Clin,. exp. Immunol. (1977) 27, 198-207.
Macrophage responses to mouldy hay dust, Micropolyspora faeni and zymosan, activators of complement by the alternative pathway H. U. SCHORLEMMER,* J. H. EDWARDSt P. DAVIESI & A. C. ALLISON* *Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex tMRC Pneumoconiosis Unit, Llandough Hospital, Penarth and t Merck Sharp and Dohme, Research Laboratories, Rahway, New Jersey, U.S.A.
(Received 2 August 1976)
SUMMARY
Mouse peritoneal macrophages in culture exposed to mouldy hay dust, Micropolyspora faeni or glycopeptide or protein/glycoprotein fractions from this organism show marked biochemical changes. For comparison the interaction of cultured macrophages with zymosan has been investigated. All these agents induce the release of hydrolytic enzymes from macrophages, even in the absence of serum in the medium. The release is time- and dose-dependent and is not associated with loss of the cytoplasmic enzyme lactate dehydrogenase or any other sign of cell death. The parallelism between the capacity of these agents to activate the complement system via the alternative pathway and to induce inflammatory responses in vivo and selective lysosomal enzyme secretion from cultures of macrophages is discussed. The in vitro phenomena seen with mouldy hay dust, M. faeni, the protein/glycoprotein and the glycopeptide derived from it, may be relevant to understanding the role of mononuclear phagocytes in the disease farmer's lung and other inflammatory reactions.
INTRODUCTION Inhalation of mouldy hay dust (MHD) produces the disease farmer's lung, an acute pulmonary inflammatory reaction that may develop into a chronic phase with permanent respiratory impairment. The main active agent in MHD is the thermophilic actinomycete Micropolyspora faeni (Wenzel et al., 1965). For some time complexes of M. faeni antigens and antibodies have been thought to be responsible for the pathogenesis of farmer's lung (Wilkie, Pauli & Gygax, 1973). More recently, evidence has accumulated that farmer's lung may be seen in individuals lacking precipitating antibodies to M. faeni antigens (Edwards, Baker & Davies, 1974). Mouldy hay dust and M. faeni in particular are efficient activators of the complement system via the alternative pathway (Edwards et al., 1974, Edwards, 1976). When M. faeni, MHD or zymosan, a well-known activator of complement by the alternative pathway, were administered to rabbits by i.t. injection, marked inflammatory reactions were observed in the lungs comparable to those in farmer's lung (Edwards, 1974; Edwards, Wagner & Seal, 1976). These reactions to M. faeni were as severe following first exposure as in animals previously immunized with the organism. These results suggest that activation of complement system via the alternative pathway may play an important role in the pathogenesis of farmer's lung. Since M. faeni constituents are ingested by alveolar macrophages soon after their inhalation, and these cells are involved in all stages of the disease, effects of mouldy hay dust, M. faeni, and fractions derived Correspondence: Dr H. U. Schorlemmer, Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
198
Macrophage response to complement-activating dust
199
from it, on cultures of macrophages have been investigated. For comparison, the interactions of cultured macrophages with zymosan are presented. MATERIALS AND METHODS Experimental animals. Swiss mice (T.O. strain) were obtained from SACI, Brentwood, Essex. Tissue culture materials. Tissue culture grade Petri dishes were obtained from Nunc Joblin Laboratories Division, Stone, Staffordshire, U.K. and MI 19 from Burroughs Wellcome, Beckenham, Kent, U.K., swine serum from Bio Cult Laboratories Ltd, Glasgow, Scotland. Biochemical reagents. Bovine serum albumin, penicillin, streptomycin, phenolphthalein glucuronic acid 0-01 M pH 7 0 and zymosan prepared from S. cerevisiae yeast were from Sigma Chemical Co., Surbiton, Surrey, p-nitrophenyl-fi-Dgalactopyranoside and p-nitrophenyl-2-acetamido-fi-n-deoxyglucopyranoside from Koch-Light Laboratories, Colnbrook, Buckinghamshire, heparin, preservative-free, from Boots, Nottingham, pyruvate and nicotinamide adenine dinucleotide from Boehringer Mannheim GmbH Germany; Triton X-100 from British Drug Houses Ltd, Poole, Dorset. Macrophage collection and culture. Mouse macrophages were obtained by peritoneal lavage of Swiss mice with 5 ml M199 containing 100 u/ml of penicillin and streptomycin and 10 iu/ml heparin. Five-millilitre 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%4 carbon dioxide and air at 370C for 1-2 hr 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 containing 10% (v/v) swine serum. The serum was heated at 56°C for 30 min before use. Cultures prepared in this way give a sheet of well-spread cells within 24 hr. 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 phosphatebuffered saline. The cells were then released by adding saline containing 0-1% (v/v) Triton X-100 and 0-1% (w/v) bovine serum albumin and scraping with sterile silicone rubber 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. fi-glucuronidase was assayed by the method of Talalay, Fishman & Huggins (1946). /J-galactosidase was assayed by the method of Conchie, Findley & Levvy (1959) using p-nitrophenyl-fl-D-galactopyranoside as substrate. N-acetyl-,8-Dglucosaminidase was assayed by the method of Woolen, Heyworth & Walker (1961) using p-nitrophenyl-2-acetamido-2-fiD-glucopyranoside as substrate dissolved in 0 1 M citrate-phosphate buffer pH 4 5. Statistical tests. Means and standard deviation 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. Mouldy hay dust. A sample of mouldy hay was obtained locally and a fraction of the dust collected by shaking the hay within a polythene bag connected to a Casella Hexlet gravimetric sampler drawing 50 1 air/min. The dust was collected in an extraction thimble and kept at 4°C until used. The concentrations were made up in saline using gentle ultrasonication to facilitate suspension. Propagation of M. faeni and preparation offractions. M. faeni organisms and antigens were prepared by the double dialysis technique (Edwards, 1971). Organisms were removed from the culture using a fine, sterile wire mesh and lyophilized. Protein/glycoprotein antigens were precipitated by adding an equal volume of ethyl alcohol, washed with 50%o alcohol and redissolved in deionized water. Trichloracetic acid was added to the alcohol supernatant and further precipitate discarded. The resulting solution was dialysed against running water and concentrated by air dialysis. Both protein/glycoprotein and glycopeptide solutions were freed from traces of the other by elution from a DEAE column equilibrated with 0-01 M Tris-HC1 buffer pH 8-0 using a continuous gradient of 0-0 5 M NaCl. Eluting fractions were monitored by immunoelectrophoresis using farmer's lung serum and appropriate fractions pooled, dialysed and filtered through sterile 220 mu filters before lyophilization. Prior to use they were redissolved in phosphate-buffered saline and filtered through sterile 220 mp filters.
RESULTS
Effects of zymosan and mouldy hay dust on macrophages Particles of zymosan and mouldy hay dust are rapidly taken up by phagocytosis, and can be seen inside cultured macrophages after 1 hr incubation at 37°C. The ingestion is followed by marked, selective release of lysosomal hydrolases into the culture medium. This effect depends on the nature and concentration of the particles used and the time of incubation (Fig. 1). After 24 hr incubation in medium
H. U. Schorlemmer et al.
200 80r.2
c
E c
40 0
c a)
201ME
0
50
25
100
200
Concentration (pg/mi)
FIG. 1. Effects of 24 hr incubation with various concentrations of zymosan ( *) and mouldy hay dust ( *) on the release of N-acetyl-fi-D-glucosaminidase and lactate dehydrogenase (o) from macrophages into the culture medium.
80F E
60K a) a)
w
40k
E
0
20k
a
_
0
3
_
6
12
24
Time (hr)
FIG. 2. The time-dependent release of N-acetyl-,f-D-glucosaminidase from macrophages exposed to 50 ,pg/ml of zymosan ( *) and 200 ,pg/ml of mouldy hay dust (A) in a serum-free medium. Controls in the absence of added particles ( *).
containing 25 jug/ml of zymosan the concentration of N-acetyl-fJ-D-glucosaminidase in the medium is significantly greater than in the controls (P
Macrophage responses to mouldy hay dust, Micropolyspora faeni and zymosan, activators of complement by the alternative pathway H. U. SCHORLEMMER,* J. H. EDWARDSt P. DAVIESI & A. C. ALLISON* *Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex tMRC Pneumoconiosis Unit, Llandough Hospital, Penarth and t Merck Sharp and Dohme, Research Laboratories, Rahway, New Jersey, U.S.A.
(Received 2 August 1976)
SUMMARY
Mouse peritoneal macrophages in culture exposed to mouldy hay dust, Micropolyspora faeni or glycopeptide or protein/glycoprotein fractions from this organism show marked biochemical changes. For comparison the interaction of cultured macrophages with zymosan has been investigated. All these agents induce the release of hydrolytic enzymes from macrophages, even in the absence of serum in the medium. The release is time- and dose-dependent and is not associated with loss of the cytoplasmic enzyme lactate dehydrogenase or any other sign of cell death. The parallelism between the capacity of these agents to activate the complement system via the alternative pathway and to induce inflammatory responses in vivo and selective lysosomal enzyme secretion from cultures of macrophages is discussed. The in vitro phenomena seen with mouldy hay dust, M. faeni, the protein/glycoprotein and the glycopeptide derived from it, may be relevant to understanding the role of mononuclear phagocytes in the disease farmer's lung and other inflammatory reactions.
INTRODUCTION Inhalation of mouldy hay dust (MHD) produces the disease farmer's lung, an acute pulmonary inflammatory reaction that may develop into a chronic phase with permanent respiratory impairment. The main active agent in MHD is the thermophilic actinomycete Micropolyspora faeni (Wenzel et al., 1965). For some time complexes of M. faeni antigens and antibodies have been thought to be responsible for the pathogenesis of farmer's lung (Wilkie, Pauli & Gygax, 1973). More recently, evidence has accumulated that farmer's lung may be seen in individuals lacking precipitating antibodies to M. faeni antigens (Edwards, Baker & Davies, 1974). Mouldy hay dust and M. faeni in particular are efficient activators of the complement system via the alternative pathway (Edwards et al., 1974, Edwards, 1976). When M. faeni, MHD or zymosan, a well-known activator of complement by the alternative pathway, were administered to rabbits by i.t. injection, marked inflammatory reactions were observed in the lungs comparable to those in farmer's lung (Edwards, 1974; Edwards, Wagner & Seal, 1976). These reactions to M. faeni were as severe following first exposure as in animals previously immunized with the organism. These results suggest that activation of complement system via the alternative pathway may play an important role in the pathogenesis of farmer's lung. Since M. faeni constituents are ingested by alveolar macrophages soon after their inhalation, and these cells are involved in all stages of the disease, effects of mouldy hay dust, M. faeni, and fractions derived Correspondence: Dr H. U. Schorlemmer, Division of Cell Pathology, MRC Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
198
Macrophage response to complement-activating dust
199
from it, on cultures of macrophages have been investigated. For comparison, the interactions of cultured macrophages with zymosan are presented. MATERIALS AND METHODS Experimental animals. Swiss mice (T.O. strain) were obtained from SACI, Brentwood, Essex. Tissue culture materials. Tissue culture grade Petri dishes were obtained from Nunc Joblin Laboratories Division, Stone, Staffordshire, U.K. and MI 19 from Burroughs Wellcome, Beckenham, Kent, U.K., swine serum from Bio Cult Laboratories Ltd, Glasgow, Scotland. Biochemical reagents. Bovine serum albumin, penicillin, streptomycin, phenolphthalein glucuronic acid 0-01 M pH 7 0 and zymosan prepared from S. cerevisiae yeast were from Sigma Chemical Co., Surbiton, Surrey, p-nitrophenyl-fi-Dgalactopyranoside and p-nitrophenyl-2-acetamido-fi-n-deoxyglucopyranoside from Koch-Light Laboratories, Colnbrook, Buckinghamshire, heparin, preservative-free, from Boots, Nottingham, pyruvate and nicotinamide adenine dinucleotide from Boehringer Mannheim GmbH Germany; Triton X-100 from British Drug Houses Ltd, Poole, Dorset. Macrophage collection and culture. Mouse macrophages were obtained by peritoneal lavage of Swiss mice with 5 ml M199 containing 100 u/ml of penicillin and streptomycin and 10 iu/ml heparin. Five-millilitre 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%4 carbon dioxide and air at 370C for 1-2 hr 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 containing 10% (v/v) swine serum. The serum was heated at 56°C for 30 min before use. Cultures prepared in this way give a sheet of well-spread cells within 24 hr. 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 phosphatebuffered saline. The cells were then released by adding saline containing 0-1% (v/v) Triton X-100 and 0-1% (w/v) bovine serum albumin and scraping with sterile silicone rubber 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. fi-glucuronidase was assayed by the method of Talalay, Fishman & Huggins (1946). /J-galactosidase was assayed by the method of Conchie, Findley & Levvy (1959) using p-nitrophenyl-fl-D-galactopyranoside as substrate. N-acetyl-,8-Dglucosaminidase was assayed by the method of Woolen, Heyworth & Walker (1961) using p-nitrophenyl-2-acetamido-2-fiD-glucopyranoside as substrate dissolved in 0 1 M citrate-phosphate buffer pH 4 5. Statistical tests. Means and standard deviation 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. Mouldy hay dust. A sample of mouldy hay was obtained locally and a fraction of the dust collected by shaking the hay within a polythene bag connected to a Casella Hexlet gravimetric sampler drawing 50 1 air/min. The dust was collected in an extraction thimble and kept at 4°C until used. The concentrations were made up in saline using gentle ultrasonication to facilitate suspension. Propagation of M. faeni and preparation offractions. M. faeni organisms and antigens were prepared by the double dialysis technique (Edwards, 1971). Organisms were removed from the culture using a fine, sterile wire mesh and lyophilized. Protein/glycoprotein antigens were precipitated by adding an equal volume of ethyl alcohol, washed with 50%o alcohol and redissolved in deionized water. Trichloracetic acid was added to the alcohol supernatant and further precipitate discarded. The resulting solution was dialysed against running water and concentrated by air dialysis. Both protein/glycoprotein and glycopeptide solutions were freed from traces of the other by elution from a DEAE column equilibrated with 0-01 M Tris-HC1 buffer pH 8-0 using a continuous gradient of 0-0 5 M NaCl. Eluting fractions were monitored by immunoelectrophoresis using farmer's lung serum and appropriate fractions pooled, dialysed and filtered through sterile 220 mu filters before lyophilization. Prior to use they were redissolved in phosphate-buffered saline and filtered through sterile 220 mp filters.
RESULTS
Effects of zymosan and mouldy hay dust on macrophages Particles of zymosan and mouldy hay dust are rapidly taken up by phagocytosis, and can be seen inside cultured macrophages after 1 hr incubation at 37°C. The ingestion is followed by marked, selective release of lysosomal hydrolases into the culture medium. This effect depends on the nature and concentration of the particles used and the time of incubation (Fig. 1). After 24 hr incubation in medium
H. U. Schorlemmer et al.
200 80r.2
c
E c
40 0
c a)
201ME
0
50
25
100
200
Concentration (pg/mi)
FIG. 1. Effects of 24 hr incubation with various concentrations of zymosan ( *) and mouldy hay dust ( *) on the release of N-acetyl-fi-D-glucosaminidase and lactate dehydrogenase (o) from macrophages into the culture medium.
80F E
60K a) a)
w
40k
E
0
20k
a
_
0
3
_
6
12
24
Time (hr)
FIG. 2. The time-dependent release of N-acetyl-,f-D-glucosaminidase from macrophages exposed to 50 ,pg/ml of zymosan ( *) and 200 ,pg/ml of mouldy hay dust (A) in a serum-free medium. Controls in the absence of added particles ( *).
containing 25 jug/ml of zymosan the concentration of N-acetyl-fJ-D-glucosaminidase in the medium is significantly greater than in the controls (P
