Immunology 1976 31 781
Effects of activated complement components on enzyme secretion by macrophages
H. U. SCHORLEM MER & A. C. ALLISON Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex
Received 18 March 1976; accepted for publication 6 May 1976
interdependent series of interacting proteins with a vital role as an effector system in the maintenance of body integrity. Physiological complement activation may proceed through at least two major pathways, both of which lead to the activation of C3 (Goetze & Muller-Eberhard, 1971; Gewurz, 1972). In this respect C3 is a central component of the complement system. Several activated components and cleavage products have the potential of mediating inflammatory tissue injury, either directly or indirectly. In addition, activation of the complement system has been associated with acute and chronic inflammatory reactions of immunological origin. Among the biologically active components of complement the low mol. wt cleavage products of C3 and C5 play the most important role in the mediation of inflammation and tissue injury. These peptides release histamine (Cochrane & Muller-Eberhard, 1968) which increases the permeability of the blood vessels near the site of complement activation. The chemotactic property of the C3a and C5a fragments promotes migration of leucocytes into the area (Ward, 1969). Goldstein, Hoffstein, Gallin & Weissmann (1973) and Becker, Showell, Henson & Hou (1974) have reported that C5a interacts with human polymorphonuclear leucocytes treated with cytochalasin B and provokes release of lysosomal enzymes from these cells. We have investigated the effects of C3 cleavage products, namely C3a and C3b, on mouse and guinea-pig peritoneal macrophages maintained in culture.
Summary. Purified cleavage products of the guineapig complement component C3, namely C3b and C3a, interact with guinea-pig and mouse macrophages in culture to induce a dose- and timedependent release of lysosmal enzymes into the medium. In the case of C3b the selectivity of the release of hydrolases, which occurs without cell killing, is shown by morphological observations and the failure of lactate dehydrogenase to appear in the medium. However, lysosomal enzyme release in the presence of C3a is accompanied by loss of cellular lactate dehydrogenase. Preincubation of C3b with anti-C3 Fab inhibits its attachment to macrophages, after which there is hardly detectable enzyme release into the medium. We have found that stimulated macrophages release enzyme(s) which can cleave C3, generating more C3b either directly or via the alternative pathway; the C3b so formed would induce further enzyme release. This amplification system may provide an explanation for the ability of macrophages to generate mediators of inflammation and cause tissue damage and degradation at sites of chronic inflammation while retaining their viability for long periods of time. INTRODUCTION The complement system comprises a complex Correspondence: Dr A. C. Allison, Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
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MATERIALS AND METHODS Experimental animals Swiss mice (T.O. strain) and guinea-pigs (strain: Hartley) were obtained from SACI, Brentwood, Essex. Tissue culture materials Tissue culture grade Petri dishes were obtained from Nunc Jobling Laboratories Division, Stane, Staffordshire, and M199 from Burroughs Wellcome, Beckenham, Kent. Biochemical reagents Bovine serum albumin, penicillin, streptomycin and phenolphthalein glucuronic acid 0 01 M pH 7 0 were from Sigma Chemical Company, Surbiton, Surrey; p-nitrophenyl-,8-D-galactopyranoside and p-nitrophenyl-2-acetamido-,6-D-deoxyglucopyranoside from Koch-Light Laboratories, Colnbrook, Bucks; heparin, preservative-free, from Boots, Nottingham; pyruvate and nicotinamide adenine dinucleotide from Boehringer, Mannheim GmbH, Germany; starch, Triton X-100 and glutaraldehyde from British Drug Houses Ltd, Poole, Dorset.
The use of a glutaraldehyde cross-linked albumin surface for culture of adherent cells 50-mm tissue culture Petri dishes were covered by a mixture of bovine serum albumin (165 mg/ml in 0-2 M acetate buffer, pH 5 0) and glutaraldehyde (16 5 mg/ml in 0-2 M acetate buffer pH 5 0). Then they were incubated overnight at 370 and washed four times with phosphate-buffered saline. These surfaces were suitable for the culture of mouse and guinea-pig peritoneal macrophages in either a serumfree or serum-containing medium. Macrophage collection and culture Guinea-pigs were injected intraperitoneally with 30 ml of 2 per cent starch gel in physiological saline and killed three days later. Cells were washed from the peritoneal cavity with 150 ml M199 containing 100 u/ml of penicillin and streptomycin and 10 i.u./ml heparin. Mouse macrophages were obtained by peritoneal lavage of Swiss mice with 5 ml M199 containing 100 u/ml of penicillin and streptomycin and 100 i.u./ml heparin. In both cases 5 ml aliquots of the peritoneal exudate cell suspension containing 0-5-10 x 106 cells/ml were distributed into 50 mm Petri dishes and incubated in a humidified atmosphere of 5 per cent carbon dioxide and air at 37° for 1-2 h to allow attachment of adherent cells. Non-
adherent cells were removed by washing four times with phosphate-buffered saline. After washing, the cells were cultured in M199. Cultures prepared in this way give a sheet of well spread cells within 24 h. In all experiments quadruplicate cultures were used and biochemical results are expressed as the mean and standard deviation. At the end of each incubation period the medium was removed and the adherent cells were washed once with phosphate-buffered saline. The cells were then released by adding saline containing 0O1 per cent (wfv) Triton X-100 and 0-1 per cent w/v bovine serum albumin and scraping with sterile siliconerubber bungs. The activities of various enzymes were assayed in both the media and cell-containing fractions. Enzyme assays All assays were conducted under conditions giving linear release of product in relation to the amount of sample used and the time of incubation. Lactate dehydrogenase was assayed by determining the rate of oxidation of reduced nicotinamide adenine dinucleotide at 340 nm, ,B-glucuronidase by the method of Talalay, Fishman & Huggins (1946). ,8-Galactosidase was assayed by the method of Conchie, Findlay & Levvy (1959) using p-nitrophenyl-fi-D-galactopyranoside as substrate. N-acetyl-fl-D-glucosaminidase was assayed by the method of Woollen, Heyworth & Walker (1961)
using p-nitrophenyl-2-acetamido-2-,8-D-glucopyranoside as substrate dissolved in 0-1 M acetate buffer, pH 4-5. Generation and purification of the complement components Guinea-pig C3 and the cleavage product C3b were prepared as previously reported (Bitter-Suermann, Hadding, Melchert & Wellensieck, 1970; Nicholson, Brade, Schorlemmer, Burger, Bitter-Suermann & Hadding, 1975). For generation and purification of C3a, highly purified C3 was incubated with the C3 cleaving complex which is formed when cobra venom factor interacts with factor B, factor D and Mg2+. This reaction mixture was passed through a Sephadex G-100 column. The fractions that mediated contraction of isolated guinea-pig terminal ileum were pooled and concentrated. Purified C3, C3a and C3b were kindly provided by Dr D. Bitter-
Suermann, Institute of Medical Microbiology Mainz, Germany.
Complement component effect on enzyme secretion Standard incubation mixture for C3 activation Equal volumes of either C4-deficient guinea-pig serum or purified C3 were incubated with appropriate dilutions of either the cell fraction or the supernatant of a stimulated or unstimulated macrophage culture for 30 min at 370 and then cooled to 40 in an ice bath. The concentrations referred to in the text, represents the final protein concentration of the complement components in this standard incubation mixture (pg/ml). Protein levels of the purified complement components were determined by the method of Lowry, Rosebrough, Farr & Randall (1951) using bovine serum albumin as a standard.
Immunoelectrophoretic analysis of C3 conversion One per cent agarose was used in all experiments. Electrophoretic separation was performed using standard conditions (veronal buffer pH 8 6). Tenmicrolitre aliquots of the incubation mixtures were placed in the wells for electrophoretic separation. Monospecific goat antiserum against guinea-pig C3 was prepared as previously reported (Konig, Bitter-Suermann, Dierich, Limbert, Schorlemmer & Hadding, 1974). Statistical tests Means and standard deviations were calculated after samples were shown to be homogeneous by calculation of coefficients of variance. The significance of differences was established by the Student's t-test. Anti-C3 Fab fragments These fragments were prepared as described by Nicholson, Brade, Lee, Shin & Mayer (1974) Briefly, the IgG fraction of goat antiserum against guinea-pig C3 was digested by pepsin in the presence of cysteine. Anti-C3 activity of the fragments was tested by inhibition of lysis of EAC1423 with
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C5-C9. The reciprocal of the antibody dilution which produced 50 per cent inhibition of haemolysis was designated as 1 unit (u) of anti-C3 activity. The final preparation used in these studies contained 500 u/ml. To check for univalence the Fab fragments were incubated with EAC 42 and subsequently C3 was added to the cells. There was no removal of C3 from the fluid phase.
Unrelated IgG Unrelated IgG was isolated from the serum of nonimmunized goats by ammonium sulphate precipitation and DEAE-cellulose chromatography. RESULTS Induction by C3b of macrophage hydrolase secretion Marked changes in the levels and distribution of the activities of acid hydrolases were induced in mouse and guinea-pig macrophages by the purified cleavage products of the complement component C3, namely C3a and C3b. Two types of experiments were performed. Firstly the effect of various concentrations of C3b and C3a on the level and distribution of enzyme activity in mouse macrophage cultures after 72 h was measured. Second the effect of a single concentration of C3b on the time course of changes in enzyme level and distribution in guinea-pig macrophage cultures was determined. In cultures grown in the presence of various concentrations of C3b for a period of 72 h the total lysosomal enzyme activity was not changed (Fig. 1); however there was a marked decrease in the intracellular acid hydrolase content, with a reciprocal and concurrent increase in the extracellular enzyme activity in the culture media (Figs 1 and 2). This redistribution of
Table 1. Lysosomal enzyme levels and distribution between cells and culture medium in mouse macrophage cultures exposed to various concentrations of C3b for 72 h
N-acetyl-,8-D-glucosaminidase (nmol product/plate/hour)
Concentration of C3b (ug/ml) o 5 10 20 30 40
Total 4003+269 3962+ 142
3843+516 4013+146 3753+299 3960+402
fi-glucuronidase
8i-galactosidase
(nmol product/plate/hour)
(nmol product/plate/hour)
Percentage in medium
Total
Percentage in medium
Total
Percentage in medium
5 0+0 8 14-7+ 1-3 24 7+ 69 37-6+2-4 50 8+ 26
184+ 13 203+8 194+ 19 208+11 202+6 209+29
10-6+2-3 19-7+0-3 33-6+ 65 500+51 63 7+25 72-1+1-0
156+3 152+ 10 175+ 11 158+15 142+4 143+4
8 5+0 7 12-7+0-8 15-4+ 1-2 292+30 49 5+ 1 5 605+1 9
63-5+1-3
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H. U. Schorlemmer and A. C. Allison 180r-
600r_ 140
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To
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a 400
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76 60k
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Ec :-
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10 20 30 Concentration (/sig/m I)
oo_
40
Figure 1. The effect of various concentrations of C3b on the levels of 8-galactosidase activity in the total culture (0) in cells (0), and in the medium (U). The cultures of mouse macrophages were assayed after incubation with C3b for 72 h.
activity was observed for all of the lysosomal hydrolases measured; these included I-glucuronidase, Ii-galactosidase and N-acetyl-fi-D-glucosaminidase as shown in Table 1. After 72 h incubation in medium containing 20 yug/ml of C3b the concentration of 8-galactosidase in the medium is significantly greater than in the controls (P
20
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0
10
0
C'
0)
F_
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A0
6
-r24
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72
Time (h)
Figure 4. The time-dependent release of 8-glucuronidase (0) and lactate dehydrogenase (A) from guinea-pig macrophages exposed to 20 pg/ml of purified guinea-pig C3b.
60
E .2
E U 50
Percentage
495+1-5
was studied in guinea-pig macrophage cultures maintained for various times up to 72 h in the presence of a single dose of 20 pig/ml of C3b. It is seen in Fig. 4 that cultures of guinea-pig macrophages exposed to C3b show no change compared to control cultures in the distribution of a representative lysosomal enzyme, 8-glucuronidase, between cells and culture medium for the first 24 h. From this time on, however, a highly significant increase (P < 0 01) in the amount of fl-glucuronidase in the medium is seen. Subsequently a rapid rise in the release of enzyme into the culture medium occurs so that at 72 h approximately 50 per cent of the total enzyme activity is found in the culture medium. While data are presented only for fl-glucuronidase, the same pattern of release was observed for other acid hydrolases measured including N-acetyl-fl-D-glucosaminidase and fl-galactosidase (Table 2). This time dependence of the selective release of lysosomal enzymes caused by C3b occurs with no detectable release of cellular lactate dehydrogenase into the culture medium, and significant increases in the cellular levels of this enzyme are observed at 72 h.
40_
g 30
1020 _10 1
0
6
24
48
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Figure 5. Inhibition of attachment of C3b to guinea-pig macrophages after preincubation of C3b with anti-C3 Fab (0) and anti-C3 IgG (A) prevents the time-dependent release of lysosomal enzymes into the medium. (0) Control of C3b preincubated with an unrelated IgG fraction.
Effects of anti-C3b on enzyme release To show conclusively that attachment of C3b to guinea-pig macrophages in culture results in a doseand time-dependent release of lysosomal enzymes into the medium, we inhibited this reaction by using an anti-C3 Fab preparation. Purified C3b (20 ,u/ml) was preincubated with anti-C3 Fab, unrelated IgG and an anti-C3 IgG fraction for 12 h at 4°. These incubation mixtures were added to the macrophage cultures and the release of lysosomal enzymes into the medium was followed. As shown in Fig. 5, there is hardly any detectable enzyme release after interaction of C3b with anti-C3 Fab. Also,
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preincubation of C3b with anti-C3 IgG partially prevented enzyme release; however, there is still a release of about 20 per cent. The unrelated IgG fraction had absolutely no effect; there is the usual rapid rise in the concentration of enzyme in the culture medium, so that by 72 h approximately 50 per cent of the total enzyme activity is released from the cells. Cleavage of C3 by enzymes secreted from stimulated macrophages Experiments were performed to ascertain whether enzymes released from stimulated guinea-pig macrophages can cleave C3 as shown by immunoelectrophoretic conversion. Supernatants from cultures of activated guinea-pig macrophages were incubated with highly purified guinea-pig C3. As shown in Fig. 6 the C3 was cleaved to generate a product with the greater anodal electrophoretic mobility. This could have been due to activation of the alternative pathway. However, direct effects of the secreted
Figure 6. Immunoelectrophoretic demonstration of the conversion of guinea-pig C3 mediated by the alternative pathway in C4-deficient guinea pig serum. (a) control, C4deficient guinea-pig serum; (b) C4-deficient guinea-pig serum incubated with the cell fraction of activated guinea-pig macrophages; (c) C4 deficient guinea-pig serum incubated with the supernatant of activated macrophages.
enzymes on purified C3 were also shown in these experiments, with generation of C3b. Studies of the biological activities of the generated components are in progress and will be reported later.
DISCUSSION Chronic inflammatory reactions are responsible for the tissue damage that occurs in several important diseases of temperate and tropical climates, including rheumatoid arthritis, rheumatic fever, chronic pulmonary disease, periodontal disease, chronic diseases of the gastro-intestinal tract, schistosomiasis and leishmaniasis. Knowledge of the pathogenesis of chronic inflammatory reactions and how they might be controlled is of considerable medical importance. It is generally accepted that mononuclear phagocytes are present in all types of chronic inflammation and that they play an essential role in the pathogenesis and resolution of this process (Allison & Davies, 1975). The control mechanism by which macrophage enzyme secretion is induced is not yet clear. The evidence presented in this paper shows that one such mechanism is mediated by cleavage products of the complement component C3. Complement interacts with mononuclear phagocytic cells in several ways. Biologically active complement components play several important roles as inflammatory mediators. The cleavage products not only display anaphylatoxic (Cochrane & Muller-Eberhard, 1968) and chemotactic activity (Ward, 1969) but are also capable of interacting with human monocytes, mouse and guinea-pig peritoneal macrophages and rabbit alveolar macrophages by the C3b receptor (Lay & Nussenzweig, 1968; Huber, Polley, Linscott, Fudenberg & Muller-Eberhard, 1968). The interaction of these phagocytic cells with complement may result in tissue damage. It has already been reported that C5a interacts with human polymorphonuclear leukocytes treated with cytochalasin B and provokes release of lysosomal enzymes from these cells (Goldstein et al., 1973; Becker et al., 1974). The experiments now reported show that guinea-pig and mouse peritoneal macrophages exposed in vitro to the purified cleavage products C3b and C3a release large amounts of lysosomal hydrolases. In the case of C3b attachment to macrophages in culture results in a dose- and time-dependent release of lysosomal enzymes into the medium. The selectivity of this release is shown by morphological observations and
Complement component effect on enzyme secretion the failure of lactate dehydrogenase to appear in the medium. The addition of C3a to macrophage cultures also results in release of acid hydrolases. However, the lysosomal enzyme release in the presence of C3a was always accompanied by loss of cellular lactate dehydrogenase, showing that the cells had been killed. As previously shown (Davies, Page & Allison, 1974) phase-contrast observations of macrophage cultures and release of lactate dehydrogenase are well correlated with other indices of cell death, includiig failure to exclude trypan blue and to split fluorescein esters. Using these criteria it is clear that incubation with C3a results in death of macrophages whereas incubation with C3b does not. The attachment of C3b to macrophages in culture and the resulting release of lysosomal enzymes into the medium can be inhibited by using an anti-C3 Fab or anti-C3 IgG. The distinct effects of C3b and C3a on mononuclear phagocytes in vitro are of interest in view of the possible differences in the ways by which they interact with membranes. The observations described provide new information on the stimulation of enzyme production by macrophages, the selective release of lysosomal hydrolases and the possible role of lysosomal hydrolase secretion in the evolution of chronic inflammatory lesions. The releasing cells appear to be healthy and viable. Three different mechanisms have been suggested for the release of acid hydrolases from cells (Weissmann et al., 1971). These include loss of integrity of the plasma and lysosomal membranes resulting in enzyme leakage and cell death; endocytosis of substances which perturb lysosomal or plasma membranes, leading to their fusion; and selective regurgitation of acid hydrolases during aborted or incomplete phagocytosis. Cells exposed to C3b do not release lactate dehydrogenase. Thus direct membrane-lytic effects are unlikely to be involved in the observed release. Likewise, regurgitation due to aborted phagocytosis does not appear to account for the release. The most probable mechanism of enzyme release by C3b is by exocytosis. This is a process similar to that seen in secretory cells where the contents of packaged secretions are discharged after the fusion of the secretory granules with the plasma membrane. Presumably interaction of C3b with its plasma membrane receptor provides the stimulus to lysosomal hydrolase release. It is now clear that both granulocytes (Goldstein et al., 1973; Becker et al., 1974) and macrophages, phagocytic cells associated with acute and chronic inflam-
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mation respectively, can selectively release lysosomal hydrolases by complement cleavage products with no detectable loss of viability. This phenomenon may provide an explanation for the ability of macrophages to cause tissue damage and degradation at sites of chronic inflammation while retaining their viability over long periods of time. This interpretation is supported by evidence that other agents inducing chronic inflammation, such as group A streptococcal cell walls (Davies et al., 1974), immune complexes (Cardella, Davies and Allison, 1974), dental plaque (Page, Davies & Allison, 1973) carageenan (Davies & Allison, 1976) and mouldy hay dust (Edwards, 1976) also cause selective release of lysosomal hydrolases from viable macrophages in vitro. All of these agents have been shown to activate complement by the alternative pathway (Bitter-Suermann et al., 1975), so generating C3b which may be the common factor mediating enzyme release from macrophages. Goldstein & Weissmann (1974) demonstrated that lysosomal enzymes from polymorphonuclear leucocytes activate factor B of the alternative pathway of complement, resulting in the cleavage of serum C5. Dourmashkin & Patterson (1975) reported that human serum C3 was converted to C3b in the presence of a lysosomal lysate fraction from polymorphonuclear leucocytes. We have found that enzymes released by activated macrophages can cleave C3 into C3a and C3b via the alternative pathway and that these enzymes can generate the split products of the purified complement component C3. These peptides can cause more lysosomal enzyme release from macrophages. This forms a self-amplifying system because C3b causes release of lysosomal enzymes which can cleave C3, generating more C3b which would induce further enzyme release. Activated C3b with factor B itself functions as a proteinase, cleaving further C3 to C3b (Nicholson et al., 1975). The role of these two amplification systems in the alternative pathway and their interaction with macrophages in chronic inflammation clearly merits detailed study.
ACKNOWLEDGMENTS We are indebted to Dr C. J. Cardella (Toronto, General Hospital) who kindly allowed us to use the method for culturing macrophages on a cross-linked albumin surface which he developed in this laboratory. We thank Miss W. Hylton for technical assist-
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ance. H.U.S. was supported by a grant (Scho 215/1) of the Deutsche Forschungsgemeinschaft., Bad Godesberg, Germany.
REFERENCES ALLISON A.C. & DAVIES P. (1975) Mononuclear Phagocytes in Immunity, Infection and Pathology, p. 487. Blackwell Scientific Publications, Oxford. BECKER E.L., SHOWELL H.J., HENSON P.M. & Hou L.S. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. I. The characteristics of the release, the importance of surfaces and the relation of enzyme release to chemotactic responsiveness. J. Immunol. 112, 2047. BITrER-SUERMANN D., HADDING U., SCHORLEMMER H.U., LIMBERT M. & DUKOR P. (1975) Activation by some Tdependent antigens and B cell mitogens of the alternative pathway of the complement system. J. Immunol. 115, 425. BITTER-SUERMANN D., HADDING U., MELCHERT F. & WELLENSIECK H.J. (1970) Independent and consecutive action of C5, C6 and C7 in immune hemolysis. I. Preparation of EAC 1-5 with purified guinea pig C3 and C5. Immunochemistry, 7, 955. CARDELLA C.J., DAVIES P. & ALLISON A.C. (1974) Immune complexes induce selective release of lysosomal hydrolases from macrophages. Nature (Lond.), 247, 46. COCHRANE C.G. & MULLER-EBERHARD H.J. (1968) The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. exp. Med. 127 CONCHIE J., FINDLAY J. & LEVVY G.A. (1959) Mammalian glycosidases. Distribution in the body. Biochem. J. 71, 318. DAVIES P. & ALLISON A.C. (1976) The Immunobiology of the Macrophage. Academic Press, London and New York. (In press.) DAVIES P., PAGE R.C. & ALLISON A.C. (1974) Changes in cellular enzyme levels and extracellular release of lysosomal acid hydrolases in macrophages exposed to group A streptococcal cell wall substance. J. exp. Med. 139, 1262. DOURMASHKIN R.R. & PATTERSON S. (1975) Complement lesions in cell membranes from joint effusions of various types of arthritis. Inflammation, 1, 155. EDWARD J.H. (1976) A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin. Allergy, 6, 19.
GEWURZ H. (1972) Biological Activities of Complement, p. 56, S. Karger, Basel. GOETZE 0. & MULLER-EBERHARD H.J. (1971) The C3activator system: an alternate pathway of complement activation. J. exp. Med. 134, 905. GOLDSTEIN I.M. & WEISSMANN G. (1974) Generation of C5derived lysosomal enzyme-releasing activity (C5a) by lysates of leukocyte lysosomes. J. Immunol. 113, 1583. 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 complement component. Proc. natl. Acad. Sci. (Wash.), 70, 2916. HUBER H., POLLEY M.J., LINSCOTT W.D., FUDENBERG H.H. & MULLER-EBERHARD H.J. (1968) Human monocytes: distinct receptor sites for the third component of complement and for immunoglobulin G. Science, 162, 1281. K6NIG W., BITTER-SUERMANN D., DIERICH M.P., LIMBERT M., SCHORLEMMER H.U. & HADDING U. (1974) DNPantigens activate the alternate pathway of the complement system. J. Immunol. 113, 501. LAY W.H. & NUSSENZWEIG W. (1968) Receptors for complement on leukocytes. J. exp. Med. 128, 991. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265. NICHOLSON A., BRADE V., SCHORLEMMER H.U., BURGER R., BITTER-SUERMANN D. & HADDING U. (1975) Interaction of C3b, B and D in the alternative pathway or complement activation. J. Immunol. 115, 1108. NICHOLSON A., BRADE V., LEE G.D., SHIN H.S. & MAYER M.M. (1974) Kinetic studies of the formation of the properdin system enzymes on zymosan: evidence that nascent C3b controls the rate of assembly. J. Immunol. 112, 1115. PAGE R.C., DAVIES P. & ALLISON A.C. (1973) Effects of dental plaque on the production and release of lysosomal hydrolases by macrophages in culture. Archs oral Biol. 18, 1481. TALALAY P., FISHMAN W.M. & HUGGINS C. (1946) Chromiogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. J. biol. Chem. 166, 757. WARD P.A. (1969) The Heterogeneity of Chemotactic Factors for Neutrophils Generated from the Complement System, p. 279. S. Karger, Basel. WEISSMANN G., DUKOR P. & ZURIER R.B. (1971) Effects of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature: New Biol. 231, 131. WOOLLEN J.W., HEYWORTH R. & WALKER P.G. (1961) Studies on glucosaminidase and N-acetyl-fl-galactosaminidase. Biochem. J. 78, 111.
Effects of activated complement components on enzyme secretion by macrophages
H. U. SCHORLEM MER & A. C. ALLISON Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex
Received 18 March 1976; accepted for publication 6 May 1976
interdependent series of interacting proteins with a vital role as an effector system in the maintenance of body integrity. Physiological complement activation may proceed through at least two major pathways, both of which lead to the activation of C3 (Goetze & Muller-Eberhard, 1971; Gewurz, 1972). In this respect C3 is a central component of the complement system. Several activated components and cleavage products have the potential of mediating inflammatory tissue injury, either directly or indirectly. In addition, activation of the complement system has been associated with acute and chronic inflammatory reactions of immunological origin. Among the biologically active components of complement the low mol. wt cleavage products of C3 and C5 play the most important role in the mediation of inflammation and tissue injury. These peptides release histamine (Cochrane & Muller-Eberhard, 1968) which increases the permeability of the blood vessels near the site of complement activation. The chemotactic property of the C3a and C5a fragments promotes migration of leucocytes into the area (Ward, 1969). Goldstein, Hoffstein, Gallin & Weissmann (1973) and Becker, Showell, Henson & Hou (1974) have reported that C5a interacts with human polymorphonuclear leucocytes treated with cytochalasin B and provokes release of lysosomal enzymes from these cells. We have investigated the effects of C3 cleavage products, namely C3a and C3b, on mouse and guinea-pig peritoneal macrophages maintained in culture.
Summary. Purified cleavage products of the guineapig complement component C3, namely C3b and C3a, interact with guinea-pig and mouse macrophages in culture to induce a dose- and timedependent release of lysosmal enzymes into the medium. In the case of C3b the selectivity of the release of hydrolases, which occurs without cell killing, is shown by morphological observations and the failure of lactate dehydrogenase to appear in the medium. However, lysosomal enzyme release in the presence of C3a is accompanied by loss of cellular lactate dehydrogenase. Preincubation of C3b with anti-C3 Fab inhibits its attachment to macrophages, after which there is hardly detectable enzyme release into the medium. We have found that stimulated macrophages release enzyme(s) which can cleave C3, generating more C3b either directly or via the alternative pathway; the C3b so formed would induce further enzyme release. This amplification system may provide an explanation for the ability of macrophages to generate mediators of inflammation and cause tissue damage and degradation at sites of chronic inflammation while retaining their viability for long periods of time. INTRODUCTION The complement system comprises a complex Correspondence: Dr A. C. Allison, Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
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MATERIALS AND METHODS Experimental animals Swiss mice (T.O. strain) and guinea-pigs (strain: Hartley) were obtained from SACI, Brentwood, Essex. Tissue culture materials Tissue culture grade Petri dishes were obtained from Nunc Jobling Laboratories Division, Stane, Staffordshire, and M199 from Burroughs Wellcome, Beckenham, Kent. Biochemical reagents Bovine serum albumin, penicillin, streptomycin and phenolphthalein glucuronic acid 0 01 M pH 7 0 were from Sigma Chemical Company, Surbiton, Surrey; p-nitrophenyl-,8-D-galactopyranoside and p-nitrophenyl-2-acetamido-,6-D-deoxyglucopyranoside from Koch-Light Laboratories, Colnbrook, Bucks; heparin, preservative-free, from Boots, Nottingham; pyruvate and nicotinamide adenine dinucleotide from Boehringer, Mannheim GmbH, Germany; starch, Triton X-100 and glutaraldehyde from British Drug Houses Ltd, Poole, Dorset.
The use of a glutaraldehyde cross-linked albumin surface for culture of adherent cells 50-mm tissue culture Petri dishes were covered by a mixture of bovine serum albumin (165 mg/ml in 0-2 M acetate buffer, pH 5 0) and glutaraldehyde (16 5 mg/ml in 0-2 M acetate buffer pH 5 0). Then they were incubated overnight at 370 and washed four times with phosphate-buffered saline. These surfaces were suitable for the culture of mouse and guinea-pig peritoneal macrophages in either a serumfree or serum-containing medium. Macrophage collection and culture Guinea-pigs were injected intraperitoneally with 30 ml of 2 per cent starch gel in physiological saline and killed three days later. Cells were washed from the peritoneal cavity with 150 ml M199 containing 100 u/ml of penicillin and streptomycin and 10 i.u./ml heparin. Mouse macrophages were obtained by peritoneal lavage of Swiss mice with 5 ml M199 containing 100 u/ml of penicillin and streptomycin and 100 i.u./ml heparin. In both cases 5 ml aliquots of the peritoneal exudate cell suspension containing 0-5-10 x 106 cells/ml were distributed into 50 mm Petri dishes and incubated in a humidified atmosphere of 5 per cent carbon dioxide and air at 37° for 1-2 h to allow attachment of adherent cells. Non-
adherent cells were removed by washing four times with phosphate-buffered saline. After washing, the cells were cultured in M199. Cultures prepared in this way give a sheet of well spread cells within 24 h. In all experiments quadruplicate cultures were used and biochemical results are expressed as the mean and standard deviation. At the end of each incubation period the medium was removed and the adherent cells were washed once with phosphate-buffered saline. The cells were then released by adding saline containing 0O1 per cent (wfv) Triton X-100 and 0-1 per cent w/v bovine serum albumin and scraping with sterile siliconerubber bungs. The activities of various enzymes were assayed in both the media and cell-containing fractions. Enzyme assays All assays were conducted under conditions giving linear release of product in relation to the amount of sample used and the time of incubation. Lactate dehydrogenase was assayed by determining the rate of oxidation of reduced nicotinamide adenine dinucleotide at 340 nm, ,B-glucuronidase by the method of Talalay, Fishman & Huggins (1946). ,8-Galactosidase was assayed by the method of Conchie, Findlay & Levvy (1959) using p-nitrophenyl-fi-D-galactopyranoside as substrate. N-acetyl-fl-D-glucosaminidase was assayed by the method of Woollen, Heyworth & Walker (1961)
using p-nitrophenyl-2-acetamido-2-,8-D-glucopyranoside as substrate dissolved in 0-1 M acetate buffer, pH 4-5. Generation and purification of the complement components Guinea-pig C3 and the cleavage product C3b were prepared as previously reported (Bitter-Suermann, Hadding, Melchert & Wellensieck, 1970; Nicholson, Brade, Schorlemmer, Burger, Bitter-Suermann & Hadding, 1975). For generation and purification of C3a, highly purified C3 was incubated with the C3 cleaving complex which is formed when cobra venom factor interacts with factor B, factor D and Mg2+. This reaction mixture was passed through a Sephadex G-100 column. The fractions that mediated contraction of isolated guinea-pig terminal ileum were pooled and concentrated. Purified C3, C3a and C3b were kindly provided by Dr D. Bitter-
Suermann, Institute of Medical Microbiology Mainz, Germany.
Complement component effect on enzyme secretion Standard incubation mixture for C3 activation Equal volumes of either C4-deficient guinea-pig serum or purified C3 were incubated with appropriate dilutions of either the cell fraction or the supernatant of a stimulated or unstimulated macrophage culture for 30 min at 370 and then cooled to 40 in an ice bath. The concentrations referred to in the text, represents the final protein concentration of the complement components in this standard incubation mixture (pg/ml). Protein levels of the purified complement components were determined by the method of Lowry, Rosebrough, Farr & Randall (1951) using bovine serum albumin as a standard.
Immunoelectrophoretic analysis of C3 conversion One per cent agarose was used in all experiments. Electrophoretic separation was performed using standard conditions (veronal buffer pH 8 6). Tenmicrolitre aliquots of the incubation mixtures were placed in the wells for electrophoretic separation. Monospecific goat antiserum against guinea-pig C3 was prepared as previously reported (Konig, Bitter-Suermann, Dierich, Limbert, Schorlemmer & Hadding, 1974). Statistical tests Means and standard deviations were calculated after samples were shown to be homogeneous by calculation of coefficients of variance. The significance of differences was established by the Student's t-test. Anti-C3 Fab fragments These fragments were prepared as described by Nicholson, Brade, Lee, Shin & Mayer (1974) Briefly, the IgG fraction of goat antiserum against guinea-pig C3 was digested by pepsin in the presence of cysteine. Anti-C3 activity of the fragments was tested by inhibition of lysis of EAC1423 with
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C5-C9. The reciprocal of the antibody dilution which produced 50 per cent inhibition of haemolysis was designated as 1 unit (u) of anti-C3 activity. The final preparation used in these studies contained 500 u/ml. To check for univalence the Fab fragments were incubated with EAC 42 and subsequently C3 was added to the cells. There was no removal of C3 from the fluid phase.
Unrelated IgG Unrelated IgG was isolated from the serum of nonimmunized goats by ammonium sulphate precipitation and DEAE-cellulose chromatography. RESULTS Induction by C3b of macrophage hydrolase secretion Marked changes in the levels and distribution of the activities of acid hydrolases were induced in mouse and guinea-pig macrophages by the purified cleavage products of the complement component C3, namely C3a and C3b. Two types of experiments were performed. Firstly the effect of various concentrations of C3b and C3a on the level and distribution of enzyme activity in mouse macrophage cultures after 72 h was measured. Second the effect of a single concentration of C3b on the time course of changes in enzyme level and distribution in guinea-pig macrophage cultures was determined. In cultures grown in the presence of various concentrations of C3b for a period of 72 h the total lysosomal enzyme activity was not changed (Fig. 1); however there was a marked decrease in the intracellular acid hydrolase content, with a reciprocal and concurrent increase in the extracellular enzyme activity in the culture media (Figs 1 and 2). This redistribution of
Table 1. Lysosomal enzyme levels and distribution between cells and culture medium in mouse macrophage cultures exposed to various concentrations of C3b for 72 h
N-acetyl-,8-D-glucosaminidase (nmol product/plate/hour)
Concentration of C3b (ug/ml) o 5 10 20 30 40
Total 4003+269 3962+ 142
3843+516 4013+146 3753+299 3960+402
fi-glucuronidase
8i-galactosidase
(nmol product/plate/hour)
(nmol product/plate/hour)
Percentage in medium
Total
Percentage in medium
Total
Percentage in medium
5 0+0 8 14-7+ 1-3 24 7+ 69 37-6+2-4 50 8+ 26
184+ 13 203+8 194+ 19 208+11 202+6 209+29
10-6+2-3 19-7+0-3 33-6+ 65 500+51 63 7+25 72-1+1-0
156+3 152+ 10 175+ 11 158+15 142+4 143+4
8 5+0 7 12-7+0-8 15-4+ 1-2 292+30 49 5+ 1 5 605+1 9
63-5+1-3
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H. U. Schorlemmer and A. C. Allison 180r-
600r_ 140
500_
OL
a)
To
10ok
a 400
a)
a-
76 60k
2
)_
300
Ec :-
0
10 20 30 Concentration (/sig/m I)
oo_
40
Figure 1. The effect of various concentrations of C3b on the levels of 8-galactosidase activity in the total culture (0) in cells (0), and in the medium (U). The cultures of mouse macrophages were assayed after incubation with C3b for 72 h.
activity was observed for all of the lysosomal hydrolases measured; these included I-glucuronidase, Ii-galactosidase and N-acetyl-fi-D-glucosaminidase as shown in Table 1. After 72 h incubation in medium containing 20 yug/ml of C3b the concentration of 8-galactosidase in the medium is significantly greater than in the controls (P
20
C
0
10
0
C'
0)
F_
0)
A0
6
-r24
48
72
Time (h)
Figure 4. The time-dependent release of 8-glucuronidase (0) and lactate dehydrogenase (A) from guinea-pig macrophages exposed to 20 pg/ml of purified guinea-pig C3b.
60
E .2
E U 50
Percentage
495+1-5
was studied in guinea-pig macrophage cultures maintained for various times up to 72 h in the presence of a single dose of 20 pig/ml of C3b. It is seen in Fig. 4 that cultures of guinea-pig macrophages exposed to C3b show no change compared to control cultures in the distribution of a representative lysosomal enzyme, 8-glucuronidase, between cells and culture medium for the first 24 h. From this time on, however, a highly significant increase (P < 0 01) in the amount of fl-glucuronidase in the medium is seen. Subsequently a rapid rise in the release of enzyme into the culture medium occurs so that at 72 h approximately 50 per cent of the total enzyme activity is found in the culture medium. While data are presented only for fl-glucuronidase, the same pattern of release was observed for other acid hydrolases measured including N-acetyl-fl-D-glucosaminidase and fl-galactosidase (Table 2). This time dependence of the selective release of lysosomal enzymes caused by C3b occurs with no detectable release of cellular lactate dehydrogenase into the culture medium, and significant increases in the cellular levels of this enzyme are observed at 72 h.
40_
g 30
1020 _10 1
0
6
24
48
76
Time (h)
Figure 5. Inhibition of attachment of C3b to guinea-pig macrophages after preincubation of C3b with anti-C3 Fab (0) and anti-C3 IgG (A) prevents the time-dependent release of lysosomal enzymes into the medium. (0) Control of C3b preincubated with an unrelated IgG fraction.
Effects of anti-C3b on enzyme release To show conclusively that attachment of C3b to guinea-pig macrophages in culture results in a doseand time-dependent release of lysosomal enzymes into the medium, we inhibited this reaction by using an anti-C3 Fab preparation. Purified C3b (20 ,u/ml) was preincubated with anti-C3 Fab, unrelated IgG and an anti-C3 IgG fraction for 12 h at 4°. These incubation mixtures were added to the macrophage cultures and the release of lysosomal enzymes into the medium was followed. As shown in Fig. 5, there is hardly any detectable enzyme release after interaction of C3b with anti-C3 Fab. Also,
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H. U. Schorlemmer and A. C. Allison
preincubation of C3b with anti-C3 IgG partially prevented enzyme release; however, there is still a release of about 20 per cent. The unrelated IgG fraction had absolutely no effect; there is the usual rapid rise in the concentration of enzyme in the culture medium, so that by 72 h approximately 50 per cent of the total enzyme activity is released from the cells. Cleavage of C3 by enzymes secreted from stimulated macrophages Experiments were performed to ascertain whether enzymes released from stimulated guinea-pig macrophages can cleave C3 as shown by immunoelectrophoretic conversion. Supernatants from cultures of activated guinea-pig macrophages were incubated with highly purified guinea-pig C3. As shown in Fig. 6 the C3 was cleaved to generate a product with the greater anodal electrophoretic mobility. This could have been due to activation of the alternative pathway. However, direct effects of the secreted
Figure 6. Immunoelectrophoretic demonstration of the conversion of guinea-pig C3 mediated by the alternative pathway in C4-deficient guinea pig serum. (a) control, C4deficient guinea-pig serum; (b) C4-deficient guinea-pig serum incubated with the cell fraction of activated guinea-pig macrophages; (c) C4 deficient guinea-pig serum incubated with the supernatant of activated macrophages.
enzymes on purified C3 were also shown in these experiments, with generation of C3b. Studies of the biological activities of the generated components are in progress and will be reported later.
DISCUSSION Chronic inflammatory reactions are responsible for the tissue damage that occurs in several important diseases of temperate and tropical climates, including rheumatoid arthritis, rheumatic fever, chronic pulmonary disease, periodontal disease, chronic diseases of the gastro-intestinal tract, schistosomiasis and leishmaniasis. Knowledge of the pathogenesis of chronic inflammatory reactions and how they might be controlled is of considerable medical importance. It is generally accepted that mononuclear phagocytes are present in all types of chronic inflammation and that they play an essential role in the pathogenesis and resolution of this process (Allison & Davies, 1975). The control mechanism by which macrophage enzyme secretion is induced is not yet clear. The evidence presented in this paper shows that one such mechanism is mediated by cleavage products of the complement component C3. Complement interacts with mononuclear phagocytic cells in several ways. Biologically active complement components play several important roles as inflammatory mediators. The cleavage products not only display anaphylatoxic (Cochrane & Muller-Eberhard, 1968) and chemotactic activity (Ward, 1969) but are also capable of interacting with human monocytes, mouse and guinea-pig peritoneal macrophages and rabbit alveolar macrophages by the C3b receptor (Lay & Nussenzweig, 1968; Huber, Polley, Linscott, Fudenberg & Muller-Eberhard, 1968). The interaction of these phagocytic cells with complement may result in tissue damage. It has already been reported that C5a interacts with human polymorphonuclear leukocytes treated with cytochalasin B and provokes release of lysosomal enzymes from these cells (Goldstein et al., 1973; Becker et al., 1974). The experiments now reported show that guinea-pig and mouse peritoneal macrophages exposed in vitro to the purified cleavage products C3b and C3a release large amounts of lysosomal hydrolases. In the case of C3b attachment to macrophages in culture results in a dose- and time-dependent release of lysosomal enzymes into the medium. The selectivity of this release is shown by morphological observations and
Complement component effect on enzyme secretion the failure of lactate dehydrogenase to appear in the medium. The addition of C3a to macrophage cultures also results in release of acid hydrolases. However, the lysosomal enzyme release in the presence of C3a was always accompanied by loss of cellular lactate dehydrogenase, showing that the cells had been killed. As previously shown (Davies, Page & Allison, 1974) phase-contrast observations of macrophage cultures and release of lactate dehydrogenase are well correlated with other indices of cell death, includiig failure to exclude trypan blue and to split fluorescein esters. Using these criteria it is clear that incubation with C3a results in death of macrophages whereas incubation with C3b does not. The attachment of C3b to macrophages in culture and the resulting release of lysosomal enzymes into the medium can be inhibited by using an anti-C3 Fab or anti-C3 IgG. The distinct effects of C3b and C3a on mononuclear phagocytes in vitro are of interest in view of the possible differences in the ways by which they interact with membranes. The observations described provide new information on the stimulation of enzyme production by macrophages, the selective release of lysosomal hydrolases and the possible role of lysosomal hydrolase secretion in the evolution of chronic inflammatory lesions. The releasing cells appear to be healthy and viable. Three different mechanisms have been suggested for the release of acid hydrolases from cells (Weissmann et al., 1971). These include loss of integrity of the plasma and lysosomal membranes resulting in enzyme leakage and cell death; endocytosis of substances which perturb lysosomal or plasma membranes, leading to their fusion; and selective regurgitation of acid hydrolases during aborted or incomplete phagocytosis. Cells exposed to C3b do not release lactate dehydrogenase. Thus direct membrane-lytic effects are unlikely to be involved in the observed release. Likewise, regurgitation due to aborted phagocytosis does not appear to account for the release. The most probable mechanism of enzyme release by C3b is by exocytosis. This is a process similar to that seen in secretory cells where the contents of packaged secretions are discharged after the fusion of the secretory granules with the plasma membrane. Presumably interaction of C3b with its plasma membrane receptor provides the stimulus to lysosomal hydrolase release. It is now clear that both granulocytes (Goldstein et al., 1973; Becker et al., 1974) and macrophages, phagocytic cells associated with acute and chronic inflam-
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mation respectively, can selectively release lysosomal hydrolases by complement cleavage products with no detectable loss of viability. This phenomenon may provide an explanation for the ability of macrophages to cause tissue damage and degradation at sites of chronic inflammation while retaining their viability over long periods of time. This interpretation is supported by evidence that other agents inducing chronic inflammation, such as group A streptococcal cell walls (Davies et al., 1974), immune complexes (Cardella, Davies and Allison, 1974), dental plaque (Page, Davies & Allison, 1973) carageenan (Davies & Allison, 1976) and mouldy hay dust (Edwards, 1976) also cause selective release of lysosomal hydrolases from viable macrophages in vitro. All of these agents have been shown to activate complement by the alternative pathway (Bitter-Suermann et al., 1975), so generating C3b which may be the common factor mediating enzyme release from macrophages. Goldstein & Weissmann (1974) demonstrated that lysosomal enzymes from polymorphonuclear leucocytes activate factor B of the alternative pathway of complement, resulting in the cleavage of serum C5. Dourmashkin & Patterson (1975) reported that human serum C3 was converted to C3b in the presence of a lysosomal lysate fraction from polymorphonuclear leucocytes. We have found that enzymes released by activated macrophages can cleave C3 into C3a and C3b via the alternative pathway and that these enzymes can generate the split products of the purified complement component C3. These peptides can cause more lysosomal enzyme release from macrophages. This forms a self-amplifying system because C3b causes release of lysosomal enzymes which can cleave C3, generating more C3b which would induce further enzyme release. Activated C3b with factor B itself functions as a proteinase, cleaving further C3 to C3b (Nicholson et al., 1975). The role of these two amplification systems in the alternative pathway and their interaction with macrophages in chronic inflammation clearly merits detailed study.
ACKNOWLEDGMENTS We are indebted to Dr C. J. Cardella (Toronto, General Hospital) who kindly allowed us to use the method for culturing macrophages on a cross-linked albumin surface which he developed in this laboratory. We thank Miss W. Hylton for technical assist-
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ance. H.U.S. was supported by a grant (Scho 215/1) of the Deutsche Forschungsgemeinschaft., Bad Godesberg, Germany.
REFERENCES ALLISON A.C. & DAVIES P. (1975) Mononuclear Phagocytes in Immunity, Infection and Pathology, p. 487. Blackwell Scientific Publications, Oxford. BECKER E.L., SHOWELL H.J., HENSON P.M. & Hou L.S. (1974) The ability of chemotactic factors to induce lysosomal enzyme release. I. The characteristics of the release, the importance of surfaces and the relation of enzyme release to chemotactic responsiveness. J. Immunol. 112, 2047. BITrER-SUERMANN D., HADDING U., SCHORLEMMER H.U., LIMBERT M. & DUKOR P. (1975) Activation by some Tdependent antigens and B cell mitogens of the alternative pathway of the complement system. J. Immunol. 115, 425. BITTER-SUERMANN D., HADDING U., MELCHERT F. & WELLENSIECK H.J. (1970) Independent and consecutive action of C5, C6 and C7 in immune hemolysis. I. Preparation of EAC 1-5 with purified guinea pig C3 and C5. Immunochemistry, 7, 955. CARDELLA C.J., DAVIES P. & ALLISON A.C. (1974) Immune complexes induce selective release of lysosomal hydrolases from macrophages. Nature (Lond.), 247, 46. COCHRANE C.G. & MULLER-EBERHARD H.J. (1968) The derivation of two distinct anaphylatoxin activities from the third and fifth components of human complement. J. exp. Med. 127 CONCHIE J., FINDLAY J. & LEVVY G.A. (1959) Mammalian glycosidases. Distribution in the body. Biochem. J. 71, 318. DAVIES P. & ALLISON A.C. (1976) The Immunobiology of the Macrophage. Academic Press, London and New York. (In press.) DAVIES P., PAGE R.C. & ALLISON A.C. (1974) Changes in cellular enzyme levels and extracellular release of lysosomal acid hydrolases in macrophages exposed to group A streptococcal cell wall substance. J. exp. Med. 139, 1262. DOURMASHKIN R.R. & PATTERSON S. (1975) Complement lesions in cell membranes from joint effusions of various types of arthritis. Inflammation, 1, 155. EDWARD J.H. (1976) A quantitative study on the activation of the alternative pathway of complement by mouldy hay dust and thermophilic actinomycetes. Clin. Allergy, 6, 19.
GEWURZ H. (1972) Biological Activities of Complement, p. 56, S. Karger, Basel. GOETZE 0. & MULLER-EBERHARD H.J. (1971) The C3activator system: an alternate pathway of complement activation. J. exp. Med. 134, 905. GOLDSTEIN I.M. & WEISSMANN G. (1974) Generation of C5derived lysosomal enzyme-releasing activity (C5a) by lysates of leukocyte lysosomes. J. Immunol. 113, 1583. 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 complement component. Proc. natl. Acad. Sci. (Wash.), 70, 2916. HUBER H., POLLEY M.J., LINSCOTT W.D., FUDENBERG H.H. & MULLER-EBERHARD H.J. (1968) Human monocytes: distinct receptor sites for the third component of complement and for immunoglobulin G. Science, 162, 1281. K6NIG W., BITTER-SUERMANN D., DIERICH M.P., LIMBERT M., SCHORLEMMER H.U. & HADDING U. (1974) DNPantigens activate the alternate pathway of the complement system. J. Immunol. 113, 501. LAY W.H. & NUSSENZWEIG W. (1968) Receptors for complement on leukocytes. J. exp. Med. 128, 991. LOWRY O.H., ROSEBROUGH N.J., FARR A.L. & RANDALL R.J. (1951) Protein measurement with Folin phenol reagent. J. biol. Chem. 193, 265. NICHOLSON A., BRADE V., SCHORLEMMER H.U., BURGER R., BITTER-SUERMANN D. & HADDING U. (1975) Interaction of C3b, B and D in the alternative pathway or complement activation. J. Immunol. 115, 1108. NICHOLSON A., BRADE V., LEE G.D., SHIN H.S. & MAYER M.M. (1974) Kinetic studies of the formation of the properdin system enzymes on zymosan: evidence that nascent C3b controls the rate of assembly. J. Immunol. 112, 1115. PAGE R.C., DAVIES P. & ALLISON A.C. (1973) Effects of dental plaque on the production and release of lysosomal hydrolases by macrophages in culture. Archs oral Biol. 18, 1481. TALALAY P., FISHMAN W.M. & HUGGINS C. (1946) Chromiogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. J. biol. Chem. 166, 757. WARD P.A. (1969) The Heterogeneity of Chemotactic Factors for Neutrophils Generated from the Complement System, p. 279. S. Karger, Basel. WEISSMANN G., DUKOR P. & ZURIER R.B. (1971) Effects of cyclic AMP on release of lysosomal enzymes from phagocytes. Nature: New Biol. 231, 131. WOOLLEN J.W., HEYWORTH R. & WALKER P.G. (1961) Studies on glucosaminidase and N-acetyl-fl-galactosaminidase. Biochem. J. 78, 111.
