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JOURNAL OF IMMUNOPHARMACOLOGY, 1(2),
219-231 (1979)
EFFECT OF MACROPHAGE ACTIVATION BY IMWNOADJUVANTS ON SERUM LEVELS OF LYSOSOMAL HYDROLASES IN MICE Anthony W. Schrecker, Richard M. Schultz and Michael A. Chirigos Laboratory of Cellular and Molecular Biology National Cancer Institute, NIH Bethesda, Maryland 20014 ABSTRACT The effect of pyran copolymer, injected into mice bearing the M109 Madison lung carcinoma, on serum concentrations of lysozyme, B-glucuronidase, and N-acetyl-B,D-glucosaminidase was studied and compared with that of other immunoadjuvants. Increases in lysozyme levels ranging from 50 to 100% were observed after injection of pyran, BCG and Bru-Pel; increases in the levels of the other enzymes were less consistent. Other immunoadjuvants were less effective in raising serum concentrations of lysosomal enzymes. The findings were correlated with the results of previous studies on macrophage activation and antineoplastic action produced by these immunoadjuvants and suggest that serum levels of lysozyme can serve as indices of these effects. INTRODUCTION Activation of macrophages is necessary for their cytostatic Such activated
o r cytolytic effect against malignant cells (1).
macrophages secrete increased amounts of lysosomal hydrolases ( 2 ) , although increased lysozyme concentrations in serum may result from an increased number of macrophages and their mobilization rather than from increased secretion after activation (3-5). These findings suggest that serum levels of lysozyme and other lysosomal enzymes, such as B-glucuronidase and N-acetyl-B,D-glucosaminidase, could serve as markers for macrophage activation. Our report presents the results of an attempt to correlate serum levels of 219 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
SCHRECKER, SCHULTZ, AND CHIRIGOS
220
these enzymes with macrophage activation by several immunoadjuvants and with their antitumor effect. Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by McMaster University on 03/06/15 For personal use only.
NATERIALS AND METHODS
Mice. Male CDFl (Balb/c x DBA/2 F1) mice,
6-8 weeks old and weighing 23-28 g, were supplied by the Mammalian Genetics and
Animal Production Section of the National Cancer Institute (NCI), Bethesda, Md. libitum.
They received Purina laboratory chow and water
Balb/c mice were used in a few early experiments. Most
experimental groups comprised 12-15 mice. Tumor. The Madison lung carcinoma (M109), derived from a spontaneous neoplasm in a Balb/c mouse and kindly supplied by Dr. Ruth Geran of the Division of Cancer Treatment (DCT), NCI, has been maintained in o u r laboratory by serial passage in Balb/c mice. It was suspended in Roswell Park Memorial Institute (RPMI) 1640 medium and inoculated subcutaneously (s.c.) in the right inguinal region of the mice. Immunoadjuvants and Drugs. Pyran copolymer (NSC 46015), a copolymer of divinyl ether and maleic anhydride, was obtained from Dr. David Breslow of the Hercules Research Center, Wilmington, Del. Glucan, a B-1,3-polyglucose isolated from Saccharomyces cerevisiae cell walls, was donated by Dr. N.R. Di Luzio of Tulane University School of Medicine, New Orleans, La. Levamisole was obtained from Janssen Pharmaceutica, Beerse, Belgium, partially purified mouse fibroblast interferon (6) from Dr. K. Paucker, Medical College o f Pennsylvania, Philadelphia, Pa., and poly(1)poly(c) from Miles Laboratories, Elkhart, Indiana. Mycobacterium bovis, Pasteur strain (BCG) and lyophilized Corynebacterium parvum (Propionibacterium acnes) were kindly provided by Dr. S.D. Chaparas, Bureau of Biologics, FDA, Bethesda, Md. Bru-Pel, the ether extraction residue of Brucella abortus 456 ( 7 ) , was donated by D r . J . S . Youngner, University of Pittsburgh School of Medicine, Pittsburgh, Pa. All immunoadjuvants were dissolved o r suspended in sterile Dulbecco's phosphate-buffered saline (PBS), except for pyran copolymer, which was dissolved by adjusting a suspension
MACROPHAGE ACTIVATION
221
in a 0.85% NaCl to pH 7.2 with 0.1 N NaOH, and injected intra-
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peritoneally (i.p.) in a volume of 0.01 ml/g of body weight. Untreated control mice received 0.2 ml o f PBS.
I-(2-Chloroethy1)-
3-(trans-4-methylcyclohexyl)-l-nitrosourea (MeCCNU; NSC 95441) was obtained through the Drug Synthesis and Chemistry Branch, DCT,
NCI and suspended in 0.3% aqueous hydroxypropylcellulose. Lysozyme Assay. Mice were exsanguinated. The blood was allowed to clot for 0.5 hr at room temperature, then for 3-4 hr at 3', and centrifuged for 15 min at 1500 rpm. The serum was used immediately or kept frozen at -25'.
Repeated freezing and
thawing was avoided. The turbidometric assay was similar to that of Gordon, Todd and Cohn (4). Micrococcus lysodeikticus (dried cells) and the hen egg white lysozyme standard (muramidase; EC 3.2.1.17), recrystallized 3x, dialyzed and lyophilized, were bought from Sigma Chemical Co., St. Louis, Mo. suspension (0.5 mg/ml) of
M.
To 1 ml of a lysodeikticus in 0.05 M KH2P04 -
Na2HP04 pH 6 . 2 , (buffer A) was added with mixing 1 ml of serum diluted with buffer A containing 0.1 M NaCl (buffer B), or lysozyme (0.4
-
4.0 pg/ml) dissolved in buffer B.
The decrease in
absorbance at 540 nm was measured immediately for 4-5 min in a Gilford 240 spectrophotometer. Assays were performed with 5 dilutions of each serum and 8 to 10 concentrations of lysozyme standard. Initial decreases in absorbance were computed by least-square regression; they were a linear function of lysozyme concentration, again computed by regression analysis. Serum lysozyme concentratiomwere expressed in equivalents of hen egg lysozyme. B-Glucuronidase. The assay, a modification of those of Plaice (8) and Fishman (9), was performed, as described recently (lo), with Sigma Chemical Co. reagents by incubating 0.2 ml of serum (duplicates) with 3 mM phenolphthalein B,D-glucuronide i n 0.16 M sodium acetate buffer, pH 4.5, in a total volume of 1 ml for 16 hr at 37O, followed by heating for 5 min at looo, adding 5 ml of 0.1 M 2-amino-2-methyl-1-propanol
buffer, pH 11, centrifuging at
SCHRECKER, SCHULTZ, AND CHIRIGOS
222
35,000 x 8,
and measuring phenolphthalein absorbance at 550 nm.
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Substrate was added after the incubation for the blank determinations. N-Acetyl-B,D-glucosaminidase.
Chemical Co.
Reagents were obtained from Sigma
The method was similar to that of Brandt et al. (11).
Serum (25 ~ 1 )(triplicates) was incubated with 2 mM p-nitrophenylN-acetyl-B,D-glucosaminide in 0.1 M sodium citrate buffer, pH 4.3,
in a total volume of 1 ml for 1 hr at 37'.
The mixture was cooled
in ice and, after addition of 1 ml of 10% trichloroacetic acid, centrifuged at 1000 x g for 10 min. The supernatant solution (1.5 ml) was mixed with an equal volume of 2 M 2-amino-2-methyl1,3-propanediol, and p-nitrophenol absorbance measured at 420 nm after 10 min. A small blank absorbance (0.85% NaCl instead of serum) was subtracted, and concentrations were calculated from a p-nitrophenol standard, in which all ingredients (except serum and substrate) were present in the proportions of the assay and which gave a linear relationship between absorbance
(E
= 15,000) and
concentration. RESULTS
Table 1 shows that lysozyme, 6-glucuronidase and N-acetyl-B,Dglucosaminidase levels were higher in the serum of mice bearing the Madison lung carcinoma than in normal controls, in agreement with findings (12) that mobilization of macrophages i n tumorbearing animals produced increased serum lysozyme levels. Pyran copolymer, an immunoadjuvant shown to activate macrophages (13), led to an increase in serum lysozyme in both the normal and tumorbearing mice; lesser increases were observed with the two lysosomal acid hydrolases. In most of our experiments, pyran copolymer was injected at a dose of 25 mg/kg 6-8 days after tumor inoculation, and the blood collected another 6 days later because previous studies had shown that this was the most effective schedule for macrophage activation and therapeutic effect (14, 15). Indeed, in preliminary experiments, it was found that elevation of serum lysozyme reached a peak 6 days after treatment.
223
MACROPHAGE ACTIVATION
TABLE 1
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Lysosomal Enzymes in Serum of Mice Treated with Pyran Copolymer Treatment of Mice
Lysozyme (pg/ml ? S.E.aJ
No tumor control
4.4 k 0.2
32.3 f 0 . 1
4.17 ? 0 . 0 2
No tumor pyran
6.8 5 0 . 3
35.7
0.2
5.94 c 0 . 0 1
M109 control
5.0
0.3
38.0 f 0 . 6
5.49
M109 pyran
7.7 ? 0.3
39.7 2 0 . 1
6.11 ? 0.02
*
6-Glucuronidase (nmoles/ml/hr)
?
N-Acetyl-B,Dglucosaminidase (pmoles/ml/hr)
L 0.02
CDFl mice were inoculated with M109 tumor ( l o 6 cells) on day 0 o r received sham inoculations with RPMI 1640 medium. They were injected i.p. with 0.2 ml of PBS (controls) o r with 25 mg/kg of pyran copolymer on day 8, and were bled on day 14. a Standard error.
Table 2 indicates that the effect of pyran copolymer varied In 14 different
little between the doses of 1 2 . 5 and 50 mg/kg.
experiments, carried out under the conditions described, lysozyme levels varied between 4 . 3 and 6 . 3 (average 5 . 3
?
0.2 S.E.) in
untreated tumor-bearing mice, and between 6 . 1 and 1 1 . 5 (av. 8.8 2 0 . 5 ) in those treated i.p. with 25 mg/kg of pyran copolymer. Increases in lysozyme levels after treatment ranged from 30 to 115%, with an average increase of 67 f 8%. Multiple treatments with pyran copolymer were found to be no more effective for macrophage activation than a single injection ( 1 4 , l S ) . This was also true f o r the effect on serum lysozyme, as indicated by an experiment (Table 3 ) , which also showed that levamisole, which fails to activate macrophages significantly ( 1 , 1 6 ) , had little effect on serum lysozyme levels and on the
survival of tumor-bearing mice. The effect o f pyran copolymer on serum levels of lysosomal enzymes was compared with that of several other immunoadjuvants known to activate macrophages. The results are summarized in
224
SCHRECKER, SCHULTZ, AND CHIRIGOS
TABLE 2
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Serum Levels o f Lysozyme a s a Function o f Pyran Copolymer Dose Pyran Copolymer (mg/kgl
% of Control
Lysozyme (ug/ml) 5 . 2 f 0.2 a
0
100
12.5
10.5 f 0 . 7
202
25
10.9 ? 0.6
210
50
10.3
0.5
198
?
Balb/c mice were i n o c u l a t e d w i t h M109 tumor (2 x lo5 c e l l s ) on day 0 , i n j e c t e d i . p . w i t h pyran copolymer on day 6 , and b l e d on day 1 2 . a Standard e r r o r .
TABLE 3 Serum Lysozyme and S u r v i v a l Times o f Tumor-bearing Mice T r e a t e d w i t h Pyran Copolymer o r Levamisole Drug
Dose (mg/kg)
Control Pyran Levami s o 1e
25 5
Days o f Treatment
Day of Bleeding
6 6,13,20
12 26
6 6,13,20
12 26
1 0 . 1 & 0.2 9 . 3 f 0.2
37.5
6 6,13,20
12 26
6.7 f 0.3 6.9 f 0.1
30.5
Lysozyme (pg/ml) 6.2 ? 0 . Z a 5.9 f 0 . 1
MST (Days)
30
6 CDFl mice, i n o c u l a t e d w i t h M109 tumor (10 c e l l s ) on day 0, were i n j e c t e d i . p . on day 6 o r on days 6, 13, and 20, and bled. 6 days a f t e r t h e l a s t t r e a t m e n t . C o n t r o l mice r e c e i v e d 0.2 m l of PBS on t h e days of t r e a t m e n t . A d d i t i o n a l mice (10 p e r group) were i n j e c t e d on days 6 , 13, and 20, and k e p t u n t i l t h e y d i e d , i n o r d e r t o d e t e r m i n e median s u r v i v a l t i m e (MST). a Standard e r r o r .
225
MACROPHAGE ACTIVATION
Tables 4 and 5.
Interferon (6), poly(1) -poly(C), which induces
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interferon (6,16), and Corynebacterium parvum (Propionibacterium acnes) (1) had only minor effects. The effects of BCG (1,16) and of Bru-Pel, an ether extraction residue from Brucella abortus ( 7 ) , were comparable to those of pyran copolymer. The increase in Bglucuronidase serum levels was in general lesser than that of lysozyme, which was to be expected because cel s other than macrophages and neutrophils produce B-glucuronidase
The elevation of
N-acetyl-B,D-glucosaminidase was comparable to that of lysozyme,
but less reproducible.
TABLE 4 Effect of Immunoadjuvants on Serum Lysozyme in Tumor-bearing Mice Drug
PBS control Pyran Bru-Pel
Lysozyme (ug/ml)
Dose
0.2 ml
4.4
25 mg/kg 100 mg/kg
BCG
106 organisms
Interferon
4 1 0 units
POlY (1) .POlY(C)
10 mg/kg
?
% of Contr o 1
0.2 a
7.0 I 0.2 9.3 I 0.3 7.5 0.2 4.8 ? 0.2 5.4 ? 0.2
*
100 160 210 170
110 120
CDFl mice were inoculated with M109 tumor (2 x lo5 cells) on day 0, injected i.p. with PBS or the drugs on day 6, and bled on day 12. a Standard error.
Glucan, a B-1,3-polyglucose isolated from Saccharomyces cerevisiae, has been found to be a potent macrophage activator and to produce 7-fold increased lysozyme levels in rats after repeated intravenous (i.v.) administration
(17).
We failed, perhaps
because of the species difference, to reproduce this effect in
25 mg/kg
0 . 2 ml
Dose
1 mg/mouse 120
*
5.8
0.2
28.3 f 0.2
110
140
170
8.0 f 0.3
35.1 f 0.3
140
34.5 f 0.1
*
200
0.4
*
9.5 f 0 . 3
9.7
100
130
0.1
C
25.5 f 0 . 3
3.67
?
0.01
4.38 f 0.02
7.17 f 0 . 0 1
7.70 f 0.04
3.34 f 0.08
110
130
215
230
100
% of N-Acetyl-B,D% of Control glucosaminidase Control (pmo1es/ml/hr)
33.4 f 0 . 3
6-Glucuronidase (nmoles/ml/hr)
100
a
% of
Control
200
4.8
Lysozyme (pg/ml)
CDFl mice were inoculated with M109 tumor (106 cells) on day 0, treated i.p. with PBS or the drugs on day 8, and b l e d on day 1 4 . a Standard error.
C. parvum -
Bru-Pel 100 mg/kg 6 BCG 10 organisms
Pyran
PBS control
Drug
Lysosomal Hydrolases in Serum of Tumor-bearing Mice Treated with Immunoadjuvants
TABLE 5
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N
H
E0
c)
m
v)
227
MACROPHAGE ACTIVATION
mice bearing the M109 tumor (Tables 6,7),
although we used the
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same dose and time schedule that was reported (17).
By comparison,
pyran copolymer was effective by both the i.p. (Table 6 ) and i.v. (Table 7) routes in raising serum levels of lysosomal enzymes. Again, multiple treatments had no advantage over a single injection TABLE 6 Lysosomal Hydrolases i n Serum of Tumor-bearing Mice Treated Intraperitoneally with Pyran Copolymer or Glucan Drug
Day of Sacrifice
Lysozyme (ug/ml ? S.E.) 0.1
8-Glucuronidase N-Acetyl-B,D(nmoles/ml/hr f glucosaminidase S.E.) ( pmo 1es/ml/ hr)
N.D.~ N.D. N.D.
N.D. N.D. N.D.
PBS control
11
4.9
Pyran
11
6.6 f 0.3
Glucan
11
4.8
PBS control
12
5.7 f 0.3
26.4
?
1.0
5.88
Pyran
12
10.4 f 0.3
32.6
?
0.4
7.27 f 0.01
Glucan
12
30.4
?
0.1
5 . 2 7 t 0.05
7.1
?
jI
f
0.3
0.1
?
0.06
CDF1 mice, inoculated with M109 tumor (2 x lo5 cells) on day . 0 , were injected intraperitoneally on days 6, 8 and 10 with PBS ( 0 . 2 ml), pyran copolymer (12.5 mg/kg), or glucan (10 mg/kg). Hal.f the mice were bled on day 11, the other half on day 12. a Not determined.
Pyran copolymer has been found to potentiate the therapeutic effect of MeCCNU against the Lewis lung carcinoma (18). However, as shown in Table 8, macrophage activation by pyran, as reflected in serum lysozyme levels, was lessened when MeCCNU was injected prior to the copolymer. Gallily et al. (19) have shown that treatment of mice with MER, the methanol extraction residue of BCG, activated their
peritoneal macrophages and produced elevated levels of lysozyme
228
SCHRECKER, SCHULTZ, AND CHIRIGOS
TABLE 7
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Lysosomal Hydrolases in Serum of Tumor-bearing Mice Treated Intravenously with Pyran Copolymer o r Glucan Drug
Lysozyme (ug/ml f S.E.)
B-Glucuronidase (nmoles/ml/hr f S.E.)
PBS control
5.2 f 0.1
29.1
1.2
f
N-Acetyl-B,Dglucosaminidase (umo1es/ml/hr) 4.52 f 0.02
Pyran
9.7 5 0.2
54.6 f 0.5
7.81
?
0.01
Glucan
6.1 f 0.2
26.9
f
0.3
4.83
?
0.01
PBS control
4.5 f 0.2
27.0
f
0.1
?
0.01
28.5 f 0.4
3.63 5.71
f
0.02
24.8
3.79
?
0.01
Pyran
7.6 f 0.4
Glucan
5.8
f
0.2
?1
0.1
CDFl mice, inoculated with M109 tumor (106 cells) on day 0, were injected intravenously on days 8, 11 and 13 with PBS (0.2 ml), pyran copolymer (12.5 mg/kg), or glucan (10 mg/kg), then bled on day 14. The Table lists the results of 2 different experiments. TABLE 8 Serum Lysozyme Levels of Tumor-bearing Mice after Combination Treatment with MeCCNU and Pyran Copolymer Treatment on Day 4
Treatment on Day 9
Day o f Sacrifice
Lysozyme (;g/mi f
% of
Control
S.E.)
MeCCNU
MeCCNU
MeCCNU
-
8
5.5 f 0.1
100
8
6.1
0.2
110
-
15
5.5 f 0.4
100
15
6.3 f 0.2
115
Pyran
15
11.9 f 0.4
200
Pyran
15
8.2 f 0.4
150
?1
CDFl mice were inoculated with M109 tumor (2 x 105 cells) on day 0, injected i’ntraperitoneally with 1-(2-~hloroethyl)-3-(trans4-methylcyclohexyl)-l-nitrosourea (MeCCNU) (30 mg/kg) and pyran copolymer (25 mg/kg) on the days listed, and bled on the day of sacrifice. Untreated controls received injections of PBS (0.2 ml) on days 4 and 9.
MACROPHAGE ACTIVATION
229
and 6-glucuronidase in these cells.
In an attempt to detem.ine whether pyran copolymer had a similar effect, we injected CDFl Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by McMaster University on 03/06/15 For personal use only.
mice with 25 mg/kg of pyran i.p. and isolated the macrophages 6 days later by the method described previously (1,15,16). number of cells (2 x
lo7
The same
from 30 mice) was obtained from both the
treated and control animals.
The cells were suspended in buffer B
and disrupted by freezing and thawing 5 times. The same amount of lysozyme (2.6
2 0.1
7
vg/lO
cells) was found in the macrophages
from the treated and control animals. An attempt to determine whether there was increased secretion of lysozyme by macrophages cultured in RPMI-1640 medium (10% fetal calf serum) f o r 3 days following isolation after pyran treatment was not conclusive because the number of macrophages in the culture medium decreased during the incubation in the case of the untreated mice. DISCUSSION The present data show that serum levels of lysosomal enzymes, and especially of lysozyme, can indeed serve as indices of macrophage activation by immunoadjuvants and be correlated with their antitumor effect.
As lysozyme and N-acetyl-B,D-glucosaminidase
are also produced by neutrophils (20), which are not subject to activation, and 6-glucuronidase by additional cell types, the increases in serum levels probably represent only a fraction of the increase in synthesis by macrophages. Moreover, it is likely that the degree of macrophage activation by immunoadjuvants and the changes in enzyme synthesis depend on the different location of the several populations of these cells (circulating monocytes, peritoneal, alveolar, histiocytes, etc.).
The findings do suggest
that measuring serum levels of lysosomal hydrolases can serve as a test for the efficacy of immunotherapy. ACKNOWLEDGMENTS We wish to thank Jake R. Fullen for skillfully inoculating,
injecting, and bleeding the animals. REFERENCES 1.
Schultz, R.M., Papamatheakis, J.D., Stylos, W.A., and Chirigos, M.A., Augmentation of Specific Macrophage-Mediated Cytotoxicity: Correlation with Agents Which Enhance Antitumor Resistance, Cell. Immunol., 25:309, 1976.
230
SCHRECKER, SCHULTZ, AND CHIRIGOS
Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by McMaster University on 03/06/15 For personal use only.
2. Fantalone, R., and Page, R.C., Enzyme Production and Secretion by Lymphokine-Activated Macrophages, RES, J. Reticuloendothel. SOC., 21:343, 1977. 3. Heise, E.R., and Myrvik, Q.N., Secretion of Lysozyme by Rabbit Alveolar Macrophages In Vitro, RES, J. Reticuloendothel. SOC., 4:510, 1967. 4. Gordon, S., Todd, J., and Cohn, Z.A., In Vitro Synthesis and Secretion of Lysozyme by Mononuclear Phagocytes, J. E x p . Med., 139:1228, 1974. 5. Unanue, E.R., Secretory Function of Mononuclear Phagocytes: A Review, Am. J. Pathol., 83:396, 1976. 6. Schultz, R.M., Papamatheakis, J.D., and Chirigos, M.A., Interferon: An Inducer o f Macrophage Activation by Polyanions, Science, 197:674, 1977. 7. Youngner, J.S., Keleti, G., and Feingold, D.S., Antiviral Activity of an Ether-Extracted Nonviable Preparation of Brucella abortus, Infect, Immun., 10:1202, 1974. 8.
Plaice, C.H.J., A note on the determination of serum betaglucuronidase activity, J. Clin. Pathol., 14:661, 1961.
9. Fishman, W.H., Kato, K., Anstiss, C.L., and Green S.,
Human Serum 6-Glucuronidase: Its Measurement and Some of Its Properties, Clin. Chim. Acta, 15:435, 1967. 10. Schrecker, A.W., and Chirigos, M.A., Improved Determination o f 6-Glucuronidase in Serum, Clin. Chim. Acta, 88:413, 1978. 11. Brandt, E.J., Elliott, R.W., and Swank, R.T., Defective Lysosomal Enzyme Secretion in Kidneys of Chediak-Higashi (Beige) Mice, J. Cell Biol., 67:774, 1975. 12.
Currie, G.A., and Eccles, S.A., Serum Lysozyme as a Marker of Host Resistance. I. Production by Macrophages Resident in Rat Sarcomata, Br. J. Cancer, 33:51, 1976.
13.
Schultz, R.M., Papamatheakis, J.D., and Chirigos, M.A., Direct Activation In Vitro of Mouse Peritoneal Macrophages by Pyran Copolymer (NSC 46015), Cell. Immunol., 29:403, 1977.
14. Papamatheakis, J.D., Chirigos, M.A., and Schultz, R.M., Effect of Dose, Route, and Timing o f Pyran Copolymer Therapy Against the Madison Lung Carcinoma, in Immune-Modulation and Control of Neoplasia by Adjuvant Therapy, edited by M.A. Chirigos, p 427, Raven Press, New York, 1978.
MACROPHAGE ACTIVATION
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15.
231
Schultz, R.M., Papamatheakis, J.D., Luetzeler, J., Ruiz, P., and Chirigos, M.A., Macrophage Involvement in the Protective Effect of Pyran Copolymer Against the Madison Lung Carcinoma (M109), Cancer Res., 37:358, 1977.
16. Schultz, R.M., Papamatheakis, J.D., Luetzeler, J., and Chirigos, M.A., Association of Macrophage Activation with Antitumor Activity by Synthetic and Biological Agents, Cancer Res., 37:3338, 1977. 17. Kokoshis, P.L., Williams, D.L., Cook, J.A., and Di Luzio, N.R., Increased Resistance to Staphylococcus aureus Infection and Enhancement in Serum Lysozyme Activity by Glucan, Science, 199:1340, 1978. 18.
Mohr, S.J., Chirigos, M.A., Fuhrman, F.S., and Pryor, J.W., Pyran Copolymer as an Effective Adjuvant to Chemotherapy Against a Murine Leukemia and Solid Tumor, Cancer Res., 35: 3750, 1975.
19. Gallily, R., Yagel, S., and Weiss, D.W., Potentiated Lysosomal Enzyme, Bacteriostatic and Bactericidal Activities of Peritoneal Macrophages of Mice Treated with the MER Fraction of Tubercle Bacilli, Adv. E x p . Med. Biol., 73A:351, 1976. 20.
Hansen, N.E., Plasma Lysozyme - A Measure of Neutrophil Turnover. An Analytical Review, Ser. Haematol., 7:1, 1974.
Received Accepted
-- July 2 4 , -- November
1978 3, 1 9 7 8
JOURNAL OF IMMUNOPHARMACOLOGY, 1(2),
219-231 (1979)
EFFECT OF MACROPHAGE ACTIVATION BY IMWNOADJUVANTS ON SERUM LEVELS OF LYSOSOMAL HYDROLASES IN MICE Anthony W. Schrecker, Richard M. Schultz and Michael A. Chirigos Laboratory of Cellular and Molecular Biology National Cancer Institute, NIH Bethesda, Maryland 20014 ABSTRACT The effect of pyran copolymer, injected into mice bearing the M109 Madison lung carcinoma, on serum concentrations of lysozyme, B-glucuronidase, and N-acetyl-B,D-glucosaminidase was studied and compared with that of other immunoadjuvants. Increases in lysozyme levels ranging from 50 to 100% were observed after injection of pyran, BCG and Bru-Pel; increases in the levels of the other enzymes were less consistent. Other immunoadjuvants were less effective in raising serum concentrations of lysosomal enzymes. The findings were correlated with the results of previous studies on macrophage activation and antineoplastic action produced by these immunoadjuvants and suggest that serum levels of lysozyme can serve as indices of these effects. INTRODUCTION Activation of macrophages is necessary for their cytostatic Such activated
o r cytolytic effect against malignant cells (1).
macrophages secrete increased amounts of lysosomal hydrolases ( 2 ) , although increased lysozyme concentrations in serum may result from an increased number of macrophages and their mobilization rather than from increased secretion after activation (3-5). These findings suggest that serum levels of lysozyme and other lysosomal enzymes, such as B-glucuronidase and N-acetyl-B,D-glucosaminidase, could serve as markers for macrophage activation. Our report presents the results of an attempt to correlate serum levels of 219 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
SCHRECKER, SCHULTZ, AND CHIRIGOS
220
these enzymes with macrophage activation by several immunoadjuvants and with their antitumor effect. Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by McMaster University on 03/06/15 For personal use only.
NATERIALS AND METHODS
Mice. Male CDFl (Balb/c x DBA/2 F1) mice,
6-8 weeks old and weighing 23-28 g, were supplied by the Mammalian Genetics and
Animal Production Section of the National Cancer Institute (NCI), Bethesda, Md. libitum.
They received Purina laboratory chow and water
Balb/c mice were used in a few early experiments. Most
experimental groups comprised 12-15 mice. Tumor. The Madison lung carcinoma (M109), derived from a spontaneous neoplasm in a Balb/c mouse and kindly supplied by Dr. Ruth Geran of the Division of Cancer Treatment (DCT), NCI, has been maintained in o u r laboratory by serial passage in Balb/c mice. It was suspended in Roswell Park Memorial Institute (RPMI) 1640 medium and inoculated subcutaneously (s.c.) in the right inguinal region of the mice. Immunoadjuvants and Drugs. Pyran copolymer (NSC 46015), a copolymer of divinyl ether and maleic anhydride, was obtained from Dr. David Breslow of the Hercules Research Center, Wilmington, Del. Glucan, a B-1,3-polyglucose isolated from Saccharomyces cerevisiae cell walls, was donated by Dr. N.R. Di Luzio of Tulane University School of Medicine, New Orleans, La. Levamisole was obtained from Janssen Pharmaceutica, Beerse, Belgium, partially purified mouse fibroblast interferon (6) from Dr. K. Paucker, Medical College o f Pennsylvania, Philadelphia, Pa., and poly(1)poly(c) from Miles Laboratories, Elkhart, Indiana. Mycobacterium bovis, Pasteur strain (BCG) and lyophilized Corynebacterium parvum (Propionibacterium acnes) were kindly provided by Dr. S.D. Chaparas, Bureau of Biologics, FDA, Bethesda, Md. Bru-Pel, the ether extraction residue of Brucella abortus 456 ( 7 ) , was donated by D r . J . S . Youngner, University of Pittsburgh School of Medicine, Pittsburgh, Pa. All immunoadjuvants were dissolved o r suspended in sterile Dulbecco's phosphate-buffered saline (PBS), except for pyran copolymer, which was dissolved by adjusting a suspension
MACROPHAGE ACTIVATION
221
in a 0.85% NaCl to pH 7.2 with 0.1 N NaOH, and injected intra-
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peritoneally (i.p.) in a volume of 0.01 ml/g of body weight. Untreated control mice received 0.2 ml o f PBS.
I-(2-Chloroethy1)-
3-(trans-4-methylcyclohexyl)-l-nitrosourea (MeCCNU; NSC 95441) was obtained through the Drug Synthesis and Chemistry Branch, DCT,
NCI and suspended in 0.3% aqueous hydroxypropylcellulose. Lysozyme Assay. Mice were exsanguinated. The blood was allowed to clot for 0.5 hr at room temperature, then for 3-4 hr at 3', and centrifuged for 15 min at 1500 rpm. The serum was used immediately or kept frozen at -25'.
Repeated freezing and
thawing was avoided. The turbidometric assay was similar to that of Gordon, Todd and Cohn (4). Micrococcus lysodeikticus (dried cells) and the hen egg white lysozyme standard (muramidase; EC 3.2.1.17), recrystallized 3x, dialyzed and lyophilized, were bought from Sigma Chemical Co., St. Louis, Mo. suspension (0.5 mg/ml) of
M.
To 1 ml of a lysodeikticus in 0.05 M KH2P04 -
Na2HP04 pH 6 . 2 , (buffer A) was added with mixing 1 ml of serum diluted with buffer A containing 0.1 M NaCl (buffer B), or lysozyme (0.4
-
4.0 pg/ml) dissolved in buffer B.
The decrease in
absorbance at 540 nm was measured immediately for 4-5 min in a Gilford 240 spectrophotometer. Assays were performed with 5 dilutions of each serum and 8 to 10 concentrations of lysozyme standard. Initial decreases in absorbance were computed by least-square regression; they were a linear function of lysozyme concentration, again computed by regression analysis. Serum lysozyme concentratiomwere expressed in equivalents of hen egg lysozyme. B-Glucuronidase. The assay, a modification of those of Plaice (8) and Fishman (9), was performed, as described recently (lo), with Sigma Chemical Co. reagents by incubating 0.2 ml of serum (duplicates) with 3 mM phenolphthalein B,D-glucuronide i n 0.16 M sodium acetate buffer, pH 4.5, in a total volume of 1 ml for 16 hr at 37O, followed by heating for 5 min at looo, adding 5 ml of 0.1 M 2-amino-2-methyl-1-propanol
buffer, pH 11, centrifuging at
SCHRECKER, SCHULTZ, AND CHIRIGOS
222
35,000 x 8,
and measuring phenolphthalein absorbance at 550 nm.
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Substrate was added after the incubation for the blank determinations. N-Acetyl-B,D-glucosaminidase.
Chemical Co.
Reagents were obtained from Sigma
The method was similar to that of Brandt et al. (11).
Serum (25 ~ 1 )(triplicates) was incubated with 2 mM p-nitrophenylN-acetyl-B,D-glucosaminide in 0.1 M sodium citrate buffer, pH 4.3,
in a total volume of 1 ml for 1 hr at 37'.
The mixture was cooled
in ice and, after addition of 1 ml of 10% trichloroacetic acid, centrifuged at 1000 x g for 10 min. The supernatant solution (1.5 ml) was mixed with an equal volume of 2 M 2-amino-2-methyl1,3-propanediol, and p-nitrophenol absorbance measured at 420 nm after 10 min. A small blank absorbance (0.85% NaCl instead of serum) was subtracted, and concentrations were calculated from a p-nitrophenol standard, in which all ingredients (except serum and substrate) were present in the proportions of the assay and which gave a linear relationship between absorbance
(E
= 15,000) and
concentration. RESULTS
Table 1 shows that lysozyme, 6-glucuronidase and N-acetyl-B,Dglucosaminidase levels were higher in the serum of mice bearing the Madison lung carcinoma than in normal controls, in agreement with findings (12) that mobilization of macrophages i n tumorbearing animals produced increased serum lysozyme levels. Pyran copolymer, an immunoadjuvant shown to activate macrophages (13), led to an increase in serum lysozyme in both the normal and tumorbearing mice; lesser increases were observed with the two lysosomal acid hydrolases. In most of our experiments, pyran copolymer was injected at a dose of 25 mg/kg 6-8 days after tumor inoculation, and the blood collected another 6 days later because previous studies had shown that this was the most effective schedule for macrophage activation and therapeutic effect (14, 15). Indeed, in preliminary experiments, it was found that elevation of serum lysozyme reached a peak 6 days after treatment.
223
MACROPHAGE ACTIVATION
TABLE 1
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Lysosomal Enzymes in Serum of Mice Treated with Pyran Copolymer Treatment of Mice
Lysozyme (pg/ml ? S.E.aJ
No tumor control
4.4 k 0.2
32.3 f 0 . 1
4.17 ? 0 . 0 2
No tumor pyran
6.8 5 0 . 3
35.7
0.2
5.94 c 0 . 0 1
M109 control
5.0
0.3
38.0 f 0 . 6
5.49
M109 pyran
7.7 ? 0.3
39.7 2 0 . 1
6.11 ? 0.02
*
6-Glucuronidase (nmoles/ml/hr)
?
N-Acetyl-B,Dglucosaminidase (pmoles/ml/hr)
L 0.02
CDFl mice were inoculated with M109 tumor ( l o 6 cells) on day 0 o r received sham inoculations with RPMI 1640 medium. They were injected i.p. with 0.2 ml of PBS (controls) o r with 25 mg/kg of pyran copolymer on day 8, and were bled on day 14. a Standard error.
Table 2 indicates that the effect of pyran copolymer varied In 14 different
little between the doses of 1 2 . 5 and 50 mg/kg.
experiments, carried out under the conditions described, lysozyme levels varied between 4 . 3 and 6 . 3 (average 5 . 3
?
0.2 S.E.) in
untreated tumor-bearing mice, and between 6 . 1 and 1 1 . 5 (av. 8.8 2 0 . 5 ) in those treated i.p. with 25 mg/kg of pyran copolymer. Increases in lysozyme levels after treatment ranged from 30 to 115%, with an average increase of 67 f 8%. Multiple treatments with pyran copolymer were found to be no more effective for macrophage activation than a single injection ( 1 4 , l S ) . This was also true f o r the effect on serum lysozyme, as indicated by an experiment (Table 3 ) , which also showed that levamisole, which fails to activate macrophages significantly ( 1 , 1 6 ) , had little effect on serum lysozyme levels and on the
survival of tumor-bearing mice. The effect o f pyran copolymer on serum levels of lysosomal enzymes was compared with that of several other immunoadjuvants known to activate macrophages. The results are summarized in
224
SCHRECKER, SCHULTZ, AND CHIRIGOS
TABLE 2
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Serum Levels o f Lysozyme a s a Function o f Pyran Copolymer Dose Pyran Copolymer (mg/kgl
% of Control
Lysozyme (ug/ml) 5 . 2 f 0.2 a
0
100
12.5
10.5 f 0 . 7
202
25
10.9 ? 0.6
210
50
10.3
0.5
198
?
Balb/c mice were i n o c u l a t e d w i t h M109 tumor (2 x lo5 c e l l s ) on day 0 , i n j e c t e d i . p . w i t h pyran copolymer on day 6 , and b l e d on day 1 2 . a Standard e r r o r .
TABLE 3 Serum Lysozyme and S u r v i v a l Times o f Tumor-bearing Mice T r e a t e d w i t h Pyran Copolymer o r Levamisole Drug
Dose (mg/kg)
Control Pyran Levami s o 1e
25 5
Days o f Treatment
Day of Bleeding
6 6,13,20
12 26
6 6,13,20
12 26
1 0 . 1 & 0.2 9 . 3 f 0.2
37.5
6 6,13,20
12 26
6.7 f 0.3 6.9 f 0.1
30.5
Lysozyme (pg/ml) 6.2 ? 0 . Z a 5.9 f 0 . 1
MST (Days)
30
6 CDFl mice, i n o c u l a t e d w i t h M109 tumor (10 c e l l s ) on day 0, were i n j e c t e d i . p . on day 6 o r on days 6, 13, and 20, and bled. 6 days a f t e r t h e l a s t t r e a t m e n t . C o n t r o l mice r e c e i v e d 0.2 m l of PBS on t h e days of t r e a t m e n t . A d d i t i o n a l mice (10 p e r group) were i n j e c t e d on days 6 , 13, and 20, and k e p t u n t i l t h e y d i e d , i n o r d e r t o d e t e r m i n e median s u r v i v a l t i m e (MST). a Standard e r r o r .
225
MACROPHAGE ACTIVATION
Tables 4 and 5.
Interferon (6), poly(1) -poly(C), which induces
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interferon (6,16), and Corynebacterium parvum (Propionibacterium acnes) (1) had only minor effects. The effects of BCG (1,16) and of Bru-Pel, an ether extraction residue from Brucella abortus ( 7 ) , were comparable to those of pyran copolymer. The increase in Bglucuronidase serum levels was in general lesser than that of lysozyme, which was to be expected because cel s other than macrophages and neutrophils produce B-glucuronidase
The elevation of
N-acetyl-B,D-glucosaminidase was comparable to that of lysozyme,
but less reproducible.
TABLE 4 Effect of Immunoadjuvants on Serum Lysozyme in Tumor-bearing Mice Drug
PBS control Pyran Bru-Pel
Lysozyme (ug/ml)
Dose
0.2 ml
4.4
25 mg/kg 100 mg/kg
BCG
106 organisms
Interferon
4 1 0 units
POlY (1) .POlY(C)
10 mg/kg
?
% of Contr o 1
0.2 a
7.0 I 0.2 9.3 I 0.3 7.5 0.2 4.8 ? 0.2 5.4 ? 0.2
*
100 160 210 170
110 120
CDFl mice were inoculated with M109 tumor (2 x lo5 cells) on day 0, injected i.p. with PBS or the drugs on day 6, and bled on day 12. a Standard error.
Glucan, a B-1,3-polyglucose isolated from Saccharomyces cerevisiae, has been found to be a potent macrophage activator and to produce 7-fold increased lysozyme levels in rats after repeated intravenous (i.v.) administration
(17).
We failed, perhaps
because of the species difference, to reproduce this effect in
25 mg/kg
0 . 2 ml
Dose
1 mg/mouse 120
*
5.8
0.2
28.3 f 0.2
110
140
170
8.0 f 0.3
35.1 f 0.3
140
34.5 f 0.1
*
200
0.4
*
9.5 f 0 . 3
9.7
100
130
0.1
C
25.5 f 0 . 3
3.67
?
0.01
4.38 f 0.02
7.17 f 0 . 0 1
7.70 f 0.04
3.34 f 0.08
110
130
215
230
100
% of N-Acetyl-B,D% of Control glucosaminidase Control (pmo1es/ml/hr)
33.4 f 0 . 3
6-Glucuronidase (nmoles/ml/hr)
100
a
% of
Control
200
4.8
Lysozyme (pg/ml)
CDFl mice were inoculated with M109 tumor (106 cells) on day 0, treated i.p. with PBS or the drugs on day 8, and b l e d on day 1 4 . a Standard error.
C. parvum -
Bru-Pel 100 mg/kg 6 BCG 10 organisms
Pyran
PBS control
Drug
Lysosomal Hydrolases in Serum of Tumor-bearing Mice Treated with Immunoadjuvants
TABLE 5
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N
H
E0
c)
m
v)
227
MACROPHAGE ACTIVATION
mice bearing the M109 tumor (Tables 6,7),
although we used the
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same dose and time schedule that was reported (17).
By comparison,
pyran copolymer was effective by both the i.p. (Table 6 ) and i.v. (Table 7) routes in raising serum levels of lysosomal enzymes. Again, multiple treatments had no advantage over a single injection TABLE 6 Lysosomal Hydrolases i n Serum of Tumor-bearing Mice Treated Intraperitoneally with Pyran Copolymer or Glucan Drug
Day of Sacrifice
Lysozyme (ug/ml ? S.E.) 0.1
8-Glucuronidase N-Acetyl-B,D(nmoles/ml/hr f glucosaminidase S.E.) ( pmo 1es/ml/ hr)
N.D.~ N.D. N.D.
N.D. N.D. N.D.
PBS control
11
4.9
Pyran
11
6.6 f 0.3
Glucan
11
4.8
PBS control
12
5.7 f 0.3
26.4
?
1.0
5.88
Pyran
12
10.4 f 0.3
32.6
?
0.4
7.27 f 0.01
Glucan
12
30.4
?
0.1
5 . 2 7 t 0.05
7.1
?
jI
f
0.3
0.1
?
0.06
CDF1 mice, inoculated with M109 tumor (2 x lo5 cells) on day . 0 , were injected intraperitoneally on days 6, 8 and 10 with PBS ( 0 . 2 ml), pyran copolymer (12.5 mg/kg), or glucan (10 mg/kg). Hal.f the mice were bled on day 11, the other half on day 12. a Not determined.
Pyran copolymer has been found to potentiate the therapeutic effect of MeCCNU against the Lewis lung carcinoma (18). However, as shown in Table 8, macrophage activation by pyran, as reflected in serum lysozyme levels, was lessened when MeCCNU was injected prior to the copolymer. Gallily et al. (19) have shown that treatment of mice with MER, the methanol extraction residue of BCG, activated their
peritoneal macrophages and produced elevated levels of lysozyme
228
SCHRECKER, SCHULTZ, AND CHIRIGOS
TABLE 7
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Lysosomal Hydrolases in Serum of Tumor-bearing Mice Treated Intravenously with Pyran Copolymer o r Glucan Drug
Lysozyme (ug/ml f S.E.)
B-Glucuronidase (nmoles/ml/hr f S.E.)
PBS control
5.2 f 0.1
29.1
1.2
f
N-Acetyl-B,Dglucosaminidase (umo1es/ml/hr) 4.52 f 0.02
Pyran
9.7 5 0.2
54.6 f 0.5
7.81
?
0.01
Glucan
6.1 f 0.2
26.9
f
0.3
4.83
?
0.01
PBS control
4.5 f 0.2
27.0
f
0.1
?
0.01
28.5 f 0.4
3.63 5.71
f
0.02
24.8
3.79
?
0.01
Pyran
7.6 f 0.4
Glucan
5.8
f
0.2
?1
0.1
CDFl mice, inoculated with M109 tumor (106 cells) on day 0, were injected intravenously on days 8, 11 and 13 with PBS (0.2 ml), pyran copolymer (12.5 mg/kg), or glucan (10 mg/kg), then bled on day 14. The Table lists the results of 2 different experiments. TABLE 8 Serum Lysozyme Levels of Tumor-bearing Mice after Combination Treatment with MeCCNU and Pyran Copolymer Treatment on Day 4
Treatment on Day 9
Day o f Sacrifice
Lysozyme (;g/mi f
% of
Control
S.E.)
MeCCNU
MeCCNU
MeCCNU
-
8
5.5 f 0.1
100
8
6.1
0.2
110
-
15
5.5 f 0.4
100
15
6.3 f 0.2
115
Pyran
15
11.9 f 0.4
200
Pyran
15
8.2 f 0.4
150
?1
CDFl mice were inoculated with M109 tumor (2 x 105 cells) on day 0, injected i’ntraperitoneally with 1-(2-~hloroethyl)-3-(trans4-methylcyclohexyl)-l-nitrosourea (MeCCNU) (30 mg/kg) and pyran copolymer (25 mg/kg) on the days listed, and bled on the day of sacrifice. Untreated controls received injections of PBS (0.2 ml) on days 4 and 9.
MACROPHAGE ACTIVATION
229
and 6-glucuronidase in these cells.
In an attempt to detem.ine whether pyran copolymer had a similar effect, we injected CDFl Immunopharmacology and Immunotoxicology Downloaded from informahealthcare.com by McMaster University on 03/06/15 For personal use only.
mice with 25 mg/kg of pyran i.p. and isolated the macrophages 6 days later by the method described previously (1,15,16). number of cells (2 x
lo7
The same
from 30 mice) was obtained from both the
treated and control animals.
The cells were suspended in buffer B
and disrupted by freezing and thawing 5 times. The same amount of lysozyme (2.6
2 0.1
7
vg/lO
cells) was found in the macrophages
from the treated and control animals. An attempt to determine whether there was increased secretion of lysozyme by macrophages cultured in RPMI-1640 medium (10% fetal calf serum) f o r 3 days following isolation after pyran treatment was not conclusive because the number of macrophages in the culture medium decreased during the incubation in the case of the untreated mice. DISCUSSION The present data show that serum levels of lysosomal enzymes, and especially of lysozyme, can indeed serve as indices of macrophage activation by immunoadjuvants and be correlated with their antitumor effect.
As lysozyme and N-acetyl-B,D-glucosaminidase
are also produced by neutrophils (20), which are not subject to activation, and 6-glucuronidase by additional cell types, the increases in serum levels probably represent only a fraction of the increase in synthesis by macrophages. Moreover, it is likely that the degree of macrophage activation by immunoadjuvants and the changes in enzyme synthesis depend on the different location of the several populations of these cells (circulating monocytes, peritoneal, alveolar, histiocytes, etc.).
The findings do suggest
that measuring serum levels of lysosomal hydrolases can serve as a test for the efficacy of immunotherapy. ACKNOWLEDGMENTS We wish to thank Jake R. Fullen for skillfully inoculating,
injecting, and bleeding the animals. REFERENCES 1.
Schultz, R.M., Papamatheakis, J.D., Stylos, W.A., and Chirigos, M.A., Augmentation of Specific Macrophage-Mediated Cytotoxicity: Correlation with Agents Which Enhance Antitumor Resistance, Cell. Immunol., 25:309, 1976.
230
SCHRECKER, SCHULTZ, AND CHIRIGOS
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2. Fantalone, R., and Page, R.C., Enzyme Production and Secretion by Lymphokine-Activated Macrophages, RES, J. Reticuloendothel. SOC., 21:343, 1977. 3. Heise, E.R., and Myrvik, Q.N., Secretion of Lysozyme by Rabbit Alveolar Macrophages In Vitro, RES, J. Reticuloendothel. SOC., 4:510, 1967. 4. Gordon, S., Todd, J., and Cohn, Z.A., In Vitro Synthesis and Secretion of Lysozyme by Mononuclear Phagocytes, J. E x p . Med., 139:1228, 1974. 5. Unanue, E.R., Secretory Function of Mononuclear Phagocytes: A Review, Am. J. Pathol., 83:396, 1976. 6. Schultz, R.M., Papamatheakis, J.D., and Chirigos, M.A., Interferon: An Inducer o f Macrophage Activation by Polyanions, Science, 197:674, 1977. 7. Youngner, J.S., Keleti, G., and Feingold, D.S., Antiviral Activity of an Ether-Extracted Nonviable Preparation of Brucella abortus, Infect, Immun., 10:1202, 1974. 8.
Plaice, C.H.J., A note on the determination of serum betaglucuronidase activity, J. Clin. Pathol., 14:661, 1961.
9. Fishman, W.H., Kato, K., Anstiss, C.L., and Green S.,
Human Serum 6-Glucuronidase: Its Measurement and Some of Its Properties, Clin. Chim. Acta, 15:435, 1967. 10. Schrecker, A.W., and Chirigos, M.A., Improved Determination o f 6-Glucuronidase in Serum, Clin. Chim. Acta, 88:413, 1978. 11. Brandt, E.J., Elliott, R.W., and Swank, R.T., Defective Lysosomal Enzyme Secretion in Kidneys of Chediak-Higashi (Beige) Mice, J. Cell Biol., 67:774, 1975. 12.
Currie, G.A., and Eccles, S.A., Serum Lysozyme as a Marker of Host Resistance. I. Production by Macrophages Resident in Rat Sarcomata, Br. J. Cancer, 33:51, 1976.
13.
Schultz, R.M., Papamatheakis, J.D., and Chirigos, M.A., Direct Activation In Vitro of Mouse Peritoneal Macrophages by Pyran Copolymer (NSC 46015), Cell. Immunol., 29:403, 1977.
14. Papamatheakis, J.D., Chirigos, M.A., and Schultz, R.M., Effect of Dose, Route, and Timing o f Pyran Copolymer Therapy Against the Madison Lung Carcinoma, in Immune-Modulation and Control of Neoplasia by Adjuvant Therapy, edited by M.A. Chirigos, p 427, Raven Press, New York, 1978.
MACROPHAGE ACTIVATION
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15.
231
Schultz, R.M., Papamatheakis, J.D., Luetzeler, J., Ruiz, P., and Chirigos, M.A., Macrophage Involvement in the Protective Effect of Pyran Copolymer Against the Madison Lung Carcinoma (M109), Cancer Res., 37:358, 1977.
16. Schultz, R.M., Papamatheakis, J.D., Luetzeler, J., and Chirigos, M.A., Association of Macrophage Activation with Antitumor Activity by Synthetic and Biological Agents, Cancer Res., 37:3338, 1977. 17. Kokoshis, P.L., Williams, D.L., Cook, J.A., and Di Luzio, N.R., Increased Resistance to Staphylococcus aureus Infection and Enhancement in Serum Lysozyme Activity by Glucan, Science, 199:1340, 1978. 18.
Mohr, S.J., Chirigos, M.A., Fuhrman, F.S., and Pryor, J.W., Pyran Copolymer as an Effective Adjuvant to Chemotherapy Against a Murine Leukemia and Solid Tumor, Cancer Res., 35: 3750, 1975.
19. Gallily, R., Yagel, S., and Weiss, D.W., Potentiated Lysosomal Enzyme, Bacteriostatic and Bactericidal Activities of Peritoneal Macrophages of Mice Treated with the MER Fraction of Tubercle Bacilli, Adv. E x p . Med. Biol., 73A:351, 1976. 20.
Hansen, N.E., Plasma Lysozyme - A Measure of Neutrophil Turnover. An Analytical Review, Ser. Haematol., 7:1, 1974.
Received Accepted
-- July 2 4 , -- November
1978 3, 1 9 7 8
