Clin. exp. Immunol. (1979) 37, 162-168.

Proteolysis of human IgG by human polymorphonuclear leucocyte elastase produces an Fc fragment with in vitro biological activity H. E. PR I NCE, J. D. FOLD S & J. K. S P I TZNAGEL Department of Bacteriology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27514, USA

(Accepted for publication 24 January 1979)

SUMMARY

The Fc fragment derived by proteolysis of human IgG by human polymorphonuclear leucocyte (PMN) elastase was tested for in vitro biological activity. This fragment could attach to the specific IgG receptor of Staphylococcus aureus Cowan I cells (protein A) and could be eluted from the cells with dissociating buffer. Taking advantage of this attachment, it was shown that the Fc fragment is capable of attaching to the antigen-combining site of an IgM rheumatoid factor and can bind to the Fc receptor of human PMN. A similar fragment produced in vivo at sites of inflammation could play a role in regulating the inflammatory response.

INTRODUCTION Human polymorphonuclear leucocytes (PMN) contain a variety of proteases in their lysosomal granules (Folds, Welsh & Spitznagel, 1972; Spitznagel et al., 1974; Dewald et al., 1975). These enzymes are no doubt involved in the breakdown of phagocytozed immune complexes (Baggioline et al., 1978); however, they are also released extracellularly into the tissues during the inflammatory response (Weissmann, Zurier & Hoffstein, 1972). In this environment, proteases active near neutrality, such as PMN elastase, may degrade both tissue and serum components (Ohlsson et al., 1976). Accordingly, Malemud & Janoff (1975) have shown that cartilage proteoglycan is degraded by elastase. Others have demonstrated that serum kininogen (Movat, Habal & MacMorine, 1976) and complement components (Johnson, Ohlsson Olsson, 1976; Taylor, Crawford & Hugh, 1977) can be cleaved by elastase. We have recently shown that human IgG can be digested by PMN elastase in vitro with the production of an Fab fragment still capable of binding to antigen (Folds, Prince & Spitznagel, 1978). An Fc fragment was also revealed by immunoelectrophoresis of the degradation products. It occurred to us that should a similar degradation of IgG by extracellular elastase take place at the inflammatory site, a biologically active Fc fragment may be involved in regulating the inflammatory response. We have, therefore, examined the in vitro biological activity of the Fc fragment produced by the action of elastase on IgG. Our findings show that the Fc fragment is capable of binding to staphylococcal protein A, IgM rheumatoid factor and Fc receptors on human PMN.

Correspondence: Dr J. D. Folds, Department of Bacteriology and Immunology, University of North Carolina, Chapel Hill, North Carolina 27514, USA. 0099-9104/79/0070-0162$02.00 (D 1979 Blackwell Scientific Publications

162

Biological activity of elastase-produced IgG-Fc

163

MATERIALS AND METHODS Preparation of human PMN elastase. Elastase was purified from human PMN as described previously (Folds et al., 1978). Briefly, granules were prepared from chronic myelogenous luekemia PMN by differential centrifugation. Granules were extracted with 0-2M acetate buffer, pH 4 5, and the extracts eluted sequentially through Sephedex G 100 (Pharmacia, Piscataway, New Jersey) and Bio Rex 70 (Bio Rad, Rockville Centre, New York). Collected fractions were assayed for elastase activity using the synthetic substrate N-benzyloxycarbonyl-L-alanine-Z-naphthyl ester, and those fractions containing the elastase peak were pooled. Preparation of IgG and IgG fragments. The IgG fraction of human anti-tetanus immune globulin (Cutter Laboratories, Berkeley, California) was obtained by elution across DEAE-cellulose (Pharmacia) in 0 01 M phosphate buffer, pH 8-0. IgG was then dialysed against 0 1 M phosphate buffered saline (PBS), pH 8-0. Human PMN elastase and IgG were combined at an enzyme: substrate ratio of 1: 150 (w/w) and incubated for 14 hr at 370C (Folds et al., 1978). The digestion mixture was eluted through a Bio-Gel A-1-5 M (Bio-Rad) column equilibrated in PBS, and two protein peaks were obtained. The second peak contained protein with a molecular weight of about 50,000 Daltons, as judged by its point of elution. This protein material was concentrated to 1-2 mg/ml using an Amicon (Lexington, Massachusetts) PM-10 membrane and was designed Fragment I. A portion of this material was radiolabeled with 1251 (New England Nuclear, Boston Massachusetts) by the lactoperoxidase method (Marchalonis, 1969) at a specific activity of about 2000 cpm/pug. IgG was digested with papain (Calbiochem, La Jolla, California) according to established procedures (Porter, 1959). Fragment I binding to staphylococcal protein A. 100 p1l of a 10% suspension of heat-killed formalin-fixed Staphylococcus aureus Cowan I strain bacterial cells were centrifuged at 3000 rev/min for 5 min. The cell pellet was then resuspended in 50 ,ul Fragment I and incubated for 15 min at room temperature. After centrifugation, the supernatant was saved and the cells washed once with PBS. The cells were then resuspended in 50,ul 3 M NaSCN and incubated for 15 min at room temperature. Following centrifugation this supernatant was also saved. To study the ability of various proteins to inhibit Fragment I binding to protein A on staphylococcal cells, the bacteria were incubated with 50 pl of the inhibitory protein for 15 min before the addition of approximately 10 pg (20,000 cpm) '2"I-Fragment I. Bacteria were then spun and washed, and the two supernatants combined. The combined supernatants and cell pellet were counted in a Packard autogamma counter. The percentage inhibition of binding was calculated using the formula: 100-

(percentage total cpm bound in the presence of inhibitory protein X100) percentage total cpm bound in the absence of inhibitory protein

Inhibition of rheumatoidfactor activity. The titre of a Bio-Gel A-Sm purified monoclonal IgM rheumatoid factor (2 mg/ml) was found to be 64 using a modified Waaler-Rose procedure (Rheumaton, Wampole Laboratories, Cranbury, New Jersey). 5 p1 of this IgM was added to 15 p1 PBS, giving a 1:4 dilution. 5 p1 of this dilution was combined with 45 ul PBS or protein to be tested for inhibition of rheumatoid factor activity, giving a final dilution of 1:40 for the IgM. After incubation for 15 min at room temperature, the samples were tested for reactivity in the Rheumaton assay. Inhibition of PMN rosettes. A modification of the method of Henson (1969) was employed. 50 ,pl human venous blood was placed on round glass coverslips (13 mm diameter) mounted on moist gauze in petri dishes and incubated for 45 min at 37°C. The clots were then removed with forceps and the monolayers rinsed with warm saline to remove non-adherent cells. Monolayers were then covered with 50 ,pl of the protein material to be tested for rosette inhibition, followed by incubation at room temperature for 30 min. Without removing the pre-incubation material, monolayers were covered with 200 p1u of a 1% sheep red blood cell (SRBC) suspension (Grand Island Biological, Grand Island, New York) treated with a subagglutinating dose of sheep cell haemolysin (Difco, Detroit, Michigan). Following incubation for 30 min at room temperature, monolayers were rinsed well with warm saline and inverted onto glass slides containing one drop of 1% crystal violet in saline. Each monolayer was then examined at 440 x magnification and the percentage of leucocytes having two or more SRBC attached (PMN rosettes) was determined. Values represent the average of three experiments.

Analytical procedures. The antigenic characteristics of Fragment I were examined by Ouchterlony analysis using antisera specific for either the Fab portion or the Fc postion of human IgG (Miles, Elkhart, Indiana). Agarose (Fisher, Raleigh, North Carolina) was 0.75% in 0 05 M Tris-HCI, pH 8-6. Electrophoresis of Fragment I in 0-1% sodium dodecyl suphate- 5% polyacrylamide gels (10 cm length) was performed at 8 mA/gel for 6 hr under non-reducing conditions (Wever & Osborne, 1969). Gels were then frozen in cold acetone and sliced using a Bio-Rad model 190 gel slicer (2 mm per slice). Each slice was placed in a plastic tube and counted in a Packard gamma counter.

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H. E. Prince, 3. D. Folds & 3. K. Spitznagel

RESULTS I Fragment binding to Siaphylococcus aureus Human anti-tetanus toxoid IgG was digested by human PMN elastase and a fragment (Fragment I) with a molecular weight of approximately 50,000 Daltons (Folds et al., 1978) was recovered. Double diffusion analysis of Fragment I (F, Fig. 1) revealed two distinct protein components-one reacting with anti-Fab (A, Fig. 1) and the other reacting with anti-Fc (B, Fig. 1) antisera. The crossing precipitin lines indicated that the Fab antigenic determinants were on a molecule distinct from the molecule bearing Fc antigenic determinants. Accordingly, these two molecules were referred to as Fab and Fc, respectively. Intact IgG (G, Fig. 1), bearing both Fab and Fc antigenic determinants on the same molecule, appeared to be identical when analyzed by anti-Fab and anti-Fc antisera.

FIG. 1. Binding of IgG fragment (Fragment I) to 5, aureus cells rich in protein A. Fragments from IgG digestion by PMN elastase (F) contain an Fab fragment and an Fc fragment as shown by reactivity with fragmentspecific antisera anti-Fab (A) and anti-Fc (B). IgG shows a reaction of identity using these two antisera. Protein remaining after absorption of Fragment I with S. aureus cells (1) contains only Fab. Protein eluted by NaSCN from bacterial cells used for absorption (2) contains Fc.

When Fragment I was absorbed with S. aureus Cowan strain I cells containing protein A, the remaining supernatant (1, Fig. 1) contained only Fab. Fc was recovered by treating the cells with the dissociating buffer 3 m NaSCN (2, Fig. 1). Thus, Fc bound to S. aureus protein A, whereas Fab did not. To illustrate further that Fab and Fc were distinct molecules, and that the cell-adherent protein was not contaminating undigested IgG, a portion of Fragment I was radiolabelled, and part of this was absorbed with bacterial cells. Unabsorbed, absorbed and cell-adherent "'I1-Fragment I were then analysed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions. Unabsorbed Fragment I, as expected, contained two proteins with similar but separable mobilities (Fig. 2a). Little, if any, contaminating IgG was present. Fragment I absorbed with S. aureus protein A contained only the faster migrating protein (Fig. 2b). Material obtained by treating bacterial cells with dissociating buffer contained only the slower migrating protein (Fig. 2c). To demonstrate that Fc bound to the same S. aureus surface receptor as intact IgG, binding inhibition studies were performed using "'2I-~Fragment I. Table 1 shows that approximately 40o% of Fragment

Biological activity of elastase-produced IgG-Fc (a)

igG

8 4 (b )

(b)

10

4

E2"

ca.29

20 30 Sl ice number

FIG. 2. Characterization of IgG fragment (Fragment I) by SDS-PAGE. (a) '251-Fragment I before absorption with S. aureus cells contains protein peaks with slightly different mobilities. No contaminating undigested IGg is observed. (b) Material obtained after absorption with S. aureus cells co-migrates with the faster migrating protein seen in (a). (c) Material eluted from cells with NaSCN co-migrates with the slower migrating protein seen in (a).

TABLE 1. Inhibition of binding of '25I-Fragment I to S. aureus Cowan I cells

Pre-incubation material (mg/ml) PBS, p1-I 8 0 BSA (2) IgG (2) Fragments from papain digest of IgG

Percentage total cpm unbound

Percentage total cpm cell-bound

Percentage inhibition of binding

59 7 59 6 84 2 85-0

40 3 404 15 8 15 0

-

0 60-8 62 8

59 1

40 9

0

(1) IgM (3)

S. aureus cells rich in cell surface protein A were incubated with the indicated material for 30 min before the addition of '251-Fragment I. mixtures were spun, washed and the supernatants and cell pellets counted separately for radioactivity. Only IgG and papain-derived IgG fragments inhibited 125I-Fragment I binding. L

165

H. E. Prince, D. Folds & I. K. Spitznagel 166 I was bound to bacterial cells, and that this binding was uninhibited by bovine serum albumin (BSA) or IgM. Only intact IgG and fragments from a papain digest of IgG (i.e., papain Fab and Fc fragments) blocked the attachment. Fragment I was tested for the ability to block the attachment of a monoclonal IgM rheumatoid factor to rabbit IgG-sensitized SRBC. As shown in Table 2, IgM rheumatoid factor pre-incubated with Fragment I was non-reactive using the Rheumaton test. Pre-incubation with BSA or Fragment I previously absorbed with S. aureus cells failed to abrogate the rheumatoid factor activity of the IgM. Inhibition of PMN rosettes Table 3 demonstrates that Fragment I inhibited the attachment of haemolysin-coated SRBC to a monolayer of human PMN. This inhibitory effect was also observed when leucocytes were first incubated with undigested IgG and fragments from papain digestion of IgG. However, the inhibitory capacity was removed by prior absorption of Fragment I and papain fragments with S. aureus cells. J.

TABLE 2. Inhibition of IgM rheumatoid factor activity by Fragment I

Pre-incubation material (mg/ml)

Rheumaton reaction

PBS, pH 8-0 BSA (2) Fragment I (2) Fragment I absorbed with S. aureus cells

+ + +

IgM rheumatoid factor was incubated with the indicated material before being tested for Rheumaton reactivity. IgM incubated with Fragment I was nonreactive in the Rheumaton test; absorption of Fragment I with protein A-containing S. aureus cells removed the factor blocking rheumatoid activity. TABLE 3. Inhibition of PMN rosettes by fragment I

Pre-incubation material (mg/ml)

PBS, pH 80 BSA (2) IgG (2) Papain IgG fragments (2) Papain IgG fragments absorbed with S. aureus cells Fragment I (2) Fragment I absorbed with S. aureus cells

Percentage of PMNs rosetted

Percentage inhibition of rosette formation

730 65.4 28-3

10-4 61-4

21.0

71-2

69-5 31-2

56 0

70 7

3-2

-

4-8

PMN monolayers were pre-incubated with the indicated material for 30 min. SRBC coated with a subagglutinating dose of haemolysin were then added and the proportion of rosetted PMN determined. IgG, papain-derived IgG fragments and Fragment I exhibited inhibitory activity Removal of Fc fragments by S. aureus cells containing cell surface protein A also removed the inhibitory activity.

Biological activity of elastase-produced IgG-Fc

167

DISCUSSION We have shown previously (Folds et al., 1978) that the Fab fragment resulting from the action of human PMN elastase on IgG is still capable of binding antigen, and that an Fc fragment, as shown by reactivity with anti-Fc antiserum, is also produced. The studies presented here were conducted to determine whether the Fc fragment still retains biological activity in vitro. Attempts to purify the Fc fragment by binding to, and subsequent elution from DEAE-cellulose (Isenman, Dorrington & Painter, 1975) were unsuccessful. We therefore investigated other methods for separating Fc from Fab. Accordingly, S. aureus Cowan I cells which had been heat-killed and formalinfixed were used. These cells are known to be rich in cell surface staphylococcal protein A, which binds IgG via the Fc portion of the molecule (Kronvall & Frommel, 1970). Ouchterlony analysis revealed that material remaining after the absorption of Fragment I with the bacterial cells contained only Fab determinants. The protein eluted from the cells using a dissociating buffer reacted strongly with anti-Fc antiserum. Slight reactivity with anti-Fab antiserum was also observed, but SDS-PAGE of radiolabelled Fragment I suggested that this reactivity was not due to contamination by undigested IgG. Possibily, the anti-Fab antiserum was contaminated with anti-Fc antibodies. Of primary importance, however, was the observation of anti-Fc reactivity, and thus Fc fragment in the material eluted from the S. aureus cells. Earlier studies have demonstrated the presence of two closely migrating, but distinct, protein bands upon SDS-PAGE of Fragment I under nonreducing conditions (H. E. Prince, unpublished observation). The adherence of Fc to S. aureus cells and its subsequent elution by dissociating buffer made it possible for us to determine which band on the gels corresponded to that protein, and thus which band represented Fab. Protein remaining after the absorption of '25I-Fragment I with bacterial cells co-migrated with the faster migrating protein observed on gels of unabsorbed Fragment I; this protein thus represented the Fab molecule. Similarly, protein bound to cells and removed with NaSCN co-migrated with the slower migrating protein on gels of unabsorbed Fragment I and thus represented the Fc molecule. To demonstrate that Fc attachment to S. aureus was occurring via the specific IgG receptor, protein A, a binding inhibition study was conducted. Intact IgG and fragments from a papain digest of IgG were equally active in blocking Fc attachment. Thus, the adherence of Fc is receptor-specific. These data show that the Fc fragment produced by elastase degradation of IgG retains enough of its three-dimensional structure to react with staphylococcal protein A. We examined the ability of Fc to interfere with the attachment to IgG-coated SRBC of an IgM rheumatoid factor, whose antigen-combining site recognized the Fc portion of IgG. Fragment I was able to block the agglutinating action of the IgM when analysed by the Rheumaton assay. This blocking activity was removed by absorbing Fragment I with S. aureus cells, showing the Fc molecule to be responsible for the blocking activity. Using an assay similar to that demonstrating inhibition of Fc binding to protein A-containing bacterial cells, we found that Fc could attach to the Fc receptor of human PMN, thus blocking the binding of haemolysin-coated SRBC. This inhibitory activity was abrogated by absorbing Fragment I with the bacteria. A very similar blocking pattern was observed using absorbed and non-absorbed papain fragments, suggesting the two Fc fragments to be similar with respect to Fc receptor binding activity. These observations also suggest that our elastase-derived Fc fragment is composed of the C2 and C3 domains of IgG, as is the papain Fc fragment. Foster, Dorrington & Painter (1978) have shown that neither the C2 nor the C3 domains alone can bind to PMN Fc receptors; only a fragment composed of both domains can do so. Several workers have shown that crude preparations of proteases from PMN can degrade IgG to Fab and Fc fragments in vitro (Ghetie & Sulica, 1970; LoSpalluto, Fehr & Ziff, 1970; Bolton, 1974; Menninger, Fehr & Otto, 1976). It has also been shown that elastase and other leucocyte enzymes are released extracellularly at sites of inflammation (Weissmann et al., 1972; Becker & Henson, 1973). Of the PMN proteases, elastase, having a pH optimum of 7 0-9 0 for enzymatic activity (Ohlsson et al., 1976), is most likely to be active when released extracellularly into the tissue environment. Thus, the

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H. E. Prince, J. D. Folds & J. K. Spitznagel

possibility that IgG present at the inflammatory site is degraded by leucocytic proteases released extracellularly during phagocytosis or cell death appears to be a very real one. In support of this concept Waller (1974) has demonstrated the presence of Fab and Fc fragments in abscess fluids. The in vivo production of biologically active Fc fragments at sites of inflammation and their relationship, if any, to the regulation of inflammatory processes are still matters for speculation. It is conceivable however, that Fc fragments produced in vivo by the action of extracellular elastase on IgG might possess biological activities similar to those demonstrated in vitro by these experiments. This hypothesis is complicated by the interactions of plasma protease inhibitors with proteases released at sites of inflammation and the biological activities of IgG fragments in relation to those of undigested IgG. Thus, the roles of PMN, their proteases and plasma components in regulating inflammation are much more complex than has been realized and deserve further study. The authors wish to thank Mr M. Lynes for supplying the Staphylococcus aureus cells and Dr M. Modrzakowski for his assistance in graphics. This work was supported by National Institutes of Health Grants AI 02430 and 5-T32-CA09057-04.

REFERENCES of immunoglobulins by intracellular proteases in the range BAGGIOLINI, M., BRETZ, U., DEWALD, B. & FEIGENSON, of neutral pH. 7. Immunol. 105, 886. M.E. (1978) The polymorphonuclear leukocyte. Agents and Actions, 8, 3. MIALEMUD, C.F. & JANOFF, A. (1975) Human polymorphonuclear leukocyte elastase and cathepsin G mediate the BECKER, E.L. & HENSON, P.M. (1973) In vitro studies of degradation of lapine articular cartilage proteoglycan. immunologically induced secretion of mediators from Ann. N.Y. Acad. Sci. 256, 254. cells and related phenomena. Adv. Immunol., 17, 43. BOLTON, C.E. (1974) In vitro studies of IgG catabolism by MARCHALONIS, J.J. (1969) An enzymic method for the trace iodination of immunoglobulin and other proteins. human leukocytes. Immunochemistry, 11, 599. Bioch. . 113, 299. DEWALD, B., RINDER-LuDWIG, R., BRETZ, U. & BAGGIOLINI, M. (1975) Subcellular localization and hetero- MENNINGER, H., FEHR, K. & OTTO, K. (1976) Digestion of human immunoglobulin G by bovine cathepsin B1. geneity of neutral proteases of neutrophilic polymorImmunochemistry, 13, 633. phonuclear leukocytes. 7. exp. Med. 141, 709. FOLDS, J.D., PRINCE, H. & SPITZNAGEL, J.K. (1978) Limited MOVAT, H.Z., HABAL, F.M. & MACMORINE, D.R.L. (1976) Neutral proteases of human polymorphonuclear leukocleavage of human immunoglobulins by elastase of human cytes with kininogenase activity. Int. Arch. Allergy appl. neutrophil polymorphonuclear granulocytes: possible Immunol. 50, 257. modulator of immune complex disease. Lab. Invest. 39, OHLSSON. K., OLSSON, I., DELSHAMMER, M. & SCHIESSLER, 313. H. (1976) Elastase from human and canine granulocytes. FOLDS, J.D., WELSH, I.R H & SPITZNAGEL, J K (1972) I. Some proteolytic and esterolytic properties. HoppeNeutral proteases confined to one class of lysosomes of Syler's Z. physiol. Chem. 357, 1245. human polymorphonuclear leukocytes Proc Soc exp PORTER, R.R. (1959) The hydrolysis of rabbit gamma gloBiol Med, 139, 461 bulin and antibodies with crystalline papain. Bioch. ]. 73, FOSTER, D.E.B., DORRINGTON, K.J. & PAINTER, R.H. (1978) 119. Structure and function of immunoglobulin domains VIII. Studies on the structural requirements of human immuno- PRINCE, H.E., FOLDS, J.D. & SPITZNAGEL, J.K. (1979) Interaction of human polymorphonuclear leukocyte elasglobulin G for granulocyte binding. j. Immunol. 120, 1952. tase with human IgM. In vitro production of a Fabp-like GHETIE, V. & SULIcA, A. (1970) Uptake and breakdown of fragment. Immunochemistry, (in press). rabbit IgG by guinea pig peritoneal polymorphonuclear SPITZNAGEL, J.K., DALLDORF, F.G., LEFFELL, M.S., FOLDS, leukocytes. Immunochemistry, 71, 175. J.D., WELSH, I.R.H., COONEY, M.H. & MARTIN, L.E. HENSON, P.M. (1969) The adherence of leukocytes and (1974) Character of azurophil and specific granules puriplatelets induced by fixed IgG antibody or complement. fied from human polymophonuclear leukocytes. Lab. Immunology, 16, 107. Invest. 30, 774. ISENMAN, D. E., DORRINGTON, K.J. & PAINTER, R.H. (1975) The structure and function of immunoglobulin domains. TAYLOR, J.C., CRAWFORD, J.P. & HUGLI, T.E (1977) Limited degradation ofthe third component (C3) ofhuman II. The importance of interchain disulfide bonds and the complement by human leukocyte elastase: partial characpossible role of molecular flexibility in the interaction terization of C3 fragments Biochemistry, 16, 3390 between Immunoglobulin G and complement. J. Immunol. WALLER, M. (1974) IgG hydrolysis in abcesses. I. A study 114, 1726. of the IgG in human abscess fluid. Immunology, 25, 725. JOHNSON, U., OHLSSON, K. & OLSSON, I. (1976) Effects of granulocyte neutral proteases on complement components. WEBER, K. & OSBORNE, M. (1969). The reliability of molecular weight determinations by dodecyl sulfate-polyScand. J. Immunol. 5, 421. acrylamide gel electrophoresis _7 biol. Chem. 244, 4406. KRONVALL, G. & FROMMEL, D. (1970) Definition of staphylococcal protein A reactivity for human immunoglobulin WEISSMANN, G., ZuRiER, R.B. & HOFFSTEIN, S. (1972) Leukocytic proteases and the immunologic release of G fragments. Immunochemistry, 7, 124. lysosomal enzymes. Amer. 7. Path. 68, 539. LOSPALLUTO, J.J., FEHR, K. & ZIFF, M. (1970) Degradation