attempting to control for the ability of organophosphorus inhibitors to act in complex biological systems as other than irreversible inhibitors of serine esterases. This has been done only in some instances. In addition to the studies of neutrophil locomotion and phagocytosis already alluded to, we have shown that di-isopropyl methyl phosphate and phenyl ethyl pentyl phosphonate are without effect on the stimulation of the locomotion of mouse B lymphocytes by anti-mouse immunoglobulin (Becker & Unanue, 1976) and on the stimulus-specific activation of the platelet release reaction (Henson et al., 1976), whereas the corresponding phosphorylating analogues markedly inhibit both kinds of reaction. Dr Teruko Ishizaka has informed one of us (ELB) that in unpublished work she has found that diisopropyl methyl phosphate has no effect on the immunologically induced release of histamine from human basophils. However, di-isopropyl methyl phosphate, the phenyl ethyl phosphosphonates or similar compounds have not been tested in other biological reactions where a role for esterase activation has been suggested. These reactions include lysosomal enzyme secretion from neutrophils induced by aggregated IgG on non-phagocytosable surfaces (Henson, 1972), inhibition of concanavalin A-induced capping of human neutrophils (Mandel, Spilberg & Lichtman, 1977), antibody-mediated lymphocyte killing and the induced release of histamine from human and guinea-pig lung or rat mast cells (reviewed in Musson & Becker, 1976). It is obvious that as a minimum, this sort of evidence is required before there can be a fuller acceptance of the hypothesis that esterase activation is required in these reactions.
ACKNOWLEDGMENTS We wish to thank Mr Henry J. Showell for his assistance and advice. The work was supported in part by U.S.P.H.S. Research Grants Al 09648 and TO1-A10043.
REFERENCES ALDRIDGE W.N. & REINER E. (1972) Enzyme Inhibitors as
Substrates. North-Holland Publishing Co., Amsterdam, Netherlands.
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ASWANIKUMAR S., CORCORAN B., SCHIFFMANN E., DAY A.R., FREER R.J., SHOWELL H.J., BECKER E.L. & PERT C.B. (1977) Demonstration of a receptor on rabbit neutrophils for chemotactic peptides. Biochem. biophys. Res. Commun. 74, 710. BECKER E.L. (1971) Phosphonate inhibition of the accumulation and retention of K+ by rabbit neutrophils in relation to chemotaxis. J. Immunol. 106, 689. BECKER E.L., FUKUTO T., BOONE B., CANHAM D. & BOGER E. (1963) The relationship of enzyme inhibitory activity to the structure of n-alkylphosphonate and phenylalkylphosphonate esters. Biochemistry, 2, 72. BECKER E.L. & SHOWELL H.J. (1972) Effects of Ca2+ and Mg2+ on the chemotactic responsiveness of rabbit polymorphonuclear leukocytes. Z. Immunitaetsforsch. Exp. Klin. Immunol. 143, 466. BECKER E.L. & SHOWELL H.J. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. 11. The mechanism of release. J. Immunol. 112, 2055. BECKER E.L., SHOWELL H.J., HENSON P.M. & Hsu L. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. 1. The characteristics of the release, the importance of surfaces and the relationship of enzyme release to chemotactic responsiveness. J. Immunol. 112, 1047. BECKER E.L. & UNANUE E.R. (1976) The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes. J. Immunol. 117, 27. FERLUGA J., ASHERSON G.L. & BECKER E.L. (1972) The effect of organophosphorus inhibitors, p-nitrophenol and cytochalasin B on cytotoxic killing of tumour cells by immune spleen cells and the effect of shaking. Immunology, 23, 577. GOLDSTEIN I., HOFFSTEIN S., GALLIN J. & WEISSMANN G. (1973) Mechanisms of lysosomal enzyme release from human leukocytes: Microtubule assembly and membrane fusion induced by a component of complement. Proc. nat. Acad. Sci. U.S.A. 70, 2916. GOLDSTEIN I.M., HOFFSTEIN S.T. & WEISSMANN G. (1975) Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes. J. Immunol. 115, 665. HENSON P.M. (1972) Pathologic mechanisms in neutrophilmediated injury. Am. J. Path. 68, 593. HENSON P.M., GOULD D. & BECKER E.L. (1976) Activation of stimulus specific serine esterases (proteases) in the initiation of platelet secretion. 1. Demonstration with organophosphorus inhibitors. J. exp. Med. 144, 1657. KUBA E., ALBUQUERQUE E.X. & HARRIS E.A. (1973) Diisopropylfluorophosphate: Suppression of ionic conductance of the cholinergic receptor. Science, 181, 853. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDAL R.J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MANDEL B.F., SPILBERG I. & LICHTMAN J. (1977) Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor. J. Immunol. 118, 1375. MUSSON R.A. & BECKER E.L. (1977) The role of an activatable esterase in immune dependent phagocytosis by human neutrophils. J. Immunol. 118, 1354. NACCACHE P.H., SHOWELL H.J., BECKER E.L. & SHA'AFI R.I. (1977) Changes in ionic movements across rabbit
polymorphonuclear leukocyte membranes during lyso-
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somal enzyme release: Possible ionic basis for lysosomal enzyme release. J. Cell. Biol. (In Press). SCHIFFMANN E., SHOWELL H.J., CORCORAN B.A., WARD P.A., SMITH E. & BECKER E.L. (1975) The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J. Immunol. 114, 1831. SHOWELL H.J., FREER R.J., ZIGMOND S.H., SCHIFFMANN E., ASWANIKUMAR S., CORCORAN B. & BECKER E.L. (1976) The structure activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J. exp. Med. 143, 1154.
SHOWELL H.J., NACCACHE P.H., SHA'AFI R.I. & BECKER E.L. (1977) The effects of extracellular K+, Na+ and Ca2+ on lysosomal enzyme secretion from polymorphonuclear leukocytes. J. Immunol. 119, 803. WOODIN A.M. & HARRIs A. (1973) The inhibition of locomotion of the polymorphonuclear leukocyte by organophosphorus compounds. Exp. Cell Res. 77, 41. WOODIN A.M. & WIENEKE A.A. (1970) Action of DFP on the leucocyte and the axon. Nature (Lond.), 277, 460.
Organophosphorus inhibition of lysosomal enzyme secretion from polymorphonuclear leucocytes EVIDENCE OF A LACK OF A REQUIREMENT FOR ESTERASE ACTIVATION
E. L. BECKER, E. P. KOZA & M. SIG MAN Department of Pathology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
Received 17 November 1977; accepted for publication 21 December 1977
Summary. Previously, di-isopropylphosphorofloridate (DFP) was shown to inhibit the release of lysosomal enzymes induced by chemotactic factors. It was suggested that the inhibition was due to the phosphorylation of a cell bound serine esterase activated by the chemotactic factor. However, as shown here, di-isopropyl methyl phosphate, a nonphosphorylating analogue of DFP, inhibits just as well as DFP, the release of lysozyme and 8 glucuronidase from rabbit polymorphonuclear leucocytes induced by chemotactic factor in the presence of cytochalasin B. Similarly, the poorly phosphorylating phenyl ethyl pentylphosphonate and phenyl ethyl phenylpropylphosphonate inhibit enzyme release, induced under the same conditions, as well as or better than the corresponding good phosphorylators, p-nitrophenyl ethyl pentylphosphonate and pnitrophenyl ethyl phenylpropylphosphonate. Phenyl ethyl pentylphosphonate inhibits at least as well or probably better than p-nitrophenyl ethyl pentyl phosphonate, the chemotactic factor induced release of lysozyme from polymorphonuclear leucocytes
spread on Millipore filters in the absence of cytochalasin B. We conclude that, under the circumstances tested, there is no evidence that the release of lysosomal enzymes from rabbit peritoneal polymorphonuclear leucocytes induced by chemotactic factor involves the activation of an esterase. INTRODUCTION Chemotactic factors in the presence of cytochalasin B induce lysosomal enzyme release from human (Goldstein, Hoffstein, Gallin & Weissmann, 1973) or rabbit (Becker, Showell, Henson & Hsu, 1974) polymorphonuclear leucocytes in suspension or, in the absence of cytochalasin B, when rabbit peritoneal polymorphonuclear leucocytes are spread on filters. Di-isopropyl phosphorofluoridate (DFP) inhibits the lysosomal enzyme secretion from rabbit peritoneal leucocytes induced by chemotactic factors in the presence of cytochalasin B (Becker & Showell, 1974). The inhibition only occurs if DFP is present during the time the chemotactic factor is reacting with the cells. If DFP is used to pretreat the leucocytes and the DFP removed before the addition of cytochalasin B and chemotactic factor, no
Correspondence: Dr E. L. Becker, Department of Pathology, University of Connecticut Health Center, Farmington, CT 06032, U.S.A.
0019-2805/78/0800-0373So2.00 ©) 1978 Blackwell Scientific Publications
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inhibition results. These results suggested the tentative conclusion that a cell bound esterase was activated by the interaction of chemotactic factor and cell and this esterase was involved in the secretory process (Becker & Showell, 1974). This conclusion was necessarily tentative at best, because it depended on the assumption that under the experimental conditions used, DFP acted only as an irreversible inhibitor of serine esterases. Although DFP is a relatively specific, irreversible inhibitor of this class of enzymes, it is known to have other actions (Woodin & Harris, 1973; Kuba, Albuquerque & Harris, 1973). The irreversible inhibition of serine esterases by organophosphorus inhibitors like DFP occurs through the phosphorylation of the active site of these enzymes (Aldridge & Reiner, 1972). We have tested the possibility that the inhibition of lysosomal enzyme secretion by DFP is not due to the phosphorylation of the active centre of the putative neutrophil esterase by comparing several nonphosphorylating or poorly phosphorylating organophosphorus inhibitors to their structurally similar phosphorylating analogues for their ability to inhibit lysosomal enzyme secretion. In what follows, we shall show that di-isopropyl methyl phosphate, a nonphosphorylating analogue of DFP is inhibitory under the same circumstances and to the same or greater extent than the good phosphorylator, DFP. Similarly, we shall report that several phenyl ethyl phosphonates that phosphorylate very poorly, inhibit chemotactic factor-induced secretion as well as or better than the corresponding p-nitrophenyl ethyl phosphonates. These results will be contrasted with the dissimilar effects of the same agents on two other functions of the neutrophil, chemotaxis (Becker, 1971) and immune dependent erythrophagocytosis (Musson & Becker, 1977).
MATERIALS AND METHODS All the experiments reported here were performed on rabbit peritoneal polymorphonuclear leucocytes. They were obtained 12-14 h after the intraperitoneal injection of 01 % glycogen, as described (Becker & Showell, 1973). The cells were washed in Hanks's balanced salt solution containing 0 01 M tris(hydroxymethyl-amino-methane), pH 7-2 1 mg/ml of glucose and 1 mg/ml crystalline bovine serum albumin. The source of the tris (hydroxymethyl
amino-methane) and crystalline bovine serum albumin was Sigma Chemical Co., St. Louis, MO. The cytochalasin B, obtained from Aldrich Chemical Co., Milwaukee, WI, was diluted just before use from a stock solution containing 4 mg/ml dimethyl sulphoxide. The final concentration of dimethyl sulphoxide was shown to have no effect on the release. The chemotactic factor used to induce release in the presence of cytochalasin B was the pronase-sensitive fraction of an Escherichia coli culture filtrate. Its preparation has been described previously (Schiffmann, Showell, Corcoran, Ward, Smith & Becker, 1975). The chemotactic factor employed in all tests of release induced on filters in the absence of cytochalasin B was the synthetic, chemotactic oligopeptide, formyl-methionyl leucyl phenylalanine, F-Met-Leu-Phe. Its preparation and use have been previously described (Showell, Freer, Zigmond, Schiffmann, Aswanikumar, Corcoran & Becker, 1976). Di-isopropylphosphorofluoridate (DFP) was obtained from Aldrich Chemical Co., Milwaukee, WI. The di-isopropyl methyl phosphate, p-nitrophenyl ethyl phosphonates and phenyl ethyl phosphonates were synthesized at Walter Reed Army Institute of Research, Washington, D.C. and have been described previously (Becker, 1971; Becker, Fukuto, Boone, Canham & Boger, 1963; Henson, Gould & Becker, 1976; Ferluga, Asherson & Becker, 1972). The degree of hydrolysis of the p-nitrophenyl ethyl phosphonates was tested by assaying their content of free pnitrophenol spectrophotometrically at 410 pm (Becker et al., 1963). The degree of hydrolysis of the phenyl ethyl phosphonates was measured by determining their content of free phenol by the technique of Lowry, Rosebrough, Farr & Randall (1965). To test the secretory ability of leucocytes suspended in the presence of cytochalasin B, the washed peritoneal cells were resuspended at a concentration of 1 x 10'/ml in Hanks's buffer containing 2 mg/ml of crystalline bovine serum albumin. In duplicate, 0 25 ml of 20 ug/ml cytochalasin was added to 0-5 ml of cells in 12 x 75 mm test tubes and allowed to stand in the cold for 10 min. Then appropriate dilutions of inhibitor, solvent or buffer and then chemotactic factor were added to bring the final volume to 1 ml. The tubes were immediately placed at 370 for 5 min (the release at this temperature is completed within 1 min) and then centrifuged at 1400 g in the cold. The supernatants were removed
375
Inhibition of lysosomal enzyme secretion and aliquots taken for measurement of lactic dehydrogenase (LDH), lysozyme and f glucuronidase, as described (Showell et al., 1976). None of the inhibitors used affected the assay of any of the enzymes. The total concentration of enzyme was measured by lysing the cells with 01% Triton X-100 (Showell et al., 1976). The units of enzyme activity were calculated as described (Showell et al., 1976). In no instance was the release of the cytoplasmic marker enzyme, lactic dehydrogenase, above control levels. The secretion induced by F-Met-Leu-Phe from peritoneal polymorphonuclear leucocytes on filters (25 mm diameter of 0-8 pm average pore size, Millipore Corp., Bedford, MA) was carried out essentially as described (Becker & Showell, 1974) with only minor modifications. Briefly, the washed cells were resuspended to a concentration of 1-5 x 107/ml in Hanks's buffer containing 3 mg/ml of crystalline bovine serum albumin. In quadruplicate, 0 5 ml cells were added to the filters in 15 ml plastic beakers (VWR Scientific Co., Boston, MA) and appropriate concentrations of inhibitor, solvent or buffer and then chemotactic peptide were added. The final concentration of cells was 5 x 106/ml, of peptide, 1 -67 x 10-8 M, and of dimethyl sulphoxide, 0 25% or 0 5%. The beakers were incubated at 370 for 45 min. At the end of that time the beaker contents were poured into 12 x 75 mm test tubes and centrifuged at 1400 g for 5 min in the cold. They were assayed for lysozyme /1 glucuronidase and lactic dehydrogenase. The release of i glucuronidase was so low (Becker & Showell, 1974) that only the lysozyme results were used to calculate the degree of inhibition. The concentration of F-Met-Leu-Phe employed was chosen because it gave 60-80 % of the maximum enzyme released. Dimethyl sulphoxide was employed as the solvent for the inhibitors in place of acetone because with the 45 min incubation period, 0-25 % or 0 5 % dimethyl sulphoxide gave no inhibition of lysosomal enzyme secretion nor induced leakage of lactic dehydrogenase, whereas the same concentrations of acetone did both. RESULTS Inhibition by DFP and di-isopropyl methyl phosphate Figure
compares the
ability of varying
concentra-
tions of DFP and the non-phosphorylating disopropyl methyl phosphate to inhibit the secretion of
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mm Inhibitor Figure 1. Inhibition by (DFP) di-isopropyl phosphorofluoridate (0) and (DMP) di-isopropyl methyl phosphate (A) of the secretion of ft glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 11-9 ± 0-18; lower panel, total = 539 ± 7.
P8 glucuronidase (upper panel) and lysozyme (lower panel). The release was induced by a 1 : 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. As is evident, the curves of inhibition given by the two organophosphorus compounds are indistinguishable despite the profound difference in their ability to phosphorylate serine esterases. A second experiment gave the same results. Inhibition by p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate Figure 2 portrays the inhibition given by varying concentrations of p-nitrophenyl ethyl pentylphosphonate and the phenyl ethyl pentylphosphonate on the release of al glucuronidase (upper panel) and lysozyme (lower panel) induced under the same
376
E. L. Becker, E. P. Koza & M. Sigman
E
0
difference in the hydrolysis products. The phenyl compound was less hydrolysed than the p-nitrophenyl compound (1 % compared to 7%) and thus had less of the phosphonate salt, sodium ethyl phenylpropylphosphonate, common to both compounds. Phenol was found to be less inhibitory than p-nitrophenol giving no inhibition at 1 6 x 10-5 M, whereas p-nitrophenol even as low as 9 X 10-6 M gave 20% inhibition of lysozyme release. In this connection, in the experiment of Fig. 3, 2 x 10-6 M p-nitrophenol, by itself, inhibited the release of both enzymes by an insignificant 5%. Moreover, complete hydrolysis of the phenyl ethyl phenylpropylphosphonate reduced very greatly its inhibitory capacity (data not shown) even though the amount of hydrolysis products was much greater than in the experiment of Fig. 3.
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Figure 2. Inhibition by p-nitrophenyl ethyl pentylphosphonate (0) and phenyl ethyl pentylphosphonate (A) of the secretion of 6 glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 11 2 ± 0-62; lower panel, total = 700 ± 36.
0
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circumstance as the experiment of Fig. 1. No differin the curves of inhibition given by the two compounds is evident despite the phenyl derivative being a poor phosphorylator of serine esterases and the p-nitrophenyl compound a good one.
_ _
ence
0
0
Inhibition by p-nitrophenyl ethyl phenylpropylphosphonate and phenyl ethyl phenyipropylphosphonate
Figure 3 shows the inhibitory effect of p-nitrophenyl ethyl phenylpropylphosphonate and the poorly phosphorylating phenyl ethyl phenylpropylphosphonate on the release of, glucuronidase (upper panel) and lysozyme (lower panel). Unexpectedly and paradoxically, phenyl ethyl phenylpropylphosphonate inhibited the release of both enzymes much better than p-nitrophenyl ethyl phenylpropylphosphonate. The same result was seen in a second experiment carried out the same way. No explanation can be offered for the greater effectiveness of the phenyl derivative compared to the p-nitrophenyl compound. It could not be due to a
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Inhibitor 3. Inhibition Figure by p-nitrophenyl ethyl phenylpropylphosphonate (0) and phenyl ethyl phenylpropylphosphonate (A) of the secretion of glucuronidase (upper panel) and lysozyme (lower panel) induced by a 1: 1000 dilution of the pronase sensitive bacterial factor in the presence of 5 pg/ml cytochalasin B. Upper panel, total = 10-6 ± 0 16; lower panel, total = 630 ± 27. mm
Inhibition of lysosomal enzyme secretion In work not shown here, the p-nitrophenyl ethyl phenylethylphosphonate was compared to the corresponding phenyl compound. As in Fig. 3, the phenyl compound inhibited release more effectively than the p-nitrophenyl compound, although the difference between the two was distinctly less than that seen in Fig. 3.
Effect of organophosphorus compounds on release induced on filters All of the work just described was done with cells in suspension in the presence of cytochalasin B. Differences exist between the release induced in this manner compared to the release induced on filters in the absence of cytochalasin B, for example, the release on filters is much slower and shows a greater dependence on Ca2+ than the release from cells in suspension (Goldstein et al., 1973; Becker & Showell, 1974; Goldstein, Hoffstein & Weissmann, 1975; Showell, Naccache, Sha'afi & Becker, 1977). Because of these differences we compared the effects of phosphorylating and poorly phosphorylating inhibitors on release induced on filters by the chemotactic peptide, F-Met-Leu-Phe, as described in Materials and Methods. The first experiments were performed with DFP and di-isopropyl methylphosphate. These were abandoned when it was found that 1 mM-3 mm DFP, in the presence or absence of chemotactic factor, frequently caused leakage of LDH from the cells that was time and concentration dependent. As in the preceding experiments, this was not evident at 5 min. Di-isopropyl methyl phosphate was a less
377
frequent offender. In addition, DFP at the higher concentrations, occasionally gave release that was greater than that given by the controls with no inhibitor. Thus, although the inhibition by diisopropyl methyl phosphate was clearly as great as, and frequently greater than that obtained with DFP, because of the difficulties mentioned, we could not evaluate the significance of the results. We, therefore, turned to a comparison of the effects of p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate. Table 1 shows the average results of three independent experiments comparing the effects of 0 5 and 1 0 mm concentrations of the two compounds. In these experiments, the effect of 0-25% and 05 % dimethyl sulphoxide on the release was also tested. These concentrations had no effect on release and the results obtained were averaged with the controls containing neither inhibitor or solvent. The inhibitory trend is evident from Table 1, even though only the inhibition given by 1 mm phenyl ethyl pentylphosphonate is statistically significant. In any event, it is clear that the p-nitrophenyl ethyl pentylphosphonate is no more and probably less inhibitory than the phenyl ethyl pentylphosphonate. DISCUSSION In the preceding sections, we have shown that the non-phosphorylating, di-isopropyl methyl phosphate inhibits lysosomal enzyme secretion induced by chemotactic factors just as well as the very active phosphorylator, DFP (Fig. 1). Di-isopropyl methyl
Table 1. Effect of p-nitrophenyl ethyl pentylphosphonate and phenyl ethyl pentylphosphonate on lysozyme release induced by F-Met-Leu-Phe from polymorphonuclear leucocytes on filters*
Inhibitor
p-nitrophenylt
phenylt None
Concentration (mM)
Activity§ (o%)
ActivityT (O%)
05 1.0 05 1-0
17-1 ±2-0 13 2 ±2-4 13-3 ±2-6
105 ±3-3 80 ±9-0 80 11
123±16 16-3 ±-6
74±4 100
* The results reported are the average of three experiments ± s.e.m.
t p-nitrophenyl ethyl pentylphosphonate. t Phenyl ethyl pentylphosphonate.
§ Activity as percent of total lysozyme activity; total activity = 1 120 ± 47 units. ¶ Activity as percent of control with no inhibitor.
378
E. L. Becker, E. P. Koza & M. Sigman
phosphate does not inhibit serine esterases. Similarly, the poorly phosphorylating phenyl ethyl pentylphosphonate (Fig. 2, Table 1) or phenyl ethyl phenylpropylphosphonate (Fig. 3) inhibits secretion as well, or better than the structurally very similar, active phosphorylators, p-nitrophenyl ethyl pentylphosphonate or p-nitrophenyl phenylpropylphosphonate. The phenyl derivatives are 0 04-0 07 % as active as serine esterase inhibitors as the corresponding p-nitrophenyl compounds (Becker, 1971). The inhibitory activity of the non- or poorly phosphorylating organophosphorus compounds is evident whether the release is induced by chemotactic factors from rabbit peritoneal polymorphonuclear leucocytes in suspension in the presence of cytochalasin B or in the absence of cytochalasin B from cells on filters. One possible explanation for the ability of the diisopropyl methyl phosphate or the phenyl ethyl phosphonates to inhibit chemotactic factor induced secretion is that they contain small amounts of a very active phosphorylating impurity. This is very unlikely for several reasons: the purification of these compounds involved washing in strong alkali; this had the purpose of destroying the more active phosphorylating impurities. In addition, one would have to postulate that the putative impurity(s) is highly active against the supposed esterase involved in lysosomal enzyme release but poorly active, if at all, against known serine esterases like acetylcholinesterase and chymotrypsin. Moreover, this hypothesis would not explain why some compounds are inactive in other responses of the neutrophil and other cells where DFP and the phosphorylating phosphonates inhibit (see below). Rather, the results imply that inhibition by DFP or the p-nitrophenyl ethyl phosphonates is occurring through a mechanism other than the phosphorylation of an esterase. What the mechanism of the inhibition is, is unclear but from the results we must conclude that at present we have no evidence that esterase activation is involved in lysosomal enzyme release induced by chemotactic factors. The inhibition by organophosphorus compounds of chemotactic factor-induced lysosomal enzyme release from neutrophils on surfaces seems to occur through the same non-phosphorylating mechanism as that occurring from cells suspended with cytochalasin B (Table 1). It is unknown why the phenyl ethyl pentylphosphonate and the p-nitrophenyl analogue gave much less inhibition of the release
induced from leucocytes on filters (0-25% inhibition, Table 1) than when the cells were in suspension in the presence of cytochalasin B (80% inhibition, Fig. 2). It is unlikely to be due to the use of different chemotactic factors for the two reactions. We have shown that the pronase-sensitive bacterial chemotactic factor reacts with the same receptor on the peritoneal neutrophil as the synthetic, chemotactic peptides like F-Met-Leu-Phe (Aswanikumar, Corcoran, Schiffmann, Day, Free, Showell, Becker & Pert, 1977). The chemotactic factor-induced secretion of cells in suspension and presumably also of cells on surfaces is related to an influx of Ca2+ and possibly of Na+ (Naccache, Showell, Becker & Sha'afi, 1977). On this basis, we speculate that the susceptibility of the stimulated transport of Ca2+ and possibly Na+ to the inhibitory action of the organophosphorus compounds might be greater when the cells are in suspension with cytochalasin B than when they are on surfaces in its absence.* We have not tested the ability of poorly phosphorylating organophosphorus compounds to inhibit the release induced by the CSa chemotactic factor. In view of the essential similarity of the release process induced by CSa and the simple peptides or the bacterial chemotactic factor it is unlikely that a difference will be found in this respect. The non- or poorly phosphorylating organophosphorus compounds are not able to inhibit all neutrophil functions inhibited by the good phosphorylators. Phenyl ethyl butylphosphonate has no effect on the locomotion of rabbit peritoneal polymorphonuclear leucocytes stimulated by chemotactic factors even though, under the same conditions, p-nitrophenyl ethyl butylphosphonate is distinctly inhibitory (Becker, 1971). Similarly, di-isopropyl methyl phosphate is without effect on immune dependent erythrophagocytosis, whereas DFP inhibits the same reaction very significantly (Musson & Becker, 1977). Thus, there seems to be a clear-cut divergence with respect to esterase activation in the mechanism of these latter two neutrophil responses compared to chemotactic factor induced lysosomal enzyme secretion. The basis for this difference is unknown. This and other work (Woodin & Wieneke, 1970; * It is to be noted that in this sentence we have selfconsciously refrained from using the cliche 'It is tempting to speculate . . .'. This noble self denial arises from the realization that in this instance, if in no other, many that do use it follow Oscar Wilde in being able to resist everything but temptation.
Inhibition of lysosomal enzyme secretion Woodin & Harris, 1973; Kuba, Albuquerque & Harris, 1973) underlines in stark strokes the necessity for attempting to control for the ability of organophosphorus inhibitors to act in complex biological systems as other than irreversible inhibitors of serine esterases. This has been done only in some instances. In addition to the studies of neutrophil locomotion and phagocytosis already alluded to, we have shown that di-isopropyl methyl phosphate and phenyl ethyl pentyl phosphonate are without effect on the stimulation of the locomotion of mouse B lymphocytes by anti-mouse immunoglobulin (Becker & Unanue, 1976) and on the stimulus-specific activation of the platelet release reaction (Henson et al., 1976), whereas the corresponding phosphorylating analogues markedly inhibit both kinds of reaction. Dr Teruko Ishizaka has informed one of us (ELB) that in unpublished work she has found that diisopropyl methyl phosphate has no effect on the immunologically induced release of histamine from human basophils. However, di-isopropyl methyl phosphate, the phenyl ethyl phosphosphonates or similar compounds have not been tested in other biological reactions where a role for esterase activation has been suggested. These reactions include lysosomal enzyme secretion from neutrophils induced by aggregated IgG on non-phagocytosable surfaces (Henson, 1972), inhibition of concanavalin A-induced capping of human neutrophils (Mandel, Spilberg & Lichtman, 1977), antibody-mediated lymphocyte killing and the induced release of histamine from human and guinea-pig lung or rat mast cells (reviewed in Musson & Becker, 1976). It is obvious that as a minimum, this sort of evidence is required before there can be a fuller acceptance of the hypothesis that esterase activation is required in these reactions.
ACKNOWLEDGMENTS We wish to thank Mr Henry J. Showell for his assistance and advice. The work was supported in part by U.S.P.H.S. Research Grants Al 09648 and TO1-A10043.
REFERENCES ALDRIDGE W.N. & REINER E. (1972) Enzyme Inhibitors as
Substrates. North-Holland Publishing Co., Amsterdam, Netherlands.
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ASWANIKUMAR S., CORCORAN B., SCHIFFMANN E., DAY A.R., FREER R.J., SHOWELL H.J., BECKER E.L. & PERT C.B. (1977) Demonstration of a receptor on rabbit neutrophils for chemotactic peptides. Biochem. biophys. Res. Commun. 74, 710. BECKER E.L. (1971) Phosphonate inhibition of the accumulation and retention of K+ by rabbit neutrophils in relation to chemotaxis. J. Immunol. 106, 689. BECKER E.L., FUKUTO T., BOONE B., CANHAM D. & BOGER E. (1963) The relationship of enzyme inhibitory activity to the structure of n-alkylphosphonate and phenylalkylphosphonate esters. Biochemistry, 2, 72. BECKER E.L. & SHOWELL H.J. (1972) Effects of Ca2+ and Mg2+ on the chemotactic responsiveness of rabbit polymorphonuclear leukocytes. Z. Immunitaetsforsch. Exp. Klin. Immunol. 143, 466. BECKER E.L. & SHOWELL H.J. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. 11. The mechanism of release. J. Immunol. 112, 2055. BECKER E.L., SHOWELL H.J., HENSON P.M. & Hsu L. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. 1. The characteristics of the release, the importance of surfaces and the relationship of enzyme release to chemotactic responsiveness. J. Immunol. 112, 1047. BECKER E.L. & UNANUE E.R. (1976) The requirement for esterase activation in the anti-immunoglobulin-triggered movement of B lymphocytes. J. Immunol. 117, 27. FERLUGA J., ASHERSON G.L. & BECKER E.L. (1972) The effect of organophosphorus inhibitors, p-nitrophenol and cytochalasin B on cytotoxic killing of tumour cells by immune spleen cells and the effect of shaking. Immunology, 23, 577. GOLDSTEIN I., HOFFSTEIN S., GALLIN J. & WEISSMANN G. (1973) Mechanisms of lysosomal enzyme release from human leukocytes: Microtubule assembly and membrane fusion induced by a component of complement. Proc. nat. Acad. Sci. U.S.A. 70, 2916. GOLDSTEIN I.M., HOFFSTEIN S.T. & WEISSMANN G. (1975) Influence of divalent cations upon complement-mediated enzyme release from human polymorphonuclear leukocytes. J. Immunol. 115, 665. HENSON P.M. (1972) Pathologic mechanisms in neutrophilmediated injury. Am. J. Path. 68, 593. HENSON P.M., GOULD D. & BECKER E.L. (1976) Activation of stimulus specific serine esterases (proteases) in the initiation of platelet secretion. 1. Demonstration with organophosphorus inhibitors. J. exp. Med. 144, 1657. KUBA E., ALBUQUERQUE E.X. & HARRIS E.A. (1973) Diisopropylfluorophosphate: Suppression of ionic conductance of the cholinergic receptor. Science, 181, 853. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDAL R.J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265. MANDEL B.F., SPILBERG I. & LICHTMAN J. (1977) Inhibition of polymorphonuclear leukocyte capping by a chemotactic factor. J. Immunol. 118, 1375. MUSSON R.A. & BECKER E.L. (1977) The role of an activatable esterase in immune dependent phagocytosis by human neutrophils. J. Immunol. 118, 1354. NACCACHE P.H., SHOWELL H.J., BECKER E.L. & SHA'AFI R.I. (1977) Changes in ionic movements across rabbit
polymorphonuclear leukocyte membranes during lyso-
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somal enzyme release: Possible ionic basis for lysosomal enzyme release. J. Cell. Biol. (In Press). SCHIFFMANN E., SHOWELL H.J., CORCORAN B.A., WARD P.A., SMITH E. & BECKER E.L. (1975) The isolation and partial characterization of neutrophil chemotactic factors from Escherichia coli. J. Immunol. 114, 1831. SHOWELL H.J., FREER R.J., ZIGMOND S.H., SCHIFFMANN E., ASWANIKUMAR S., CORCORAN B. & BECKER E.L. (1976) The structure activity relations of synthetic peptides as chemotactic factors and inducers of lysosomal enzyme secretion for neutrophils. J. exp. Med. 143, 1154.
SHOWELL H.J., NACCACHE P.H., SHA'AFI R.I. & BECKER E.L. (1977) The effects of extracellular K+, Na+ and Ca2+ on lysosomal enzyme secretion from polymorphonuclear leukocytes. J. Immunol. 119, 803. WOODIN A.M. & HARRIs A. (1973) The inhibition of locomotion of the polymorphonuclear leukocyte by organophosphorus compounds. Exp. Cell Res. 77, 41. WOODIN A.M. & WIENEKE A.A. (1970) Action of DFP on the leucocyte and the axon. Nature (Lond.), 277, 460.
