Clin. exp. Immunol. (1990) 81, 352-356
Stimulation of neutrophil elastase and myeloperoxidase release by IgG fragments I.
ECKLE, G. KOLB, C. HEISER & K. HAVEMANN Centre for Internal Medicine, Philipps Marburg, West Germany
University,
(Acceptedfor publication 8 March 1990)
SUMMARY Human leucocyte elastase (HLE) cleaves IgG into Fab and Fc fragments. The Fc fragment bears an elastase-specific antigen and has previously been reported to be found in synovial fluid during rheumatoid arthritis. In addition, biological activity of elastase-specific Fc fragments has been described in modulating granulocyte oxidative metabolism. To investigate further regulatory effects of the elastase-induced IgG cleavage products, we tested the elastase and myeloperoxidase release of granulocytes. IgG fragments induce no enzyme release of unstimulated neutrophils. But elastase and myeloperoxidase release of cytochalasin b/FMLP-treated neutrophils is stimulated in a dosedependent manner by the Fab fragments. The extent of stimulation depends on stimulus concentration and is at its maximum for low (e.g. 2 5 10-8 M) FMLP concentration. Ten nanomoles Fab/4 x 106 PMN augment elastase release to 206% and myeloperoxidase release to 155% after prestimulation with 2 5 10-8 M FMLP. Fc fragments stimulate elastase release to 162% but no MPO release. Untreated IgG1 and analog Fab and Fc fragments produced by papain cleavage react similarly. Elastase-generated IgG fragments may therefore up-regulate their concentration by stimulating elastase release. The concomitantly stimulated release of myeloperoxidase may influence bactericidal activity and termination of oxidative burst. x
x
Keywords elastase myeloperoxidase IgG fragments release neutrophils
Reactive oxygen metabolites like superoxideanion and hydrogenperoxide are produced by the membrane bound NADPH oxidase (Gabig & Babior, 1979). Hydrogenperoxide together with halogenid and the released MPO constitute a system with high oxidizing potency (Klebanoff, 1975). Stimulation is produced in vivo by the invading microorganisms and bacterial products or activated serum components. The down-regulation has been only poorly understood until now; it is possibly caused partly by substances produced by the PMN themselves. We have measured elastase and MPO release of neutrophils dependent on the elastase-generated IgG fragments. The Fc fragments are already shown to have an inhibitory effect on 02generation of prestimulated neutrophils (Eckle, Kolb & Havemann, 1990). We therefore looked for possible regulation mechanisms of the fragments on elastase release. We also tested MPO which constitutes the second powerful oxidative system beside the 02-forming NADPH oxidase in stimulated neutrophils.
INTRODUCTION The acute inflammatory response is characterized by a rapid influx of polymorphonuclear granulocytes (PMN) under the influence of various chemotactic agents. At the inflammatory locus the PMN are higher stimulated to secrete lysosomal enzymes and produce activated oxygen radicals (Bender et al., 1983). The lysosomal enzymes of the neutrophil are stored in azurophil (1) and specific (2°) granules. The azurophil granules contain elastase, cathepsin G, myeloperoxidase (MPO), figlucuronidase, and lysozyme; the specific granules lysozyme, collagenase, lactoferrin and receptors for FMLP and C3bi (Ohlsson, Olsson & Spitznagel, 1977; Boxer & Smolen, 1988). Elastase is a neutral proteinase with broad substrate specificity. In plasma the released enzyme is rapidly complexed by inhibitors like al-proteinaseinhibitor and a2-macroglobulin (Ohlsson & Olsson, 1974). Nevertheless, protein degradation by elastase occurs in vivo, as specific split-products of IgG and fibrinogen have been detected (Kolb et al., 1988; Weitz et al., 1986).
MATERIALS AND METHODS Preparation of IgG fragments Neutrophil elastase HLE was isolated as described by Baugh & Travis (1976), starting from neutrophils of chronic myeloid
Correspondence: Ilsebill Eckle, PhD, Zentrum Innere Medizin, Philipps-Universitdt, Baldingerstrasse, D-355 Marburg, West Germany.
352
Stimiulation o 'release leukaemia patients. The purity of the enzyme tested by disk electrophoresis (Reisfeld, Lewis & Williams. 1962). IgG I isolated from plasma of plasmocytoma patients by anion-exchange chromatography and tested for purity by immunodiffusion (Mancini. Carbonara & Heremans. 1965) and SDS -PAGE (Weber & Osborn. 1969). IgG I fragments were generated by incubation of IgG I with purified clastase and separation of the incubation mixture by size-exclusion chromatography (Sepharose CL 6B in 100 m\i sodium phosphate buffer. pH 6 5). anion-exchange chromatography (DEAE Sepharose CL 4B in 20 m.\ triethanolamin. pH 7 5; sodium chloride gradient 0-1 xi) and affinity chromatography on protein A Sepharose (100 mxi sodium phosphate buffer. pH 7 0; desorption xxith 100 mnt acetic acid) (Eckle ct al.. 1990). Chromatographic material was provided by Pharmacia LKBI Bromma, Sweden. Isolation of papain generated fragments xxas performed in the same way after incubation of IgGI with papain (Serxa. Heidelberg. FRG), according to Porter (1959). The purity of the fragments was tested by SDS-PAGE and ultra-thin isoelectric focusing (Pharmacia LKB). Protein determination was done by dye-binding assay (BioRad Laboratories. Munich. FRG).
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0) 10-6), the Fc fragments showed neutral isoelectric points (HLE-Fc 6-6-7-3; Pap-Fc 5-8-6 5). Incubation of neutrophils with IgG I or IgG l fragments in a concentration range of 0-2 to 20 nmol/4 x 106 PMN for up to 1 h resulted in no measurable release of HLE or MPO. Release of LDH by PMN exposed to IgG fragments was not different than that of cells exposed to buffer alone (maximal
4%), and there was no additional release of LDH by stimulated cells pre-incubated with fragments. This indicates that IgG fragments were not toxic to PMN at the concentrations employed in these experiments. Simultaneous incubation of FMLP-stimulated cells with HLE-generated Fab fragments resulted in a concentrationdependent augmentation of HLE and MPO release. Figure la shows HLE release by different FMLP concentrations. Mean values of three different cell donors done in duplicate are given. Addition of HLE-induced Fab (10 nmol/4 x 106 PMN) results in an increase of HLE release over nearly the entire FMLP concentration range. Maximum over-stimulation is obtained by low FMLP concentrations, e.g. 2-5 x 10-8 M from 27 5% to 55.6% of the total HLE content. In Fig. lb the analogous data for MPO release are shown. This is only stimulated by HLE-Fab (10 nmol/4 x 106 PMN) in the concentration range of 10-8-10-7 M FMLP at its maximum from 5 2% to 23 8% of the total MPO content. Figure 2 shows stimulation of HLE and MPO release by 2-5 x 10-8 M FMLP versus concentration of added Fab. The Fab-stimulated release is expressed as percentage of release values by FMLP only. HLE release is already stimulated by about I nmol Fab/4 x 106 PMN, while an equal stimulation of MPO release is achieved by about 10 nmol Fab. The stimulating effect of Fab depends on the sequence of addition. Maximum stimulation is obtained by simultaneous addition of fragments and stimulus to the cells. Pre-incubation of fragments with cells gives lower values and addition of fragments after FMLP has no effect. Incubation of released HLE and MPO with Fab fragments results in no change of enzyme activity, so an influence on the test system can be excluded.
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Stimulation of release Figure 3 compares the effects of HLE-generated Fab with Fc fragments, with papain-produced fragments and with native IgGI in identical concentrations of 10 nmol/4 x 106 PMN. The stimulated release is expressed as percentage of release by FMLP alone. As a control, BSA is included, which does not stimulate HLE or MPO release. HLE-Fc stimulates only HLE release, to 160%, but not MPO release. Native IgGI and IgG fragments by papain cleavage stimulate HLE and MPO release between 155 and 215%.
DISCUSSION HLE cleaves IgG into Fab and Fc fragments beside small molecular peptides (Kolb et al., 1982). The fragments have similar molecular weights (43 kD versus 54 kD) but can be isolated by ion-exchange chromatography because of their widely different isoelectric points (Eckle et al., 1990). The analogous Fab and Fc fragments by papain cleavage (molar mass 51 kD versus 47 kD) with correspondent isoelectric points can be isolated in the same way. Stimulation of isolated neutrophils with FMLP after pretreatment with cytochalasin b results in a concentrationdependent release of azurophil granules. Maximum release by 5 x 10-7 M FMLP is 61% of HLE and 49% of MPO content. Although the enzymes are both stored in azurophil granules, heterogeneity of the granule contents (Damiano et al., 1988; Pember, Shapira & Kincade, 1983) can result in a different enzyme share (Bentwood & Benson, 1980). Self-deactivation of released MPO (Edwards, Nurcombe & Hart, 1987) was not measured after stimulating for up to 30 min. Released HLE is known to be resistant to oxidative inactivation (Vissers & Winterton, 1987). IgGl or IgGl fragments did not induce degranulation of unstimulated cells (Treadway et al., 1979; Henson, Johnson & Speigelberg, 1972). Addition of IgGl and its fragments to FMLP-stimulated cells resulted in a potent increase of elastase and MPO release. The amount of over-stimulation depended on the applied FMLP concentration: the smaller the stimulus concentration, the greater the additional effect of the fragments, and maximally stimulated cells could not be further degranulated (Fig. la, b). HLE release was already stimulated by smaller Fab concentrations than MPO release (Fig. 2). This can also be a consequence of granule heterogeneity (Damiano et al., 1988). The addition of fragments together with FMLP resulted in maximum stimulation, addition after FMLP had no effect as the release is already terminated within several seconds (Smolen, Korchack & Weismann, 1983). Augmented enzyme release by cell damage induced by IgG fragments could be excluded, as there was no higher LDH release after incubation with FMLP, with or without protein. An influence of the fragments on the test system for HLE or MPO could be excluded by measurements of enzyme activity after addition of fragments which induced no change. Both HLE and MPO release were maximally influenced by Fab fragments: HLE-Fab stimulated HLE and papain-Fab MPO release to 205 and 215% of values without addition of fragments respectively. IgG1 and Fc fragments showed smaller effects. Moreover, by HLE cleavage two molecules of Fab and one Fc are produced from one IgG, therefore a quantitatively higher effect on release is achieved by IgG cleavage. As papain-
generated IgGI fragments reacted analogous to the HLEinduced Fab fragments, the effect of the IgG fragments on the enzyme release is not specific for the elastase cleavage. This is in contrast to the effects of IgG fragments on oxidative burst (Eckle et al., 1990), where only HLE-generated Fc fragments show an effect. FMLP stimulates enzyme release after binding to a specific receptor via at least two pathways: 10 augmentation of intracellular calcium concentration by release from intracellular stores, or by enhanced influx of extracellular calcium and 2° by activation of protein kinase C. The mechanism of enhanced FMLP stimulation by IgG fragments may therefore be explained through modulation of calcium metabolism or protein kinase C activity (White et al., 1984; Wilson et al., 1987; Salzer, Gerard & McCall, 1987; Kokot et al., 1987), as already discussed for the inhibitory effects of IgG fragments on oxidative burst. There is an inverse relation between oxidative burst and enzyme release, as scavengers of oxygen-derived reactants potentiate degranulation (Skosey et al., 1981). MPO itself may play a role in terminating the oxidative burst of neutrophils, e.g. by inhibiting the 02-producing NADPH oxidase (Jandl et al., 1978; Edwards & Swain, 1986). This was also demonstrated by the prolonged oxidative burst of MPO-deficient neutrophils (Rosen & Klebanoff, 1976; Nauseef, Metcalf & Root, 1983). Oxidative inactivation of chemotactic factors by MPO also results in down-regulation of PMN function (Clark, 1982). However, oxidative inactivation of enzyme inhibitors like al proteinase inhibitor by the MPO/H202/halide system may potentiate the effects of HLE and thereby stimulate proteolysis (Carp & Janoff, 1980; Shock & Baum, 1988). HLE-generated IgGI fragments have a stimulating effect on HLE and MPO release. The released HLE may further degrade IgG, thereby potentiating the effect of the fragments. Released MPO may enhance HLE action by inactivation of plasma inhibitors on the one hand act synergistically with the IgG fragments in the down-regulation of dAidative metabolism on the other hand. ACKNOWLEDGEMENT Supported by the Bundesministerium fur Forschung und Technologie, grant no. 01 VM 8613.
REFERENCES BAUGH, R.J. & TRAVIS, J. (1976) Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry, 15, 836. BENDER, J.G., MCPHAIL, L.C. & VAN Epps, D.E. (1983) Exposure of human neutrophils to chemotactic factors potentiates activation of the respiratory burst enzyme. J. Immunol. 130, 2316. BENTWOOD, B.J. & HENSON, P.M. (1980) The sequential release of granule constituents from human neutrophils. J. Immunol. 124, 855. BERGMEYER, H.U. (1983) Methods in Enzymatic Analysis (ed. by H. U. Bergmeyer) p. 737. Academic Press, New York. BOYUM, A. (1967) IV. Isolation of mononuclear cells and granulocytes from blood-isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation of 1g. Scand. J. clin. Lab. Invest. 21, (Suppl. 97), 77. BOXER, L.A. & SMOLEN, J. E. (1988) Neutrophil granule constituents and their release in health and disease. Hematol. Oncol. Clinics North Am. 2, 101. CARP, H. & JANOFF, A. (1980) Potential mediator of inflammation.
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Phagocyte-derived oxidans suppress the elastase-inhibitory capacity of alphal-proteinase inhibitor in vitro. J. clin. Invest. 66, 987. CLARK, R.A. (1982) Chemotactic factors trigger their own oxidative inactivation by human neutrophils. J. Immunol. 129, 2725. DAMIANO, V.V., KUCICH, U., MURER, E., LAUDENSLAGER, N. & WEINBAUM, G. (1988) Ultrastructural quantitation ofperoxidase- and elastase-containing granules in human neutrophils. Am. J. Pathol. 131, 235. ECKLE, I., KOLB, G. & HAVEMANN, K. (1990) Inhibition of neutrophil oxidative burst by elastase generated IgG fragments. Biol. Chem. Hoppe-Seyler, 371, 69. EDWARDS, S.W. & SWANN, T.F. (1986) Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils. Biochem. J. 237, 601. EDWARDS, S.W., NURCOMBE, H.L. & HART, C.A. (1987) Oxidative inactivation of myeloperoxidase released from human neutrophils. Biochem. J. 245, 925. FANTOZZI, R., BRUNELLESCHI, S., SOLDAINI, G.B., BLANDINI, P., MASINI, E., RAIMONDI, D. & MANNAIONI, P.F. (1983) N-formylmethionylleucyl-phenylalanin: different releasing effects on human neutrophils and rat mast cells. Agents Actions, 13, 218. GABIG, T.G. & BABIOR, B.M. (1979) The 02-forming oxidase responsible for the respiratory burst in human neutrophils. Properties of the solubilized enzyme. J. biol. Chem. 254, 9070. GANZ, T. (1987) Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect. Immun. 55, 568. HENSON, P.M., JOHNSON, H.B. & SPIEGELBERG, H.L. (1972) The release of granule enzymes from human neutrophils stimulated by aggregated Immunoglobulins of different classes and subclasses. J. Immunol. 109, 1182. HENSON, P.M., ZANOLARI, B., SCHWARTZMAN, N.A. & HONG, S.R. (1978) Intracellular control of human neutrophil secretion I C5ainduced stimulus specific desensitization and the effects of cytochalasin b. J. Immunol. 121, 851. JANDL, R.C., ANDRt-SCHWARTZ, J., BORGES-DU Bois, L., KIPNES, R.S., MCMURRICH, B.J. & BABIOR, B.M. (1978) Termination of the respiratory burst in human neutrophils. J. clin. Invest. 61, 1176. KLEBANOFF, S.J. (1975) Antimicrobial systems in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12, 117. KOLB, G., ECKLE, I., HEIDTMANN, H.-H., NEURATH, F. & HAVEMANN, K. (1988) Neoantigenic group on Fc fragments in rheumatoid arthritis synovial fluid. Scand. J. Rheumatol. 75S, 179. KOLB, G., KOPPLER, H., GRAUSE, M. & HAVEMANN, K. (1982) Cleavage of IgG by elastase-like protease (ELP) of human polymorphonuclear leukocytes (PMN): isolation and characterization of Fab and Fc fragments and low-molecular weight peptides. Immunobiology, 161, 507. KOKOT, K., TESCHNER, M., SCHAEFER, R.M. & HEIDLAND, A. (1987) Stimulation and inhibition of elastase release from human neutrophils dependent on the calcium messenger system. Mineral Electrolyte Metab. 13, 133. KRAMPS, J.A., VAN TwISK, C.H. & VAN DER LINDEN, A.C. (1983) L-
pyroglutamyl-L-prolyl-L-valine-p-nitroanalide, a highly specific substrate for granulocyte elastase. Scand. J. clin. Lab. Invest. 43, 427. MANCINI, G., CARBONARA, A.0. & HEREMANS, J.F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235.
NAUSEEF, W.M., METCALF, J.A. & ROOT, R.K. (1983) Role of myeloperoxidase in the respiratory burst of human neutrophils. Blood, 61, 483. OHLSSON, K. & OLSSON, I. (1974) Neutral proteases of human granulocytes. III. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand. J. clin. Invest. 34, 349. OHLSSON, K., OLSSON, I. & SPITZNAGEL, J.K. (1977) Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes. Hoppe-Seyler's Z. Physiol. Chem. 358, 361. PEMBER, S.O., SHAPIRA, R. & KINKADE, J.M. (1983) Multiple forms of myeloperoxidase from human neutrophilic granulocytes: Evidence for differences in compartmentalization, enzymatic activity, and subunit structure. Arch. Biochem. Biophys. 221, 391. PORTER, R.R. (1959) The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochem. J. 73, 119. REISFELD, R.H., LEWIS, K.J. & WILLIAMS, D.E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamidgels. Nature, 195, 281. ROSEN, H. & KLEBANOFF, S.J. (1976) Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes. J. clin. Invest. 58, 50. SALZER, W., GERARD, C. & MCCALL, C. (1987) Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation. Biochem. Biophys. Res. Commun. 148, 747. SHOCK, A. & BAUM, H. (1988) Inactivation of alpha-l-proteinase inhibitor in serum by stimulated human polymorphonuclear leukocytes. Evidence for a myeloperoxidase-dependent mechanism. Cell. Biochem. Funct. 6, 13. SKOSEY, J.C., CHOW, D.C., NUSINOW, S., MAY, J., GESTAUTAS, V. & NIWA, Y. (1981) Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis. J. cell. Biol. 88, 358. SMOLEN, J.E., KORCHAK, H.M. & WEISSMANN, G. (1983) The kinetics of lysosomal degranulation of human neutrophils as measured by 9aminoacridine quenching. Biochim. biophys. Acta, 762, 145. TREADWAY, W., COLLINS, R.L., TURNER, R.A. & DECHATELET, L. (1979) Polymorphonuclear leukocyte secretory and metabolic responses to immunoglobulin aggregates. J. Rheumatol. 6, 636. VISSERS, M.C.M. & WINTERBOURN, C.C. (1987) Myeloperoxidasedependent oxidative inactivation of neutrophil neutral proteinases and microbicidal enzymes. Biochem. J. 245, 277. WEBER, K. & OSBORN, M. (1969) The reliability of molecular weight determination by dodecyl sulfate polyacrylamidgel electrophoresis. J. Biochem. 244, 4406. WEITZ, J.I., LANDMAN, S.L., CROWLEY, K.A., BIRKEN, S. & MORGAN, F.J. (1986) Development of an assay for in vivo human neutrophil elastase activity. J. clin. Invest. 78, 155. WHITE, J.R., HUANG, G.K., HILL, J.M., NACCHACHE, P.U., BECKER, E.L. & SHA'AFI, R.J. (1984) Effect of phorbol-12-myristate-13-acetate and its analogue 4a-phorbol- 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils. J. biol. Chem. 259, 8605. WILSON, E., RICE, W.G., KINKADE, J.M., MERRILL, A.H., ARNOLD, R.R. & LAMBETH, J.D. (1987) Protein Kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils. Arch. Biochem. Biophys. 259, 204.
Stimulation of neutrophil elastase and myeloperoxidase release by IgG fragments I.
ECKLE, G. KOLB, C. HEISER & K. HAVEMANN Centre for Internal Medicine, Philipps Marburg, West Germany
University,
(Acceptedfor publication 8 March 1990)
SUMMARY Human leucocyte elastase (HLE) cleaves IgG into Fab and Fc fragments. The Fc fragment bears an elastase-specific antigen and has previously been reported to be found in synovial fluid during rheumatoid arthritis. In addition, biological activity of elastase-specific Fc fragments has been described in modulating granulocyte oxidative metabolism. To investigate further regulatory effects of the elastase-induced IgG cleavage products, we tested the elastase and myeloperoxidase release of granulocytes. IgG fragments induce no enzyme release of unstimulated neutrophils. But elastase and myeloperoxidase release of cytochalasin b/FMLP-treated neutrophils is stimulated in a dosedependent manner by the Fab fragments. The extent of stimulation depends on stimulus concentration and is at its maximum for low (e.g. 2 5 10-8 M) FMLP concentration. Ten nanomoles Fab/4 x 106 PMN augment elastase release to 206% and myeloperoxidase release to 155% after prestimulation with 2 5 10-8 M FMLP. Fc fragments stimulate elastase release to 162% but no MPO release. Untreated IgG1 and analog Fab and Fc fragments produced by papain cleavage react similarly. Elastase-generated IgG fragments may therefore up-regulate their concentration by stimulating elastase release. The concomitantly stimulated release of myeloperoxidase may influence bactericidal activity and termination of oxidative burst. x
x
Keywords elastase myeloperoxidase IgG fragments release neutrophils
Reactive oxygen metabolites like superoxideanion and hydrogenperoxide are produced by the membrane bound NADPH oxidase (Gabig & Babior, 1979). Hydrogenperoxide together with halogenid and the released MPO constitute a system with high oxidizing potency (Klebanoff, 1975). Stimulation is produced in vivo by the invading microorganisms and bacterial products or activated serum components. The down-regulation has been only poorly understood until now; it is possibly caused partly by substances produced by the PMN themselves. We have measured elastase and MPO release of neutrophils dependent on the elastase-generated IgG fragments. The Fc fragments are already shown to have an inhibitory effect on 02generation of prestimulated neutrophils (Eckle, Kolb & Havemann, 1990). We therefore looked for possible regulation mechanisms of the fragments on elastase release. We also tested MPO which constitutes the second powerful oxidative system beside the 02-forming NADPH oxidase in stimulated neutrophils.
INTRODUCTION The acute inflammatory response is characterized by a rapid influx of polymorphonuclear granulocytes (PMN) under the influence of various chemotactic agents. At the inflammatory locus the PMN are higher stimulated to secrete lysosomal enzymes and produce activated oxygen radicals (Bender et al., 1983). The lysosomal enzymes of the neutrophil are stored in azurophil (1) and specific (2°) granules. The azurophil granules contain elastase, cathepsin G, myeloperoxidase (MPO), figlucuronidase, and lysozyme; the specific granules lysozyme, collagenase, lactoferrin and receptors for FMLP and C3bi (Ohlsson, Olsson & Spitznagel, 1977; Boxer & Smolen, 1988). Elastase is a neutral proteinase with broad substrate specificity. In plasma the released enzyme is rapidly complexed by inhibitors like al-proteinaseinhibitor and a2-macroglobulin (Ohlsson & Olsson, 1974). Nevertheless, protein degradation by elastase occurs in vivo, as specific split-products of IgG and fibrinogen have been detected (Kolb et al., 1988; Weitz et al., 1986).
MATERIALS AND METHODS Preparation of IgG fragments Neutrophil elastase HLE was isolated as described by Baugh & Travis (1976), starting from neutrophils of chronic myeloid
Correspondence: Ilsebill Eckle, PhD, Zentrum Innere Medizin, Philipps-Universitdt, Baldingerstrasse, D-355 Marburg, West Germany.
352
Stimiulation o 'release leukaemia patients. The purity of the enzyme tested by disk electrophoresis (Reisfeld, Lewis & Williams. 1962). IgG I isolated from plasma of plasmocytoma patients by anion-exchange chromatography and tested for purity by immunodiffusion (Mancini. Carbonara & Heremans. 1965) and SDS -PAGE (Weber & Osborn. 1969). IgG I fragments were generated by incubation of IgG I with purified clastase and separation of the incubation mixture by size-exclusion chromatography (Sepharose CL 6B in 100 m\i sodium phosphate buffer. pH 6 5). anion-exchange chromatography (DEAE Sepharose CL 4B in 20 m.\ triethanolamin. pH 7 5; sodium chloride gradient 0-1 xi) and affinity chromatography on protein A Sepharose (100 mxi sodium phosphate buffer. pH 7 0; desorption xxith 100 mnt acetic acid) (Eckle ct al.. 1990). Chromatographic material was provided by Pharmacia LKBI Bromma, Sweden. Isolation of papain generated fragments xxas performed in the same way after incubation of IgGI with papain (Serxa. Heidelberg. FRG), according to Porter (1959). The purity of the fragments was tested by SDS-PAGE and ultra-thin isoelectric focusing (Pharmacia LKB). Protein determination was done by dye-binding assay (BioRad Laboratories. Munich. FRG).
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0) 10-6), the Fc fragments showed neutral isoelectric points (HLE-Fc 6-6-7-3; Pap-Fc 5-8-6 5). Incubation of neutrophils with IgG I or IgG l fragments in a concentration range of 0-2 to 20 nmol/4 x 106 PMN for up to 1 h resulted in no measurable release of HLE or MPO. Release of LDH by PMN exposed to IgG fragments was not different than that of cells exposed to buffer alone (maximal
4%), and there was no additional release of LDH by stimulated cells pre-incubated with fragments. This indicates that IgG fragments were not toxic to PMN at the concentrations employed in these experiments. Simultaneous incubation of FMLP-stimulated cells with HLE-generated Fab fragments resulted in a concentrationdependent augmentation of HLE and MPO release. Figure la shows HLE release by different FMLP concentrations. Mean values of three different cell donors done in duplicate are given. Addition of HLE-induced Fab (10 nmol/4 x 106 PMN) results in an increase of HLE release over nearly the entire FMLP concentration range. Maximum over-stimulation is obtained by low FMLP concentrations, e.g. 2-5 x 10-8 M from 27 5% to 55.6% of the total HLE content. In Fig. lb the analogous data for MPO release are shown. This is only stimulated by HLE-Fab (10 nmol/4 x 106 PMN) in the concentration range of 10-8-10-7 M FMLP at its maximum from 5 2% to 23 8% of the total MPO content. Figure 2 shows stimulation of HLE and MPO release by 2-5 x 10-8 M FMLP versus concentration of added Fab. The Fab-stimulated release is expressed as percentage of release values by FMLP only. HLE release is already stimulated by about I nmol Fab/4 x 106 PMN, while an equal stimulation of MPO release is achieved by about 10 nmol Fab. The stimulating effect of Fab depends on the sequence of addition. Maximum stimulation is obtained by simultaneous addition of fragments and stimulus to the cells. Pre-incubation of fragments with cells gives lower values and addition of fragments after FMLP has no effect. Incubation of released HLE and MPO with Fab fragments results in no change of enzyme activity, so an influence on the test system can be excluded.
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Stimulation of release Figure 3 compares the effects of HLE-generated Fab with Fc fragments, with papain-produced fragments and with native IgGI in identical concentrations of 10 nmol/4 x 106 PMN. The stimulated release is expressed as percentage of release by FMLP alone. As a control, BSA is included, which does not stimulate HLE or MPO release. HLE-Fc stimulates only HLE release, to 160%, but not MPO release. Native IgGI and IgG fragments by papain cleavage stimulate HLE and MPO release between 155 and 215%.
DISCUSSION HLE cleaves IgG into Fab and Fc fragments beside small molecular peptides (Kolb et al., 1982). The fragments have similar molecular weights (43 kD versus 54 kD) but can be isolated by ion-exchange chromatography because of their widely different isoelectric points (Eckle et al., 1990). The analogous Fab and Fc fragments by papain cleavage (molar mass 51 kD versus 47 kD) with correspondent isoelectric points can be isolated in the same way. Stimulation of isolated neutrophils with FMLP after pretreatment with cytochalasin b results in a concentrationdependent release of azurophil granules. Maximum release by 5 x 10-7 M FMLP is 61% of HLE and 49% of MPO content. Although the enzymes are both stored in azurophil granules, heterogeneity of the granule contents (Damiano et al., 1988; Pember, Shapira & Kincade, 1983) can result in a different enzyme share (Bentwood & Benson, 1980). Self-deactivation of released MPO (Edwards, Nurcombe & Hart, 1987) was not measured after stimulating for up to 30 min. Released HLE is known to be resistant to oxidative inactivation (Vissers & Winterton, 1987). IgGl or IgGl fragments did not induce degranulation of unstimulated cells (Treadway et al., 1979; Henson, Johnson & Speigelberg, 1972). Addition of IgGl and its fragments to FMLP-stimulated cells resulted in a potent increase of elastase and MPO release. The amount of over-stimulation depended on the applied FMLP concentration: the smaller the stimulus concentration, the greater the additional effect of the fragments, and maximally stimulated cells could not be further degranulated (Fig. la, b). HLE release was already stimulated by smaller Fab concentrations than MPO release (Fig. 2). This can also be a consequence of granule heterogeneity (Damiano et al., 1988). The addition of fragments together with FMLP resulted in maximum stimulation, addition after FMLP had no effect as the release is already terminated within several seconds (Smolen, Korchack & Weismann, 1983). Augmented enzyme release by cell damage induced by IgG fragments could be excluded, as there was no higher LDH release after incubation with FMLP, with or without protein. An influence of the fragments on the test system for HLE or MPO could be excluded by measurements of enzyme activity after addition of fragments which induced no change. Both HLE and MPO release were maximally influenced by Fab fragments: HLE-Fab stimulated HLE and papain-Fab MPO release to 205 and 215% of values without addition of fragments respectively. IgG1 and Fc fragments showed smaller effects. Moreover, by HLE cleavage two molecules of Fab and one Fc are produced from one IgG, therefore a quantitatively higher effect on release is achieved by IgG cleavage. As papain-
generated IgGI fragments reacted analogous to the HLEinduced Fab fragments, the effect of the IgG fragments on the enzyme release is not specific for the elastase cleavage. This is in contrast to the effects of IgG fragments on oxidative burst (Eckle et al., 1990), where only HLE-generated Fc fragments show an effect. FMLP stimulates enzyme release after binding to a specific receptor via at least two pathways: 10 augmentation of intracellular calcium concentration by release from intracellular stores, or by enhanced influx of extracellular calcium and 2° by activation of protein kinase C. The mechanism of enhanced FMLP stimulation by IgG fragments may therefore be explained through modulation of calcium metabolism or protein kinase C activity (White et al., 1984; Wilson et al., 1987; Salzer, Gerard & McCall, 1987; Kokot et al., 1987), as already discussed for the inhibitory effects of IgG fragments on oxidative burst. There is an inverse relation between oxidative burst and enzyme release, as scavengers of oxygen-derived reactants potentiate degranulation (Skosey et al., 1981). MPO itself may play a role in terminating the oxidative burst of neutrophils, e.g. by inhibiting the 02-producing NADPH oxidase (Jandl et al., 1978; Edwards & Swain, 1986). This was also demonstrated by the prolonged oxidative burst of MPO-deficient neutrophils (Rosen & Klebanoff, 1976; Nauseef, Metcalf & Root, 1983). Oxidative inactivation of chemotactic factors by MPO also results in down-regulation of PMN function (Clark, 1982). However, oxidative inactivation of enzyme inhibitors like al proteinase inhibitor by the MPO/H202/halide system may potentiate the effects of HLE and thereby stimulate proteolysis (Carp & Janoff, 1980; Shock & Baum, 1988). HLE-generated IgGI fragments have a stimulating effect on HLE and MPO release. The released HLE may further degrade IgG, thereby potentiating the effect of the fragments. Released MPO may enhance HLE action by inactivation of plasma inhibitors on the one hand act synergistically with the IgG fragments in the down-regulation of dAidative metabolism on the other hand. ACKNOWLEDGEMENT Supported by the Bundesministerium fur Forschung und Technologie, grant no. 01 VM 8613.
REFERENCES BAUGH, R.J. & TRAVIS, J. (1976) Human leukocyte granule elastase: rapid isolation and characterization. Biochemistry, 15, 836. BENDER, J.G., MCPHAIL, L.C. & VAN Epps, D.E. (1983) Exposure of human neutrophils to chemotactic factors potentiates activation of the respiratory burst enzyme. J. Immunol. 130, 2316. BENTWOOD, B.J. & HENSON, P.M. (1980) The sequential release of granule constituents from human neutrophils. J. Immunol. 124, 855. BERGMEYER, H.U. (1983) Methods in Enzymatic Analysis (ed. by H. U. Bergmeyer) p. 737. Academic Press, New York. BOYUM, A. (1967) IV. Isolation of mononuclear cells and granulocytes from blood-isolation of mononuclear cells by one centrifugation and of granulocytes by combining centrifugation and sedimentation of 1g. Scand. J. clin. Lab. Invest. 21, (Suppl. 97), 77. BOXER, L.A. & SMOLEN, J. E. (1988) Neutrophil granule constituents and their release in health and disease. Hematol. Oncol. Clinics North Am. 2, 101. CARP, H. & JANOFF, A. (1980) Potential mediator of inflammation.
356
I. Eckle et al.
Phagocyte-derived oxidans suppress the elastase-inhibitory capacity of alphal-proteinase inhibitor in vitro. J. clin. Invest. 66, 987. CLARK, R.A. (1982) Chemotactic factors trigger their own oxidative inactivation by human neutrophils. J. Immunol. 129, 2725. DAMIANO, V.V., KUCICH, U., MURER, E., LAUDENSLAGER, N. & WEINBAUM, G. (1988) Ultrastructural quantitation ofperoxidase- and elastase-containing granules in human neutrophils. Am. J. Pathol. 131, 235. ECKLE, I., KOLB, G. & HAVEMANN, K. (1990) Inhibition of neutrophil oxidative burst by elastase generated IgG fragments. Biol. Chem. Hoppe-Seyler, 371, 69. EDWARDS, S.W. & SWANN, T.F. (1986) Regulation of superoxide generation by myeloperoxidase during the respiratory burst of human neutrophils. Biochem. J. 237, 601. EDWARDS, S.W., NURCOMBE, H.L. & HART, C.A. (1987) Oxidative inactivation of myeloperoxidase released from human neutrophils. Biochem. J. 245, 925. FANTOZZI, R., BRUNELLESCHI, S., SOLDAINI, G.B., BLANDINI, P., MASINI, E., RAIMONDI, D. & MANNAIONI, P.F. (1983) N-formylmethionylleucyl-phenylalanin: different releasing effects on human neutrophils and rat mast cells. Agents Actions, 13, 218. GABIG, T.G. & BABIOR, B.M. (1979) The 02-forming oxidase responsible for the respiratory burst in human neutrophils. Properties of the solubilized enzyme. J. biol. Chem. 254, 9070. GANZ, T. (1987) Extracellular release of antimicrobial defensins by human polymorphonuclear leukocytes. Infect. Immun. 55, 568. HENSON, P.M., JOHNSON, H.B. & SPIEGELBERG, H.L. (1972) The release of granule enzymes from human neutrophils stimulated by aggregated Immunoglobulins of different classes and subclasses. J. Immunol. 109, 1182. HENSON, P.M., ZANOLARI, B., SCHWARTZMAN, N.A. & HONG, S.R. (1978) Intracellular control of human neutrophil secretion I C5ainduced stimulus specific desensitization and the effects of cytochalasin b. J. Immunol. 121, 851. JANDL, R.C., ANDRt-SCHWARTZ, J., BORGES-DU Bois, L., KIPNES, R.S., MCMURRICH, B.J. & BABIOR, B.M. (1978) Termination of the respiratory burst in human neutrophils. J. clin. Invest. 61, 1176. KLEBANOFF, S.J. (1975) Antimicrobial systems in neutrophilic polymorphonuclear leukocytes. Semin. Hematol. 12, 117. KOLB, G., ECKLE, I., HEIDTMANN, H.-H., NEURATH, F. & HAVEMANN, K. (1988) Neoantigenic group on Fc fragments in rheumatoid arthritis synovial fluid. Scand. J. Rheumatol. 75S, 179. KOLB, G., KOPPLER, H., GRAUSE, M. & HAVEMANN, K. (1982) Cleavage of IgG by elastase-like protease (ELP) of human polymorphonuclear leukocytes (PMN): isolation and characterization of Fab and Fc fragments and low-molecular weight peptides. Immunobiology, 161, 507. KOKOT, K., TESCHNER, M., SCHAEFER, R.M. & HEIDLAND, A. (1987) Stimulation and inhibition of elastase release from human neutrophils dependent on the calcium messenger system. Mineral Electrolyte Metab. 13, 133. KRAMPS, J.A., VAN TwISK, C.H. & VAN DER LINDEN, A.C. (1983) L-
pyroglutamyl-L-prolyl-L-valine-p-nitroanalide, a highly specific substrate for granulocyte elastase. Scand. J. clin. Lab. Invest. 43, 427. MANCINI, G., CARBONARA, A.0. & HEREMANS, J.F. (1965) Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry, 2, 235.
NAUSEEF, W.M., METCALF, J.A. & ROOT, R.K. (1983) Role of myeloperoxidase in the respiratory burst of human neutrophils. Blood, 61, 483. OHLSSON, K. & OLSSON, I. (1974) Neutral proteases of human granulocytes. III. Interaction between human granulocyte elastase and plasma protease inhibitors. Scand. J. clin. Invest. 34, 349. OHLSSON, K., OLSSON, I. & SPITZNAGEL, J.K. (1977) Localization of chymotrypsin-like cationic protein, collagenase and elastase in azurophil granules of human neutrophilic polymorphonuclear leukocytes. Hoppe-Seyler's Z. Physiol. Chem. 358, 361. PEMBER, S.O., SHAPIRA, R. & KINKADE, J.M. (1983) Multiple forms of myeloperoxidase from human neutrophilic granulocytes: Evidence for differences in compartmentalization, enzymatic activity, and subunit structure. Arch. Biochem. Biophys. 221, 391. PORTER, R.R. (1959) The hydrolysis of rabbit gamma globulin and antibodies with crystalline papain. Biochem. J. 73, 119. REISFELD, R.H., LEWIS, K.J. & WILLIAMS, D.E. (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamidgels. Nature, 195, 281. ROSEN, H. & KLEBANOFF, S.J. (1976) Chemiluminescence and superoxide production by myeloperoxidase-deficient leukocytes. J. clin. Invest. 58, 50. SALZER, W., GERARD, C. & MCCALL, C. (1987) Effect of an inhibitor of protein kinase C on human polymorphonuclear leukocyte degranulation. Biochem. Biophys. Res. Commun. 148, 747. SHOCK, A. & BAUM, H. (1988) Inactivation of alpha-l-proteinase inhibitor in serum by stimulated human polymorphonuclear leukocytes. Evidence for a myeloperoxidase-dependent mechanism. Cell. Biochem. Funct. 6, 13. SKOSEY, J.C., CHOW, D.C., NUSINOW, S., MAY, J., GESTAUTAS, V. & NIWA, Y. (1981) Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis. J. cell. Biol. 88, 358. SMOLEN, J.E., KORCHAK, H.M. & WEISSMANN, G. (1983) The kinetics of lysosomal degranulation of human neutrophils as measured by 9aminoacridine quenching. Biochim. biophys. Acta, 762, 145. TREADWAY, W., COLLINS, R.L., TURNER, R.A. & DECHATELET, L. (1979) Polymorphonuclear leukocyte secretory and metabolic responses to immunoglobulin aggregates. J. Rheumatol. 6, 636. VISSERS, M.C.M. & WINTERBOURN, C.C. (1987) Myeloperoxidasedependent oxidative inactivation of neutrophil neutral proteinases and microbicidal enzymes. Biochem. J. 245, 277. WEBER, K. & OSBORN, M. (1969) The reliability of molecular weight determination by dodecyl sulfate polyacrylamidgel electrophoresis. J. Biochem. 244, 4406. WEITZ, J.I., LANDMAN, S.L., CROWLEY, K.A., BIRKEN, S. & MORGAN, F.J. (1986) Development of an assay for in vivo human neutrophil elastase activity. J. clin. Invest. 78, 155. WHITE, J.R., HUANG, G.K., HILL, J.M., NACCHACHE, P.U., BECKER, E.L. & SHA'AFI, R.J. (1984) Effect of phorbol-12-myristate-13-acetate and its analogue 4a-phorbol- 12,13-didecanoate on protein phosphorylation and lysosomal enzyme release in rabbit neutrophils. J. biol. Chem. 259, 8605. WILSON, E., RICE, W.G., KINKADE, J.M., MERRILL, A.H., ARNOLD, R.R. & LAMBETH, J.D. (1987) Protein Kinase C inhibition by sphingoid long-chain bases: effects on secretion in human neutrophils. Arch. Biochem. Biophys. 259, 204.
