Inflammation, 1977, Vol. 2, No. 2, pp. 165-177.

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA X. The Role Played by Leukocyte Factors, Cationic Polyelectrolytes, and by Membrane-Damaging Agents in the Lysis of Staphylococcus aureus: Relation to Chronic Inflammatory Procesess 1 MEIR LAHAV and ISAAC GINSBURG Department of Oral Biology, Hebrew University-Hadassah School of Dental Medicine, Foundedby the Alpha Omega Fraternity, Jerusalem, Israel

Abstract--A heat-stable factor present in extracts of human blood leukocytes is capable of lysing young Staphylococcus aureus at pH 5.0. Lysis is characterized by breakdown of cell-wall components as judged by electron microscopic and biochemical analysis. The leukocyte extracts can be replaced by a variety of agents known to injure cell membranes, e.g., leukocyte cationic protein histone, polymyxin B, colimycin, phospholipase A, and lysolecithin. The mechanisms by which all these agents bring about the degradation of the staphylococcal walls was studied. By using ~4C-labeled cell walls devoid of cytoplasmic structures, it was demonstrated that none of the above-mentioned agents had a direct lytic effect on purified cell walls. On the other hand, when any of these agents first interacted with intact staphylococci, a factor (presumably an autolysin) was generated that directly lysed the cell walls. Lysis of cell wails in the presence of intact staphylococci used as a source of autolysin was strongly inhibited by a variety of anionic polyelectrolytes such as heparine and liquoid. The possible role olayed by bacterial autolysins in the generation of microbial cell-wall comp~,nents capable of triggering chronic inflammation is discussed.

In previous publications (6, 7, 11-14, 19), it was demonstrated that freezeand-thaw extracts of human blood leukocytes (ENZ) were highly bactericidal and bacteriolytic for a variety ofStaph, aureus, Staph. albus, and Streptococcus faecalis strains. It was also demonstrated that, although the lysis of Staph. This study was supported by grants from the Joint Research Fund of the Hebrew University-Hadassah School of Dental Medicine, founded by the Alpha Omega Fraternity, and the Hadassah Medical Organization; by grants from the Chief Scientist, the Ministry of Health, Government of Israel; and the Max Bogen Research Fund, obtained through the Friends of the Hebrew University in the United States. 165 This journal is copyrightedby Plenum. Each articleis availablefor $7.50 from PlenumPublishingCorporation, 227 West 17th Street, New York, N.Y. 10011.

166

LAHAV AND GINSBURG

aureus by the leukocyte extracts was not lysozyme dependent, lysis of Staph. albus and Strep. faecalis was strictly dependent on the concentration of lysozyme in the leukocyte extracts (13). Lysis of 14C-labeled Staph. aureus by ENZ, as judged by the solubilization of radioactivity and by electron microscopic analysis (11), was also found to be strictly dependent on the age of the bacterial cells. While bacteria that had been harvested from the early or midlogarithmic phases of growth were highly susceptible to the killing and lytic effects of ENZ, staphylococci harvested from the stationary phase of growth were highly resistant to both actions of ENZ. Since young staphylococci that had been heated to 60~ for 30 rain became highly resistant to bacteriolysis, and since heated ENZ continued to lyse intact bacteria, it was postulated that a relatively heatstable factor present in ENZ probably activated a heat-labile autolytic system, which subsequently led to the degradation of the bacterial cell walls. The purpose of the present communication is to further study the role played by endogenous staphylococcal leukocyte factors in bacteriolysis. The possible role played by undergraded bacterial cell wall components in the propagation of chronic inflammatory process is discussed.

MATERIALS AND METHODS Staphylococcal Strains. A penicillin-resistant coagulase-positive Staph. aureus (designated X) was isolated from a pyogenic lesion of a patient. The bacteria were maintained on blood agar plates. Preparation ofLabelledand Unlabelled Bacteria. Staphylococci, that had been cultivated overnight in brain-heart infusion broth (BHI) (Difco) were inoculated into Edenmayer flasks containing 20-50 ml of BHI in the absence or presence of 0.5 pCi/ml of uniformally labeled 14C-D-glucose, specific activity 150-250 mCi/mmol (New England Nuclear, Boston, Massachusetts). The cultures were incubated in a water bath at 37~ under constant shaking and were harvested either from the logarithmic phase of growth (approximately 300 Klett units (KU) = 0 . 3 0 D 54 filter) or from the late stationary phase of growth (approximately 1000 K U = 2 . 0 0 D ) . The bacterial growth was washed several times with saline and resuspended in 0.1 M acetate buffer, pH 5.0 or in Tris buffer, pH 7.4, to the desired density. Preparation of 14C_LabelledCell Walls (14C-C W). Staphylococci, harvested from either the early logarithmic or late stationary phase of growth, were washed in distilled water and broken in the cold, in a Braun shaker, using Ballotini glass beads (0.1 ram) as described (26). The broken cells were then treated with 2 ~ sodium lauryl sulfate, washed several times in distilled water, and then employed in the lytic experiments described. Baeteriolytic Agents. The ENZ were obtained as described previously (11, 12). The extracts were centrifuged at 30,000g for 30 rain, and the supernatant fluids were used in the various experiments. The extracts were frozen and stored for several weeks at - 2 5 ~ without loss of activity. In some experiments, we have also employed the following lytic agents: (a) "cocktail," containing 100 ttg/ml of each of crude trypsin (Difco Laboratories), lysolecithin, and lysozyme (Sigma) or its individual constituents; (b) leukocyte cationic proteins (LCP) prepared from human leukocytes as described (28); (c) nuclear histone type

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA. X

167

ll-A tSigma); (d) phospholipase A2 (Sigma); (e) polymyxin B sulfate (Neosporin) (Burroughs Wellcome Co. ; (f) colimycin sulfate (Rafa Jerusalem); (g) spermine (Fluka AG); (h) crystalline egg-white lysozyme (Sigma); (i) platelet extracts prepared as described (12); (j) normal human serum (NHS) obtained from healthy donors; (k) synovial fluids obtained from knee joints of patients having mechanical trauma or suffering from rheumatoid arthritis; and (1) Myeloperoxidase (MPO), kindly provided by Dr. I. Olsson, Department of Internal Medicine University of Lund, Dalby, Sweden. The sera were used as such or after they had been heated to 56~ for 30 rain. Effect of Lytic Agents on Bacteria and Cell Walls. 14C-labeled staphylococcal suspensions containing approximately 5 • 104 cpm/100 KU/ml (54 filter) (100 KU = 0 . 2 0 D at 540 nm) and 14C-labeled cell walls containing 25,000 cpm/100 KU were incubated with the various lytic agents for 18 h at 37~ under constant shaking in 0.1 M acetate buffer (pH 5.0) or in Tris buffer 0.1 M (pH 7.4). Labeled bacteria or cell wails incubated in buffer alone served as controls. After incubation, the reaction mixtures were centrifuged at 500020,000g for 30 rain, and 50-~1 aliquots of the supernatant fluids were assayed for soluble radioactivity in a Packard scintillation counter (11). The results were expressed as percent radioactivity solubilized by the lytic agent from a standard suspension of labeled bacteria or cell walls. In most cases, the spontaneous radioactivity lost from the labeled cell walls or suspensions did not exceed 2 ~ and 20~ of the total radioactivity, respectively. Inhibition of Bacteriolysis. The following agents were tested for their inhibitory activity on bacteriolysis: (1) heparin-sodium (Nutritional Biochemical Corporation, Cleveland, Ohio); (2) liquoid (sodium polyanethole sulfonic acid); and (3) histone type II-A (Sigma Chemical Co.). The materials were added to the lyric agents J 4 C-CW mixtures, in 0.1 M acetate, and the solubilization of radioactivity was determined after 16 h of incubation.

RESULTS

Studies on the Mechanism ofLysis ofStaph, aureus by Leukocyte Extracts. In previous publications (6, 11-13, 19), it was found that 14C-Staph. aureus incubated in buffers alone sometimes lost about 1 0 ~ - 4 0 ~ o f its total radioactivity. A l t h o u g h the source and nature o f the radioactivity that "leaked o u t " were not fully k n o w n we have postulated that it might have originated as a result o f a partial autolysis (11). Further support for this assumption came f r o m preliminary electron microscopic and chromatographic analysis, which showed breakdown o f cell-wall structures and the accumulation o f solubilized cell-wall components in the supernatants. To further determine the role played by autolysins in bacteriolysis and the role o f leukocyte factors as possible activators o f autolytic systems, we employed 14C-labeled cell walls (which lacked autolytic systems) as targets for leukocyte extracts and a variety o f other lytic agents. Table 1 shows that y o u n g (logarithmic phase) staphylococci are lysed to a large extent by relatively small amounts o f leukocyte extracts, L C P and histone, polymyxin B, platelets, cocktail, and lysolecithin as well as by N H S and synovial fluid. O n the other hand, m u c h less lysis was exerted by egg white, lysozyme, trypsin, colimycin, and spermidine. When, however, 14C-

168

LAHAV AND GINSBURG TABLE 1

Lysis of 14C Staph. aureus (logarithmic phase) by Different Lytic Agents Reaction mixture Buffer (control) + Leukocyte extracts + LCP + Lysozyme + Histone + Platelets + Polymixin + Colimicin + Spermidine + NHS + Synovial fluid + Cocktail c + Trypsin + Lysolecithin

Concentration (pg/ml) Radioactivity solubilized (~)a -50-200 50 50 50 50 500 500 500 1000 1000 -100 100

24 85-98 ~ 90 34 70 62 48 50 44 46-75 b 75 90 36 38-63 b

a The results are expressed as means of values from six separate experiments. 14C-labeled staphylococci, 100 KU/ml, containing approximately 25,000 CPM, were incubated for 18 h at 37~ in 0.1 M acetate buffer, pH 5.0, in the presence of different lyric agents. The lysis of the bacteria was determined as described in Materials and Methods section. b The degree of lysis varied in different experiments. c 100/zg/ml of each of crude trypsin, lysolecithin, and lysozyme.

C W derived from young staphylococci were treated with all the agents described in Table 1, we found that, surprisingly, no substantial lysis of the cell walls occurred. These results therefore suggested that the absence of cytoplasmic structures from the partially purified cell walls was probably responsible for the lack of lysis. To test this assumption, we analyzed the lysis of cell walls in the presence of intact staphylococci (100 KU/ml) to which were added a variety of agents known to lyse young staphylococci (Table 1). As can be seen (Table 2), leukocyte extracts, cationic polyelectrolytes, NHS, synovial fluid, platelets extract, lysolecithin, and certain antibiotics, which alone were incapable of lysing cell walls, caused a substantial lysis of these structures in the presence of intact staphylococci. It is of interest that intact staphylococci alone caused only about 30% lysis of the cell walls. The results suggested, therefore, that intact staphylococci generated a factor (presumably an autolysin) upon interaction with the various "lytic" agents, and that this autolysin (27) is responsible for the cleavage of the 14C-CW. Unlike the cationic proteins, a series of anionic polyelectrolytes (e.g.,

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA. X

169

TABLE 2

Lysis by Different Lytic Agents of ~*C-Labeled Staphylococcal Cell Walls in the Presence or Absence of Unlabeled Intact Staph. aureus Release of radioactivity from 14C-CW"

Lytic agent Buffer (control) Leukocyte extract LCP MPO c Histone Polymixin B Colimicin Spermidine Platelet extract NHS Synovial fluid Cocktaild Lysozyme Trypsin Lysolecithin

Concentration (flg/ml) -50-500 50 100 100 500 100 500 100 500 100 500 100 500 500 100 100 100 500

In absence of staphylococci

In presence of staphylococcP

2.0 6.0 7.0 4.0 2.0 4.0 2.0 5.0 3.0 6.0 1.0 4.0 5.0 3.0 4.0 7.0 4.0 8.0 1.0 3.0

30.0 90.0 85.0 78.0 83.0 40.0 50.0 85.0 75.0 92.0 35.0 65.0 83.0 75.0 70.0 80.0 35.0 35.0 70.0 85.0

"The results are expressed as means of values from five or more separate experiments. The conditions of assay were the same as described in Table 1 except that 14C-CW (50 KU/ml) were used instead of intact Staph. aureus as targets for the lytic agents. b Unlabelled bacteria 100 KU/ml. c Myeloperoxidase (MPO) was kindly supplied by Dr. I. Olsson of University Hospital, Lund, Sweden. 100/zg/ml each of crude trypsin, lysolecithin, and lysozyme.

heparin, liquoid, a n d c h o n d r o i t i n sulfate) failed to " a c t i v a t e " the autolysins. M o r e o v e r , some o f the negatively c h a r g e d polyelectrolytes m a r k e d l y suppressed b o t h the s p o n t a n e o u s leakage o f r a d i o a c t i v i t y f r o m b a c t e r i a i n c u b a t e d in buffer alone (see below), a n d the lysis o f intact b a c t e r i a i n d u c e d by the v a r i o u s lytic agents (7, 8). T o further study the role p l a y e d b y intact staphylococci in the lysis o f cell walls, we h e a t e d the bacterial suspensions at 60~ for 30 rain a n d f o u n d t h a t such h e a t e d cells failed to w o r k with the " l y t i c " agents ( E N Z , LCP,

170

LAHAV AND GINSBURG

TABLE 3 Effect of pH on the Lysis of Young and Old Staphylococcal Cell Walls by Different Agents Lysis of cell walls" Reaction mixture

Buffer and pH

Buffer (control)

Acetate, pH 5.0 Tris-HCl, pH 7.4 Acetate, pH 5.0 Tris-HC1, pH 7.4 Acetate, pH 5.0 Tris-HCl, pH 7.4 Acetate, pH 5.0 Tris-HCl, pH 7.4

Leukocyte extracts (0.2 mg) Unlabeled staphylococci Unlabeled staphylococci+ Leukocyte extracts

Youngb Old~ 2.0 4.0 6.0 10.0 30.0 67.0 88.0 78.0

3.0 5.0 8.0 10.0 27.0 43.0 46.0 56.0

"The results are expressed as means of values from five or more separate experiments. The conditions of assay were the same as in Table 1. b Young or old I~C-CW (50 KU/ml) were derived from logarithmic or stationary phases of growth, respectively.

histone, etc.) in the lysis of the 14C-CW. Furthermore, heated ENZ or LCP continued to "activate" the autolysin in intact staphylococci, indicating that the resistance of heated staphylococci to lysis by leukocyte extracts and LCP was probably caused by inactivation of the intrinsic autolysin. Since the data presented in Tables 1 and 2 were obtained with young (logarithmic phase) staphylococci, it was of interest to establish whether cell walls derived from old (stationary phase) staphylococci would also be more resistant to degradation by a mixture of ENZ or LCP with intact staphylococci. Table 3 compares the lysis of young and old staphylococcal cell walls by leukocyte extracts in the presence of intact staphylococci. As can be seen, old cell walls are more resistant to lysis by the mixtures. It was also of interest to establish whether the supply of the "autolysin" by the intact staphylococci was dependent on the age of the cells. It was found that, surprisingly, both young and old staphylococci were equally efficient in lysis of cell walls, in the presence of ENZ or LCP. Moreover, staphylococci aged at - 2 0 ~ or cells subjected to repeated freezing and thawing were better forces of "autolysin" as compared with young intact cells. Several attempts to release the "autolysin" from staphylococci by various established methods failed, and it is necessary to use either whole bacteria or bacterial fragments as a source of lyric agents.

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA. X

171

The Effect ofpH. Since all the lytic experiments were conducted in an acid medium, probably similar to that found in vivo in inflammatory sites, it was of interest to compare the lysis of young and old cell walls in acid and neutral buffers. As can be seen (Table 3), lysis of cell walls is quite significant in the presence of intact staphylococci alone when the incubation is conducted at pH 7.4. These results suggest that the autolytic system in the staphylococci acts optimally at neutral pH. The Lysis of Staphylococci and Their Cell Walls by Phospholipase A. Since a variety of cationic polyelectrolytes, which are known to injure cell membranes, have been found to work with an intrinsic staphylococcus factor in bacteriolysis (see Tables 2 and 3), and since the interaction of different bacterial species with cationic proteins of PMN resulted in the alteration of

TABLE 4

Lysis of 14C-Labeled Staph. a u r e u s and ~4C-CW by Phospholipase A2(PLA2) and by Leukocyte Cationic Proteins (LCP)

Lytic agents Buffer alone Leukocyte extracts PLA2

LCP

Concentration (/zg/ml)

Lysis of intact 14C.staphylcoccP

-100 50 100 250 50 I00 250

23.0 82.0 48.0 63.0 74.0 85.0 87.0 90.0 Lysis of 14C-CWV

Buffer alone Leukocyte extracts PLA2

LCP

a See Table 1. b See Table 2.

-100 10 50 100 10 50 100

In absence of staphylococci

In presence of staphylococci

2.0 5.0 2.0 2.5 2.5 2.0 3.0 3.0

30.0 85.0 54.0 81.0 97.0 74.0 90.0 93.0

172

LAHAV AND GINSBURG

their cell membrane phospholipids and in an increase in the permeability o f the cell membranes (29), it was of interest to examine the possibility that phospholipase A may also contribute to the lysis of intact staphylococci. Table 4 compares the lysis of intact staphylococci and their cell walls (in the presence of intact staphylococci) by PLA, lysolecithin, and LCP as well as by leukocyte extracts. As can be seen, ENZ and LCP can be effectively replaced by PLA or by lysolecithin (see Table 3) in bacteriolysis, and the lysis by the two agents is dose dependent. The Effect of Inhibitors on Bacteriolysis. In previous publications (7, 8) it was found that the lysis by leukocyte extracts of young staphylococci or of old staphylococci cultivated in the presence of penicillin (3) was markedly inhibited by relatively small amounts of anionic polyelectr01ytes (heparin, DNA, chondroitin sulfate, suramine, liquoid, poly-c-glutamic acid). Furthermore bacteriolysis was also markedly inhibited to a large extent by histone, protamine sulfate, protamine chloride, and polylysine. It was also found that histone behaved in an anolomous fashion in the bacteriolytic reaction (7). While at low concentrations (10-50 #g/ml) it readily lysed young staphylococci, concentrations greater than 100 pg/ml were not lytic by themselves, but TABLE 5

Inhibition by Basic and Acidic Polyelectrolytes of the Degradation of Cell Walls by Leukocyte Extracts Release of radioactivity from 14C-CWa

Reaction mixture

Concentration (pg/ml)

Buffer (control) Leukocyte extractsb Heparin Leukocyte extracts + Heparin Liquoid Leukocyte extracts + Liquoid DNA Leukocyte extracts+ DNA Histone Leukocyte extracts +Histone

0.25 0.25 0.1 0.1 1.0 1.0 0.5 0.5

In absence of In presence of staphylococci staphylococci 2 6 2 3 0 0 4 3 4 5

30 90 18c 9 4c 3 12~ 36 23c 29

"See Table 2. b Leukocyte extracts were employed at 200 pg/ml of protein. The inhibitors were added to the reaction mixture, and incubation was terminated after 16 h at 37~ c Note that the inhibitors suppressed the spontaneous release of radioactivity from intact staphylococci.

E F F E C T OF L E U K O C Y T E H Y D R O L A S E S ON BACTERIA. X

173

strongly inhibited the lysis of bacteria by leukocyte extracts. It was, therefore, of interest to test the effects of different negatively and positively charged polyelectrolytes on the lysis of intact staphylococci as well as of cell walls. Table 5 demonstrates that heparin, DNA, and liquoid, as well as the positively charged histone (500/~g/ml) strongly inhibited the lysis of the cell walls induced by intact staphylcocci and leukocyte extracts.

DISCUSSION The data presented describe the role played by leukocyte extracts, cationic proteins, polypeptide antibiotics, lysolecithin, and phospholipase A 2 in the lysis of intact Staph. aureus and of staphylococcal cell walls. The phenomenon of "spontaneous" leakage out of radioactivity, observed when staphylococci are incubated in buffers alone, lead us to postulate a key role that is probably played by autolysins (24, 25) in the degradation of staphylococci following treatment by leukocyte extracts. To test our hypothesis, we have approached this problem by employing partially purified cell walls, presumably lacked cytoplasmic components, as targets for the alleged lyric enzymes present in leukocyte lysosomes. The failure of such leukocyte extracts to lyse cell walls (Table 2) was surprising, and necessitated the reevaluation of the current hypothesis, which implicated leukocyte hydrolases as direct lysers of the bacterial wall structures. In our earlier publications we have already pointed out that several leukocyte factors (a heat-stable cationic protein + lysozyme) were essential for the degradation of staphyloce cci and certain streptococci, and that maximal lysis of a variety of other bacterial species involves collaboration between crude trypsin (or pancreatin) lysolecithin, phospholipase C, and lysozyme. The need for a multiplicity of enzymes in this reaction is considered to be linked with the presence, upon the bacterial surface, of components which must be removed before lysozyme-like enzymes can split the peptidoglycan and to lead to osmotic rupture of the protoplast. Since Staph. aureus was found by us to be lysed by leukocyte extracts, devoid of lysozyme, and since staphylococci are extremely resistant to lysozyme alone (17), we had to postulate the presence in staphylococci of an endogenous mechanism which is responsible for a partial lysis when the cells are aged in buffer. This autolytic system can be activated by certain factors present in leukocytes, platelets, and normal serum as well as in extracts of small intestines (6). Indeed it was found that cell walls can be readily lysed by leukocyte extracts if intact "leaking" staphylococci, which by themselves cause only marginal lysis of the cell walls, are also added. The necessity to include a heat-stable leukocyte factor suggested to us

174

LAHAV AND GINSBURG

that a cationic protein may be involved in this reaction (Table 2). Because leukocyte extracts can be effectively replaced by membrane-injuring factors, we may postulate that the membrane-injuring agents first interact with intact staphylococci to activate autolytic factors that degrade the cell walls. Further support for this assumption came from experiments that showed that the intact staphylococci lost their capacity to collaborate with leukocyte extracts in bacteriolysis if they had been preheated to 60~ for 30 min, Such heat treatment did not, however, affect the leukocyte extracts. Since we failed to isolate the autolysin from intact bacteria, the final proof on the involvement of "activated" autolysin awaits further confirmation. The inhibition by anionic polyelectrolytes of the lysis by ENZ of both intact staphylococci and cell walls supplemented by intact staphylococci, and the depression by liquoid and heparine of the spontaneous lysis of staphylococci aged in buffer alone, also point to the presence in intact staphylococci of a nascent autolytic system, which can be inhibited by negatively charged polyelectrolytes. Another intriguing finding was that, although young staphylococci were extremely susceptible to lysis by the different "lytic" agents, old staphylococci were highly resistant to bacteriolysis. Since cell walls derived from old staphylococci are much more resistant to degradation than the young cell walls, and since both young and old staphylococci are equally active as a source of autolysin in the presence of an activating agent, it can be postulated that the resistance of old staphylococci to lysis is not caused by the lack of an autolysin. It may be caused by either the much higher degree of peptide crosslinking (1, 22) or by the much thicker cell walls of stationary phase bacteria (23). It was also shown that old staphylococci can be completely lysed if incubated with leukocyte extract for 4 consecutive days (4). The finding that phospholipase Az and lysolecithin are capable of "activating" autolysins in staphylococci is of special importance in view of the studies of Elsbach et al. (2, 5, 28). These authors have shown that cationic proteins derived from rabbit PMN were capable of rapidly killing E. coli without gross structural disorganization or serious damage to the bacterial biochemical machinery (29). The mechanisms by which the cationic proteins bring about such changes have been linked with their capacity to enhance the permeability of the microbial envelopes. Extensive purification of the permeability-increasing activity has yielded fractions that contain similarly enriched phospholipase A2 and bactericidal activities (28). Since Gramnegative bacteria possess envelopes rich in phospholipids, and since most of the phospholipids of Gram-positive bacteria are associated with the protoplast membranes, it is possible that the target for phospholipase Az and lysolecithin in staphylococci may be the cell membrane. Alterations in the permeability

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA. X

175

of the cellular membrane without grossly affecting the intactness of the rigid peptidoglycan may thus be accompanied by the activation of autolysins, which subsequently leads to peptidoglycan degradation. Finally, the role played by bacterial autolysins and leukocyte factors in the initiation and propagation of chronic inflammation should be considered. It is well established that bacterial peptidoglycans and other undegraded cell wall constituents are highly phlogistic (9). Such constituents may be taken up by macrophages and can induce the selective secretion of a variety of lysosomal enzymes (16) capable of degrading the connective tissue matrix. Moreover, small cell wall fragments of a variety of Gram-positive bacteria have been shown to induce adjuvant arthritis in the rat (10); cellfree extracts (sonicates) of group A streptococci have been shown to induce limiting (18) and self-perpetuating arthritis (21) in the rabbit. Chronic arthritis can also be induced in rabbits by lipoteichoic acid (15). This surface bacterial antigen can readily be extracted from both staphylococci and streptococci by leukocyte extracts and lysozyme (20). Recent studies in our laboratory (to be published) have shown that supernatant derived from 14C-labeled group A streptococci and from Staph. aureus following treatment with leukocyte extracts and lysozyme could be resolved into two distinct radioactive peaks following gel filtration on Sephadex G-150 columns. The first peak, which comprised approximately 50~ of the total soluble radioactivity, was eluted from the column with the void volume and had a molecular weight greater than 100,000 daltons. The second peak had a molecular weight of approximately 20,000 daltons. Experiments to determine the possible phlogistic effects of the soluble fragments derived from these bacteria are underway. It is hoped that these experiments will shed more light on the biological properties of bacterial fragments released by leukcoyte factors. REFERENCES 1. ARCHIBALD,A.R. 1972. The chemistry of staphylococcal cell walls. In The staphylococci. J.O. Cohen, editor. Interscience, New York. 75-109. 2. BECKERDITE,S., C. MOONEY,J. WEISS,R. FRANSON,and P. ELSBACH.1977. Early and discrete changes in permeability of Escherichia coli and certain other Grain-negative bacteria during killing by granulocytes. J. Exp. IVied. 140:396-409. 3. EFRATI,C., T. SACKS,N. NE'EMAN,M. LAHAV,and I. GINSBURC. 1976. The effect of leukocyte hydrolases on bacteria. VIII. The combined effect of leukocyte extracts, lysozyme, enzyme "cocktails," and penicillin on the lysis of Staphylococcus aureus and group A streptococci in vitro. Inflammation 1:371-407. 4. EHRLICH, J., M. LAHAV, and M.N. SELA. 1976. The bacteriolytic effect of human dentoalveolar purulent exudates and leukocyte extracts. Submitted for publication. 5. FRANSON, S.A., P. PATRIARCA,and P. ELSBACH. 1974. Phospholipid metabolism by phagocytic cells. Phospholipases A2 associated with rabbit polyrnorphonuclear leukocyte granules. J. Lipid Res. 15:380-388.

176

LAHAV AND GINSBURG

6. GINSBURG,I., N. NE'EMAN,Z. DUCHAN, M.N. SELA,J.M. JAMES,and M. LAHAV.1975. The effect of leukocyte hydrolases on bacteria. IV. The role played by artificial enzyme "cocktails" and tissue enzymes in bacteriolysis. Inflammation 1:45-56. 7. GINSBURG, I., M. LAHAV, N. NE'EMAN, Z. DUCHAN, S. CHANES, and M.N. SELA. The interaction of leukocytes and their hydrolases with bacteria in vitro and in vivo: The modification of the bactericidal and bacteriolytic reactions by cationic and anionic macromolecular substances and by anti-inflammatoryagents. Agents Actions 6:292-305. 8. GINSBURG,I., M. LAHAV,N. NE'EMAN,and J.M. JAMES. 1974. The inhibition by basic and acidic polyelectrolytes of the degradation of bacteria by leukocyte enzymes: Relation to the persistence of microbial constituents in inflammatorysites. In Activation of Macrophages. W.H. Wagner, H. Hahn, and R. Evans, editors. Excerpta Medica, Amsterdam. 162-169. 9. GINSBURG,I., and M.N. SELA. 1976. The role of leukocytes and their hydrolases in the persistence, degradation and transport of bacterial constituents in tissues: Relation to chronic inflammatory processes in staphylococcal, streptococcal and mycobacterial infections and in chronic periodontal disease. Crit. Rev. Microbiol. 4:249-331. 10. KOGA, T., S. KOTANI, T. NAR1TA,and C.M. PEARSON. 1976. Induction of adjuvant arthritis in the rat by various bacterial ceU walls and their water-soluble components. Int. Arch. Allergy Appl. ImmunoL 51:206. 11. LAHAV,M., N. NE'EMAN,E. ADLER,and I. GINSBURG.1974. Effect of leukocyte hydrolases on bacteria. I. Degradation of 14C-labeled streptococcus and staphylococcus. J. Infect. Dis. 129:528-537. 12. LAHAV,M., N. NE'EMAN,J. JAMES,and I. GINSBt~6. 1975. The effect of leukocyte hydrolases on bacteria. III. Bacteriolysis induced by extracts of different leukocyte populations and the inhibition of lysis by macromolecular substances. J. Infect. Dis. 131:149-157. 13. ]'qE'EMAN,N., M. LAHAV,and I. GINSBURG.1974. The effect of leukocyte hydrolases on bacteria. II. The synergistic action of lysozyme and extracts of PMN, macrophages, lymphocytes and platelets in bacteriolysis. Proc. Soc. Exp. BioL Med. 146:1137-1145. 14. NE'EMAN,N., Z. DUCHAN,M. LAHAV,M.N. SELA,and I. GINSBURG.1976. The effect of leukocyte hydrolases on bacteria. VII. Bactericidal and bacteriolytic reactions mediated by leukocyte and tissue extracts and their modifications by polyelectrolytes. Inflammation 1:261-284. 15. NE'EMAN,N., and I. GINSBtmG.1972. Red cell sensitizing antigen of group A streptococci. II. Immunological and immunopathologicalproperties. Isr. J. Med. Sci. 8:18071816. 16. PACE,R.C., P. DAVIES,and A.C. ALLISON.1974. Participation of mononuclear phagocytes in chronic inflammatory diseases. J. Reticuloendothel. Soc. 15:413438. 17. SALTON,M.R.J., and J.G. PAVLIK. 1960. Studies of the bacterial cell wall. VI. Wall composition and sensitivity to lysozyme. Biochim. Biophys. Acta 39:398-407. 18. SCHWAB,J.H., W.J. CROMARTIE,S.H. OHANIAN,and J.E. CRADDOCK.1967. Association of experimental chronic arthritis with the persistence of group A streptococcal cell walls in the articular tissue. J. Bacteriol. 94:1728-1735. 19. SELA,M.N., M. LAHAV,N. NE'EMAN,Z. DUCI-IAN,and I. GINSBURG.1975. The effect of leukocyte hydrolases on bacteria. V. Modification of bacteriolysis by antiinfiammatory agents and by cationic and anionic polyelectrolytes. Inflammation 1:57-69. 20. SELA,M.N., M. LAHAV,and I. GINSBURG.1977. The effect of leukocyte hydrolases on bacteria. IX. The release of lipoteichoic acid from group A streptococci and from Strep. mutans by leukocyte extracts and by lysozyme. Inflammation 2:151-164. 21. STFIN, H., R. YAROM,S. LEVINE,T. DISHON, and I. GINSB~RG. 1973. Chronic self-

EFFECT OF LEUKOCYTE HYDROLASES ON BACTERIA. X

177

perpetuating arthritis induced in rabbits by a cell free extract of group A streptococci. Proc. Soc. Exp. Biol. Med. 143:1106-1112.

22. STROM~NCER,J.L., and GnUSEN, J.M. 1967. Mechanisms of enzymatic bacteriolysis. Science 156:213-217. 23. St3GANUMA,A., 1972. Fine structure of staphylococci: Electron microscopy. In The Staphylococci. J.O. Cohen, editor. Interscience, New York. 213-217. 24. TAKEBA,I., H.J. SINGER,E.M. WISE, and J.T. PARK. 1970. Staphylococcus aureus H. autolytic activity: General properties. J. Bacteriol. 102:14-19. 25. TIPVER, D.J. 1969. Mechanism of autolysis of isolated cell walls of Staphylococcus aureus. J. Bacteriol. 97:837. 26. WADSTR6M,T., and K. HISATSt3NE. 1970. Bacteriolytic enzymes from Staphylococcus aureus. Specificity of action of endo-fl-N-acetylglucosaminidase. Biochem. J. 120:735744. 27. WADSTR6M, T. 1973. Bacteriolytic enzymes from staphylococci. Contributions to Microbiology and Immunology. Vol. 1. Staphylococci and Staphylococcal Infections. Karger, Basel. 397-405. 28. WEISS, J., R.C. FRANSON,S. BECKERDITE,K. SCHMEIDLER,and P. ELSBACFI. 1975. Partial characterization and purification of a rabbit granulocyte factor that increases permeability of Eseherichia coll. J. Clin. Invest. 55:33-42. 29. WEISS,J., R.C. FRANSON,J. SCHMEIDLER,and P. ELSBACH. 1976. Reversible envelope effects during and after killing of Escherichia coli W by a highly purified rabbit polymorphonuclear leukocyte fraction. Biochim. Biophys. Acta 436:154-169.