- Email: [email protected]
The in vitro bactericidal activity of peritoneal and spleen cells from Listeria-resistant and -susceptible mouse strains
CELLULAR
IMMUNOLOGY
99, 160-169 (1986)
The in Vitro Bactericidal Activity of Peritoneal and Spleen Cells from Listeria-Resistant and -Susceptible Mouse Strains P. R. WOOD,* V. SPANIDIS,K. FRANGOS, AND C. CHEERS Department of Microbiology, Melbourne University, Parkville, Victoria 3052, Australia Received October 1, 1985; accepted November 21, 1985 Two days after Wisteria-resistant (Lra) C57BL/lOmicewereinfectedintraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (L?) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, ofthe enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the numberof cellsor cellular composition of theexudates. No difference in thein vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infectedperitoneal cavity of C57BL/lO mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/lO mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/lO spleen already contains high numbers of these efficient monocytes. 0 1986 Academic Pw, inc.
INTRODUCTION
Critical differences in the genetic resistance of mice to Listeria monocytogenesare expressed before the development of the T-cell-dependent specific immune response (l-3). Events during this early phase have been studied comparing susceptible BALB/c and resistant C57BL/lO (1) or susceptible A/J and resistant C57BL/6 mice (3). The initial localization of Listeriu in the liver and spleen is identical in the C57BL/ 10 and BALB/c mice (1). However, 24 hr later lo-fold more Listeria are found in the livers of BALB/c mice compared with C57BL/lO. Similar early differences are observed between A/J and C57BL/6 mice (3). Sadarangani et al. (4) found that a highly radiosensitive bone marrow-derived cell was responsible for the genetic advantage of resistant C57BL/6 mice over susceptible A/J mice in the first 48 hr of infection. Stevenson et al. (5) provided evidence that the cellular basis of genetically determined resistance to Listeria in C57BL/6 mice compared with A/J was a more rapid mobilization of monocytes to the foci of infection. This early inflammatory response has now been shown to be controlled by the Hc locus on chromosome 2 which determines the level of the Cs component of complement (6). However, they believed that another gene and another mechanism also contributed. The effects of the Listeriu resistance (Lr) gene first described by Cheers and McKenzie (7) cannot be accounted for by differences in the level of C5 as both the Listeria * Present address: CSIRO, Division of Animal Health, Parkville, Victoria 3052, Australia. 160 0008-8749186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
BACTERICIDAL
ACTIVITY
OF
SUSCEPTIBLE
AND
RESISTANT
MICE
161
resistant C57BL/lO and susceptible BALB/c and CBA are all Cs+ strains. We had previously shown (8) that macrophages from Wisteria-infected resistant (C57BL/ 10) mice had superior tumouricidal activity to macrophages from susceptible (BALB/c) mice. In this paper we examine whether differences in the bactericidal activity of macrophages could account for the Lr gene effects described by Cheers et al. (1) and whether there are any differences in the chemotactic responsiveness of cells from these strains. The bactericidal assay system used was based on a technique developed by Davies (9) which measures the release of radioactively labeled DNA from Listeria as an indicator of the bactericidal activity of cells. Chemotaxis was measured in the classical Boyden chamber ( 10). MATERIALS
AND
METHODS
Mice. Inbred C57BL/lO, BALB/c, and CBA mice were maintained under conventional but disease-free conditions by strict brother-sister mating in the Microbiology Animal Breeding Unit, University of Melbourne. Mice were used when 5-8 weeks old. Bacteria. Listeria monucytogenes,obtained from R. V. Blanden (Australian National University), was subcultured on horse blood agar (HBA) weekly and renewed from freeze-dried stock after 25 passages. Infection of mice. Twenty-four-hour actively growing cultures were washed from tryticase soy agar plates with 1% serum in distilled water. The inoculum was standardized turbidimetrically using a calorimeter (EEL International Ltd). The bacteria were injected in 0.2 ml and the dose checked retrospectively using Miles and Misra ( 11) viable counts. Preparation of peritoneal cell suspension.Mice were killed with fluothane and the peritoneal cavities washed out with 5.0 ml RPM1 1640 containing 5% fetal calf serum (FCS) and 10 units/ml heparin. Pooled cell suspensions harvested from groups of 5 mice were centrifuged, washed once with 10 ml medium, and finally suspended in 5 ml of RPM1 only. Cell yields were determined and assessed for viability by staining with 0.2% eosin. Preparation of spleen cell suspensions.Spleens were cut into fragments and gently pushed through fine stainless steel sieves into cold RPM1 medium supplemented with 5% FCS. After clumps had settled out cells were centrifuged and the pellets resuspended in 10 ml Tris-buffered ammonium chloride to lyse red cells (12). The cell suspensions were incubated at 37°C for 15 min, then washed twice with RPM1 medium. Cell pellets were finally resuspended in 10 ml RPM1 and adjusted to 2 X lo7 viable cells/ml. All assays employed cells pooled from groups of 3-4 mice. Labeling of bacteria. A lo-ml tryticase soy broth (TSB) was inoculated with lo7 glycerol preserved Listeria (lo9 organisms/ml) stored at -20°C. The broth was incubated at 25 “C for 17 hr in the presence of 10 &i/ml tritiated thymidine (Amersham). Labeled Listeria were then washed free of unincorporated label by centrifuging at 4000g for 15 min at 4°C. Following three washes the bacteria were finally resuspended in 10 ml RPMI. The number of labeled organisms was checked retrospectively by Miles and Misra (11) viable counts. These conditions were determined to yield approximately 1O9labeled organisms/ml.
162
WOOD
ET
AL.
Bactericidal assay. The assay system used was developed from that described by Davies (9). Peritoneal cells (4 X 106/ml) or spleen cells (2 X 107/ml) in 0.5 ml RPM1 medium containing 50 mg/ml cold thymidine were distributed into 1.5-ml Eppendorf tubes along with 0.5 ml of labeled Listeria (approximately 8 X 107/ml). Tubes were incubated at 37°C in a water bath for various periods oftime then microfuged ( lO,OOOg, 7 min) and 0.8 ml of supernatant sampled into 8 ml of scintillant (EP Ready Solv, Beckman) and counted in a liquid scintillation spectrometer. Results are expressed as counts per minute (cpm) f standard deviation averaged from triplicate tubes. The spontaneous release of label from bacteria incubated without cells is shown in the legend of each figure. Day-to-day variation in the cpm released was observed according to the efficiency of bacterial labeling and the exact number of bacteria added to the test so comparisons can only be made within experiments. Phagocytosis and d@rential cell counts. To determine the amount of phagocytosis of bacteria, Eppendorf tubes were set up as described above and incubated for 2 hr at 37°C. Bacteria not associated with cells were then removed by underlaying the cell suspensions with 0.5 ml FCS and centrifuging at 900g for 10 min at 4°C. This procedure was repeated and the cell pellets finally resuspended in 1 ml RPMI. A sample of the cell suspension was taken and total radioactive counts associated with cells determined. Approximately lo- 15% of the bacteria added were cell associated after 2 hr of incubation. Smears of these cell suspensions were also prepared using a cytocentrifuge, then stained with Diff-Quik stain (AHS, Australia). Differential counts were determined by counting a minimum of 200 cells. Boyden chemotaxis assay. Peritoneal cells (4 X lo6 in 200 ~1 Hank’s balanced salt solution) were placed in the upper well of a Boyden chamber (Nuclepore, Pleasanton, Calif.) and allowed to migrate across an 8-ppore-size cellulose acetate/nitrate membrane (Millipore, San Francisco) toward hydrolyzed casein at 37°C for 5 hr. The filters were fixed in alcohol, stained with Mayer’s hematoxylin, and dehydrated by immersion in absolute alcohol and then xylol, before being mounted in DPX between a microscope slide and a coverslip. The results were quantitated by the leading front method (13) in which the maximum distance the cells have migrated into the filter is measured using a micrometer guage on the fine focus knob of the microscope. For each filter 20 fields were measured, 10 from left to right and 10 from top to bottom of the filter. For each experimental group triplicate chambers were tested. The fields for each filter were averaged and a mean and standard deviation expressed for each triplicate group. RESULTS The bactericidal activity of resident peritoneal cells from uninfected BALB/c (susceptible) and C57BL/ 10 (resistant) mice was compared in at least 10 separate experiments and no consistent differences were detected in their ability to kill Listeria (data not shown). However, following infection with lo4 Listeria differences were seen in the degree of enhanced bactericidal activity of peritoneal cells from resistant and susceptible mice. Peritoneal cells from C57BL/lO mice infected i.p. with Listeria 2 days previously showed greatly increased bactericidal activity when compared with cells from i.p.-infected BALB/c mice or uninfected control animals (Fig. 1). This difference in the degree of enhanced bactericidal activity of peritoneal cells from C57BL/ 10 and BALB/c mice was not seen following i.v. infection with the same dose of Listeria (Fig.
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
163
25 SO min
190
min
20 -
IV
controt
IP
Cell
IV
Control
Type
FIG. 1. Bactericidal activity of peritoneal cells from C57BL/lO (m) and BALB/c (0) mice. Mice were uninfected or infected i.p. or iv. with lo4 Listeria 2 days prior to assay.Bactericidal activity was measured at 90 and 180 min after incubation of bacteria with cells. Spontaneous release of label at 90 min (442 f 14) and 180 min (519 + 40).
1). When a second Listeriu susceptible strain of mice (CBA) was also examined, again only peritoneal cells from i.p. infected resistant mice showed increased bactericidal activity 2 days after infection (Fig. 2). Irradiation of mice immediately prior to infection with Listeria completely ablates the superior resistance of C57BL/lO mice to Listeria (4, 14). Irradiation of mice 2 hr before i.p. infection with lo4 Listeria also completely abolished the enhanced bactericidal response of peritoneal cells from 2-day infected C57BL/lO mice (Fig. 3A). To ensure that irradiation was not acting on the bactericidal mechanism of peritoneal cells itself, a control experiment was performed in which 2-day infected mice were irradiated just before harvesting their peritoneal cells. This treatment, in contrast to irradiation prior to infection, did not affect the enhanced bactericidal activity of peritoneal cells from C57BL/lO mice (Fig. 3B). These strain differences in bactericidal activity were analyzed by considering whether differences in the degree of phagocytosis of Listeriu could account for the advantage of C5 7BL/ 10 peritoneal cells over BALB/c cells. The degree of phagocytosis of bacteria was determined by counting the number of labeled Listeria associated with macrophages after removal of unassociated organisms by sequential centrifugation through FCS gradients. No significant differences were detected in the number of bacteria phagocytosed by cells from uninfected or infected mice of either strain (Table 1). This
164
WOOD ET AL. 20
I, Infected 1 controt
Control
2
I ,
Infected
3 Time
(hrs)
FIG. 2. BactericidaJ activity of peritoneal cells from uninfected and List&a-infected C57BL/lO (kB),BALB/ c (0) and CBA (m) mice. Mice were infectd i.p. with lo4 Listeria 2 days prior to assay.Bactericidal activity was measured at 2 and 3 hr after incubation of bacteria with cells. Spontaneous release of label at 2 hr (3 10 f 58) and 3 hr (346 f 22).
finding was also confirmed by direct microscopic counting of the numbers of bacteria associated with individual cells (data not shown). We next determined whether the enhanced bactericidal response of C57BL/ 10 mice was the result of a specific response to Listeria or a more generalized response to any 40
80
-A
30
7 0 5 z
20
s
had.
had.
FIG. 3. Effect of irradiation on the bactericidal activity of peritoneal cells from C57BL/lO (IB) and BALB/c (0) mice. Control animals were uninfected. The other groups of mice were infected i.p. with IO4 Listeria 2 days prior to assay. (A) Half of the infected mice received 800 rads irradiation 2 hr prior to infection with Lisferia. (B) Half of the infected mice received 800 rads irradiation just before harvesting their peritoneal cells. Bactericidal activity was measured 3 hr after incubation ofbacteria with cells. Spontaneous release of label in (A) (469 f 40) and (B) (675 + 63).
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
165
TABLE 1 Comparison of the Ability of BALB/c and C57BL/IO Peritoneal Cells from 2-Day Infected Mice to Phagocytose Listeriu Cell type”
Total cell associated counts after a double wash*
Day 2 C57BL/lO BALB/c
2803 + 397 3458 + 418
Control C57BL/lO BALB/c
3257 I!Z209 3357 + 490
a Cells were harvested from normal and Wisteria-infected C57BL/lO and BALB/c mice. Mice were injected i.p. with lo4 Listerin48hr prior to the experiment. * Total counts in the resuspended pellets following an FCS underlay and centrifugation step repeated twice.
i.p. intrusion. Mice were injected with 1 ml of sterile irritant (10% proteose peptone) and 2 days later their peritoneal cells were assayed for bactericidal activity. Peritoneal cells from C57BL/lO mice displayed far greater bactericidal activity compared to BALB/c cells following injections of proteose peptone (Fig. 4). Chemotactic activity of peritoneal cells from the two strains of mice was compared by examining migration in vitro across a membrane toward a chemotactic stimulus 60
50
40 T 0 = s
30
t 20
10
0
Proteose Peptons
FIG. 4. Bactericidal activity of peritoneal cells from normal and protease peptone injected C57BL/lO (BJ) and BALB/c (0) mice. Mice. were injected i.p. with 1 ml of 10% protease peptone 48 hr prior to assay. Bactericidal activity was measured 3 hr after incubation of bacteria with cells. Spontaneous release of label (348 I? 29).
166
WOOD ET AL. TABLE 2 Comparison of the Chemotactic Response of Peritoneal Cells from Listeriu-Infected BALB/c and C57BL/lO Mice 24 hr after Infection Distance migrated (pm)” Strain
Noninfected
Infected
BALB/c C57BL/IO
31.1 f 18.3 24.3 f 4.2
103.6 k 18.5 115.7 f 2.3
a Distance migrated represents the mean + SD of triplicate filters.
(Table 2). No difference was seen between normal C57BL/lO and BALB/c mice. Chemotactic responsiveness was enhanced equally in the two strains 24 hr after infection. The fact that BALB/c mice were not deficient in their ability to attract cells into the peritoneal cavity was confirmed when cell numbers were examined. BALB/c mice yielded slightly higher numbers of peritoneal cells compared to C57BL/lO mice after i.p. infection with List&z (Table 3). There was also no consistent differences in the percentage of macrophages in populations of cells recovered from these two mouse strains (data not shown). When bactericidal activity of spleen cells was compared, there was a significant difference between the two mouse strains in the degree of bactericidal activity seen with spleen cells from uninfected animals in a 3-hr in vitro assay (Fig. 5). The degree of increased activity 2 days after intravenous infection was less than that described in the peritoneal cavity and occurred in both strains. During the course of this study we also examined the listericidal activity of neutrophils in the peritoneal cavity by inducing populations of cells enriched for neutrophils by injection of proteose peptone or Listeria 6 hr before harvesting. The bactericidal activity of these neutrophil enriched cell populations was however, not markedly different from that of control cell populations (Table 4). Smears of these cell populations were prepared and examined for the degree of phagocytosis by the individual cell types present. The vast majority of bacteria phagocytosed was found to be associated with macrophages (Table 4). Irradiation of mice prior to infection with Listeria was also found to increase the percentage of neutrophils in the peritoneal cavity from approximately 5 to between 50 and 80%, yet this treatment dramatically reduced the bactericidal activity of this cell population (Fig. 3A). Collectively these data suggest that TABLE 3 Total Number of Peritoneal Cells Recovered following Intraperitoneal Injection of Listerid Strain
Uninfected
C57BL/lO BALB/c
2.4 f 0.7 3.0 f 0.8
Listeria
infected
4.8 + 1.7 7.9 f 1.9
a Data expressed asmean number of cells per mouse.(X 106) + standard deviation of 13 separate experiments. In each experiment groups of 5 mice were infected i.p. with approximately lo4 Listeriu 2 days prior to harvest.
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
Control
’ Infected
AND RESISTANT
Control
167
MICE
I Infected
5
1
3 Time
(hrs)
FOG.5. Bactericidal activity of spleen cells from C57BL/lO (Q and BALB/c (Cl) mice. Mice were uninfected or infected i.v. with IO’ Listeria 2 days prior to assay.Bactericidal activity was measured at 1 and 3 hr after incubation of bacteria with cells. Spontaneous release of label at 1 hr (209 f 40) and 3 hr (236 f 6).
neutrophils contributed experiments.
only minimaly
to the bactericidal activity measured in these
DISCUSSION Peritoneal cells from intraperitoneally, but not intravenously, Listeria-infected C57BL/lO mice rapidly developed far greater bactericidal activity than the same number of cells from susceptible BALB/c and CBA mice. The induction of enhanced bactericidal activity of cells from C57BL/lO mice was sensitive to irradiation of the donor mice at the time of infection, as is the in vivo resistance to Listeria of these mice (4, 14), suggesting a role for recently divided cells in C57BL/lO and their unavailability in BALB/c mice. That expression of the enhanced bactericidal activity TABLE 4 Bactericidal and Phagocytic Activity of Neutrophil-enriched
Cell Populations”
No. of bacteriaphagocytosedper cellb Cell type
w of total population
Nil
1-5
5-10
>lO
Proteosepeptone
Macrophage Neutrophil
29 52
48 a4
15 12
6 3
31
Listeria
Macrophage Neutrophil
39 36
37 76
13 16
9 a
40 0
Control
Macrophage Neutrophil
75
33
13
15
39
-
-
-
-
Treatment
0
a Groups of 5 mice were injected i.p. with IO6 Lisferia or 1 ml proteosepeptone 6 hr prior not injected. b 200 cells were counted and the results expressed as a percentage.
1
CPM releasedC 953k
a7
1029 f 134 767 k
69
to assay.Control mice were
‘Counts per minute releasedinto supematant following a 2-hr incubation of cells with labeled Listeria.
168
WOOD
ET
AL.
was not radiosensitive was shown by the fact that irradiation of the donors just before harvesting the cells did not affect their activity. We found no evidence that differences between C57BL/lO and BALB/c mice in either the total number of peritoneal cells recovered, the percentage of macrophages in these populations or their ability to phagocytose Listeria would account for the enhanced bactericidal activity of cells from C57BL/lO mice. The finding that i.p. injection of proteose peptone also induced a higher degree of bactericidal activity in resistant mice compared to susceptible mice suggests that this phenomenon is not the result of a specific response to Listeria infection. However as with Listeria infection, the bactericidal differences observed in these mouse strains following injection of proteose peptone were not due to differences in the total numbers of macrophages recruited into their peritoneal cavities. Therefore the enhanced bactericidal activity of 2 day infected C57BL/ 10 compared with BALB/c cells is most likely due to selective recruitment to the site of infection and/or activation of recently divided bone marrow-derived monocytes/macrophages with enhanced bactericidal activity. C57BL/lO mice have twice as many monocyte precursors (macrophage colony forming cells) as BALB/c and these are rapidly depleted from the bone marrow following Listeria infection ( 15). These results may be compred with those of Gervais et al. (6), who found an enhanced inflammatory reponse in C57BL/lO (Lrx) mice compared with A/J(L?) mice 3 days post infection. This difference was genetically linked in back-cross and recombinant inbred mice with differences in Listeria resistance, the in vitro chemotactic response of peritoneal cells and the Hc gene which determines the level of the Cs component of complement. They believed that there was also a second gene acting to render the A/J mice susceptible. Czuprynski et al. (16) have confirmed that the inflammatory response of A/J mice is less efficient than that of C57BL/6 mice and have partially restored the response by supplying Cs. They found that while there were higher numbers of peritoneal cells in their i.p. infected resistant mice compared with infected susceptible mice, there was little difference in the bactericidal activity, cell for cell, between the strains. Since BALB/c and CBA mice are both Cs sufficient strains, the Hc gene cannot account for the difference in resistance we observe between them and C57BL/ 10 mice. Furthermore, they appear to differ from C57BL/lO mice, not in the numbers of cells recruited to the site of infection, but in the bactericidal efficiency of these cells. Indeed no difference was observed in the in vitro chemotactic response of the two strains. While higher numbers of cells were attracted to the peritoneal cavity of BALB/c mice following i.p. infection, this was possibly due to the attainment of higher bacterial numbers in this strain than in C57BL/lO. When spleen cell bactericidal activity was compared, resistant C57BL/lO showed greater efficiency than BALB/c, even before infection. Intravenous infection somewhat enhanced the activity of both strains, but the degree of enhancement was not as great as that seen in the peritoneal cavity of C57BL/lO mice infected i.p. It is possible that the spleen, which like the bone marrow is a hemopoietic organ, contains many less mature monocyte/macrophages which our studies in the peritoneal cavity suggest are strongly bactericidal. Spleens of uninfected C57BL/lO mice contain three times the number of colony forming cells (macrophage precursors) found in BALB/c mice ( 15). If a correspondingly high number of young, efficiently bactericidal monocytes exist
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
169
in the C57BL/lO spleens, this could account for the difference in innate bactericidal ability in the spleens of the two mouse strains. The finding that neutrophils played only a minor role in the phagocytosis and killing of Listeria in vitro disagrees with the findings of Czuprynski et al. (17), who reported that inflammatory peritoneal neutrophils killed Listeria in vitro even better than did inflammatory macrophages. These differences may be associated with the different bactericidal systems used, in particular the use of opsonins in their assay. Macrophages do not require antibody for the uptake and lysis of Listeria (18) and serum has been found to be toxic for Listeria ( 19). Therefore we excluded it from our assay system. However, we cannot discount the possibility that neutrophils have a greater requirement for opsonins that macrophages for the phagocytosis and lysis of Listeria. Finally the bactericidal assay used in this paper measures the actual lysis of bacteria by cells in contrast to other assays which quantitate the survival of bacteria following incubation with cells. The advantage of this system is that the problems of growth of extracellular bacteria are avoided thus making this assay far more sensitive. ACKNOWLEDGMENTS We thank Wayne Davies for his assistance in establishing the bactericidal assay.The work supported by the National Health and Medical Research Council of Australia.
REFERENCES 1. Cheers, C., McKenzie, I. F. C., Pavlov, H., Ward, C., and York, J., Infect. Zmmun. 19, 763, 1978. 2. Cheers, C., McKenzie, I. F. C., Mandel, T. E., and Chan, Y. Y., In “Genetic Control of Natural Resistance to Infection and Malignancy.” (E. Skamene, P. A. L., Kongshavn, and M. Landy, Eds.), p. 141. Academic Press, New York, 1980. 3. Skamene, E., and Kongshavn, P. A. L., Infect. Zmmun. 25, 345, 1979. 4. Sadarangani, C., Skamene, E., and Kongshavn, P. A. L., Infect. Zmmun. 28,381, 1980. 5. Stevenson, M. M., Kongshavn, P. A. L., and Skamene, E., J. Zmmunol. 127,402, 1981. 6. Gervais, F., Stevenson, M., and Skamene, E., J. Zmmunol. 132, 1, 1984. 7. Cheers, C., and McKenzie, I. F. C., Infect. Zmmun. 19, 755, 1978. 8. Wood, P., and Cheers, C., Immunology 54, 113, 1985. 9. Davies, W. A., J. Reticuloendoth. Sot. 32, 461, 1982. 10. Boyden, S., J. Exp. Med. 115,453, 1962. 11. Miles, A. A., and Misra, S. S., J. Hyg. 28, 732, 1938. 12. Boyle, W., Transplantation 6, 761, 1968. 13. Zigmond, S. H., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. 14. Cheers, C., and Macgeorge, J. In “NK Cells and Other Natural Effector Cells.” (R. B. Herberman, Ed.), p. 152 1. Academic Press, New York, 1982. 15. Young, A. M., and Cheers, C., Cell. Zmmunol. 97,227, 1986. 16. Czuprynski, C. J., Canons, B. P., Henson, P. M., and Campbell, P. A., Immunology 55, 5 11, 1985. 17. Czuprynski, C. J., Henson, P. M., and Campbell, P. A., J. Leuk. Biol. 35, 193, 1984. 18. Cooper, J. M., Johnson, R. B., and Rowley, D., Aust. J. Exp. Med. Sci. 61, 63, 1983. 19. Davies, W. A., J. Reticuloendoth. Sot. 34, 13 1, 1983.
IMMUNOLOGY
99, 160-169 (1986)
The in Vitro Bactericidal Activity of Peritoneal and Spleen Cells from Listeria-Resistant and -Susceptible Mouse Strains P. R. WOOD,* V. SPANIDIS,K. FRANGOS, AND C. CHEERS Department of Microbiology, Melbourne University, Parkville, Victoria 3052, Australia Received October 1, 1985; accepted November 21, 1985 Two days after Wisteria-resistant (Lra) C57BL/lOmicewereinfectedintraperitoneally with Listeria, their peritoneal macrophages demonstrated enhanced bactericidal activity beyond that seen in susceptible (L?) BALB/c or CBA mice. Intravenous infection had no effect on peritoneal cell activity. The induction, but not expression, ofthe enhanced activity was radiosensitive. There was no significant difference between the strains with respect to the numberof cellsor cellular composition of theexudates. No difference in thein vitro chemotactic response of cells from the two strains could be demonstrated. Therefore there seems to be recruitment to the infectedperitoneal cavity of C57BL/lO mice of young, efficiently bactericidal monocytes/macrophages. On the other hand, spleen cell bactericidal activity was intrinsically superior in C57BL/lO mice compared with BALB/c mice, possibly because, as a haemopoietic organ, the C57BL/lO spleen already contains high numbers of these efficient monocytes. 0 1986 Academic Pw, inc.
INTRODUCTION
Critical differences in the genetic resistance of mice to Listeria monocytogenesare expressed before the development of the T-cell-dependent specific immune response (l-3). Events during this early phase have been studied comparing susceptible BALB/c and resistant C57BL/lO (1) or susceptible A/J and resistant C57BL/6 mice (3). The initial localization of Listeriu in the liver and spleen is identical in the C57BL/ 10 and BALB/c mice (1). However, 24 hr later lo-fold more Listeria are found in the livers of BALB/c mice compared with C57BL/lO. Similar early differences are observed between A/J and C57BL/6 mice (3). Sadarangani et al. (4) found that a highly radiosensitive bone marrow-derived cell was responsible for the genetic advantage of resistant C57BL/6 mice over susceptible A/J mice in the first 48 hr of infection. Stevenson et al. (5) provided evidence that the cellular basis of genetically determined resistance to Listeria in C57BL/6 mice compared with A/J was a more rapid mobilization of monocytes to the foci of infection. This early inflammatory response has now been shown to be controlled by the Hc locus on chromosome 2 which determines the level of the Cs component of complement (6). However, they believed that another gene and another mechanism also contributed. The effects of the Listeriu resistance (Lr) gene first described by Cheers and McKenzie (7) cannot be accounted for by differences in the level of C5 as both the Listeria * Present address: CSIRO, Division of Animal Health, Parkville, Victoria 3052, Australia. 160 0008-8749186 $3.00 Copyright 0 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
BACTERICIDAL
ACTIVITY
OF
SUSCEPTIBLE
AND
RESISTANT
MICE
161
resistant C57BL/lO and susceptible BALB/c and CBA are all Cs+ strains. We had previously shown (8) that macrophages from Wisteria-infected resistant (C57BL/ 10) mice had superior tumouricidal activity to macrophages from susceptible (BALB/c) mice. In this paper we examine whether differences in the bactericidal activity of macrophages could account for the Lr gene effects described by Cheers et al. (1) and whether there are any differences in the chemotactic responsiveness of cells from these strains. The bactericidal assay system used was based on a technique developed by Davies (9) which measures the release of radioactively labeled DNA from Listeria as an indicator of the bactericidal activity of cells. Chemotaxis was measured in the classical Boyden chamber ( 10). MATERIALS
AND
METHODS
Mice. Inbred C57BL/lO, BALB/c, and CBA mice were maintained under conventional but disease-free conditions by strict brother-sister mating in the Microbiology Animal Breeding Unit, University of Melbourne. Mice were used when 5-8 weeks old. Bacteria. Listeria monucytogenes,obtained from R. V. Blanden (Australian National University), was subcultured on horse blood agar (HBA) weekly and renewed from freeze-dried stock after 25 passages. Infection of mice. Twenty-four-hour actively growing cultures were washed from tryticase soy agar plates with 1% serum in distilled water. The inoculum was standardized turbidimetrically using a calorimeter (EEL International Ltd). The bacteria were injected in 0.2 ml and the dose checked retrospectively using Miles and Misra ( 11) viable counts. Preparation of peritoneal cell suspension.Mice were killed with fluothane and the peritoneal cavities washed out with 5.0 ml RPM1 1640 containing 5% fetal calf serum (FCS) and 10 units/ml heparin. Pooled cell suspensions harvested from groups of 5 mice were centrifuged, washed once with 10 ml medium, and finally suspended in 5 ml of RPM1 only. Cell yields were determined and assessed for viability by staining with 0.2% eosin. Preparation of spleen cell suspensions.Spleens were cut into fragments and gently pushed through fine stainless steel sieves into cold RPM1 medium supplemented with 5% FCS. After clumps had settled out cells were centrifuged and the pellets resuspended in 10 ml Tris-buffered ammonium chloride to lyse red cells (12). The cell suspensions were incubated at 37°C for 15 min, then washed twice with RPM1 medium. Cell pellets were finally resuspended in 10 ml RPM1 and adjusted to 2 X lo7 viable cells/ml. All assays employed cells pooled from groups of 3-4 mice. Labeling of bacteria. A lo-ml tryticase soy broth (TSB) was inoculated with lo7 glycerol preserved Listeria (lo9 organisms/ml) stored at -20°C. The broth was incubated at 25 “C for 17 hr in the presence of 10 &i/ml tritiated thymidine (Amersham). Labeled Listeria were then washed free of unincorporated label by centrifuging at 4000g for 15 min at 4°C. Following three washes the bacteria were finally resuspended in 10 ml RPMI. The number of labeled organisms was checked retrospectively by Miles and Misra (11) viable counts. These conditions were determined to yield approximately 1O9labeled organisms/ml.
162
WOOD
ET
AL.
Bactericidal assay. The assay system used was developed from that described by Davies (9). Peritoneal cells (4 X 106/ml) or spleen cells (2 X 107/ml) in 0.5 ml RPM1 medium containing 50 mg/ml cold thymidine were distributed into 1.5-ml Eppendorf tubes along with 0.5 ml of labeled Listeria (approximately 8 X 107/ml). Tubes were incubated at 37°C in a water bath for various periods oftime then microfuged ( lO,OOOg, 7 min) and 0.8 ml of supernatant sampled into 8 ml of scintillant (EP Ready Solv, Beckman) and counted in a liquid scintillation spectrometer. Results are expressed as counts per minute (cpm) f standard deviation averaged from triplicate tubes. The spontaneous release of label from bacteria incubated without cells is shown in the legend of each figure. Day-to-day variation in the cpm released was observed according to the efficiency of bacterial labeling and the exact number of bacteria added to the test so comparisons can only be made within experiments. Phagocytosis and d@rential cell counts. To determine the amount of phagocytosis of bacteria, Eppendorf tubes were set up as described above and incubated for 2 hr at 37°C. Bacteria not associated with cells were then removed by underlaying the cell suspensions with 0.5 ml FCS and centrifuging at 900g for 10 min at 4°C. This procedure was repeated and the cell pellets finally resuspended in 1 ml RPMI. A sample of the cell suspension was taken and total radioactive counts associated with cells determined. Approximately lo- 15% of the bacteria added were cell associated after 2 hr of incubation. Smears of these cell suspensions were also prepared using a cytocentrifuge, then stained with Diff-Quik stain (AHS, Australia). Differential counts were determined by counting a minimum of 200 cells. Boyden chemotaxis assay. Peritoneal cells (4 X lo6 in 200 ~1 Hank’s balanced salt solution) were placed in the upper well of a Boyden chamber (Nuclepore, Pleasanton, Calif.) and allowed to migrate across an 8-ppore-size cellulose acetate/nitrate membrane (Millipore, San Francisco) toward hydrolyzed casein at 37°C for 5 hr. The filters were fixed in alcohol, stained with Mayer’s hematoxylin, and dehydrated by immersion in absolute alcohol and then xylol, before being mounted in DPX between a microscope slide and a coverslip. The results were quantitated by the leading front method (13) in which the maximum distance the cells have migrated into the filter is measured using a micrometer guage on the fine focus knob of the microscope. For each filter 20 fields were measured, 10 from left to right and 10 from top to bottom of the filter. For each experimental group triplicate chambers were tested. The fields for each filter were averaged and a mean and standard deviation expressed for each triplicate group. RESULTS The bactericidal activity of resident peritoneal cells from uninfected BALB/c (susceptible) and C57BL/ 10 (resistant) mice was compared in at least 10 separate experiments and no consistent differences were detected in their ability to kill Listeria (data not shown). However, following infection with lo4 Listeria differences were seen in the degree of enhanced bactericidal activity of peritoneal cells from resistant and susceptible mice. Peritoneal cells from C57BL/lO mice infected i.p. with Listeria 2 days previously showed greatly increased bactericidal activity when compared with cells from i.p.-infected BALB/c mice or uninfected control animals (Fig. 1). This difference in the degree of enhanced bactericidal activity of peritoneal cells from C57BL/ 10 and BALB/c mice was not seen following i.v. infection with the same dose of Listeria (Fig.
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
163
25 SO min
190
min
20 -
IV
controt
IP
Cell
IV
Control
Type
FIG. 1. Bactericidal activity of peritoneal cells from C57BL/lO (m) and BALB/c (0) mice. Mice were uninfected or infected i.p. or iv. with lo4 Listeria 2 days prior to assay.Bactericidal activity was measured at 90 and 180 min after incubation of bacteria with cells. Spontaneous release of label at 90 min (442 f 14) and 180 min (519 + 40).
1). When a second Listeriu susceptible strain of mice (CBA) was also examined, again only peritoneal cells from i.p. infected resistant mice showed increased bactericidal activity 2 days after infection (Fig. 2). Irradiation of mice immediately prior to infection with Listeria completely ablates the superior resistance of C57BL/lO mice to Listeria (4, 14). Irradiation of mice 2 hr before i.p. infection with lo4 Listeria also completely abolished the enhanced bactericidal response of peritoneal cells from 2-day infected C57BL/lO mice (Fig. 3A). To ensure that irradiation was not acting on the bactericidal mechanism of peritoneal cells itself, a control experiment was performed in which 2-day infected mice were irradiated just before harvesting their peritoneal cells. This treatment, in contrast to irradiation prior to infection, did not affect the enhanced bactericidal activity of peritoneal cells from C57BL/lO mice (Fig. 3B). These strain differences in bactericidal activity were analyzed by considering whether differences in the degree of phagocytosis of Listeriu could account for the advantage of C5 7BL/ 10 peritoneal cells over BALB/c cells. The degree of phagocytosis of bacteria was determined by counting the number of labeled Listeria associated with macrophages after removal of unassociated organisms by sequential centrifugation through FCS gradients. No significant differences were detected in the number of bacteria phagocytosed by cells from uninfected or infected mice of either strain (Table 1). This
164
WOOD ET AL. 20
I, Infected 1 controt
Control
2
I ,
Infected
3 Time
(hrs)
FIG. 2. BactericidaJ activity of peritoneal cells from uninfected and List&a-infected C57BL/lO (kB),BALB/ c (0) and CBA (m) mice. Mice were infectd i.p. with lo4 Listeria 2 days prior to assay.Bactericidal activity was measured at 2 and 3 hr after incubation of bacteria with cells. Spontaneous release of label at 2 hr (3 10 f 58) and 3 hr (346 f 22).
finding was also confirmed by direct microscopic counting of the numbers of bacteria associated with individual cells (data not shown). We next determined whether the enhanced bactericidal response of C57BL/ 10 mice was the result of a specific response to Listeria or a more generalized response to any 40
80
-A
30
7 0 5 z
20
s
had.
had.
FIG. 3. Effect of irradiation on the bactericidal activity of peritoneal cells from C57BL/lO (IB) and BALB/c (0) mice. Control animals were uninfected. The other groups of mice were infected i.p. with IO4 Listeria 2 days prior to assay. (A) Half of the infected mice received 800 rads irradiation 2 hr prior to infection with Lisferia. (B) Half of the infected mice received 800 rads irradiation just before harvesting their peritoneal cells. Bactericidal activity was measured 3 hr after incubation ofbacteria with cells. Spontaneous release of label in (A) (469 f 40) and (B) (675 + 63).
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
165
TABLE 1 Comparison of the Ability of BALB/c and C57BL/IO Peritoneal Cells from 2-Day Infected Mice to Phagocytose Listeriu Cell type”
Total cell associated counts after a double wash*
Day 2 C57BL/lO BALB/c
2803 + 397 3458 + 418
Control C57BL/lO BALB/c
3257 I!Z209 3357 + 490
a Cells were harvested from normal and Wisteria-infected C57BL/lO and BALB/c mice. Mice were injected i.p. with lo4 Listerin48hr prior to the experiment. * Total counts in the resuspended pellets following an FCS underlay and centrifugation step repeated twice.
i.p. intrusion. Mice were injected with 1 ml of sterile irritant (10% proteose peptone) and 2 days later their peritoneal cells were assayed for bactericidal activity. Peritoneal cells from C57BL/lO mice displayed far greater bactericidal activity compared to BALB/c cells following injections of proteose peptone (Fig. 4). Chemotactic activity of peritoneal cells from the two strains of mice was compared by examining migration in vitro across a membrane toward a chemotactic stimulus 60
50
40 T 0 = s
30
t 20
10
0
Proteose Peptons
FIG. 4. Bactericidal activity of peritoneal cells from normal and protease peptone injected C57BL/lO (BJ) and BALB/c (0) mice. Mice. were injected i.p. with 1 ml of 10% protease peptone 48 hr prior to assay. Bactericidal activity was measured 3 hr after incubation of bacteria with cells. Spontaneous release of label (348 I? 29).
166
WOOD ET AL. TABLE 2 Comparison of the Chemotactic Response of Peritoneal Cells from Listeriu-Infected BALB/c and C57BL/lO Mice 24 hr after Infection Distance migrated (pm)” Strain
Noninfected
Infected
BALB/c C57BL/IO
31.1 f 18.3 24.3 f 4.2
103.6 k 18.5 115.7 f 2.3
a Distance migrated represents the mean + SD of triplicate filters.
(Table 2). No difference was seen between normal C57BL/lO and BALB/c mice. Chemotactic responsiveness was enhanced equally in the two strains 24 hr after infection. The fact that BALB/c mice were not deficient in their ability to attract cells into the peritoneal cavity was confirmed when cell numbers were examined. BALB/c mice yielded slightly higher numbers of peritoneal cells compared to C57BL/lO mice after i.p. infection with List&z (Table 3). There was also no consistent differences in the percentage of macrophages in populations of cells recovered from these two mouse strains (data not shown). When bactericidal activity of spleen cells was compared, there was a significant difference between the two mouse strains in the degree of bactericidal activity seen with spleen cells from uninfected animals in a 3-hr in vitro assay (Fig. 5). The degree of increased activity 2 days after intravenous infection was less than that described in the peritoneal cavity and occurred in both strains. During the course of this study we also examined the listericidal activity of neutrophils in the peritoneal cavity by inducing populations of cells enriched for neutrophils by injection of proteose peptone or Listeria 6 hr before harvesting. The bactericidal activity of these neutrophil enriched cell populations was however, not markedly different from that of control cell populations (Table 4). Smears of these cell populations were prepared and examined for the degree of phagocytosis by the individual cell types present. The vast majority of bacteria phagocytosed was found to be associated with macrophages (Table 4). Irradiation of mice prior to infection with Listeria was also found to increase the percentage of neutrophils in the peritoneal cavity from approximately 5 to between 50 and 80%, yet this treatment dramatically reduced the bactericidal activity of this cell population (Fig. 3A). Collectively these data suggest that TABLE 3 Total Number of Peritoneal Cells Recovered following Intraperitoneal Injection of Listerid Strain
Uninfected
C57BL/lO BALB/c
2.4 f 0.7 3.0 f 0.8
Listeria
infected
4.8 + 1.7 7.9 f 1.9
a Data expressed asmean number of cells per mouse.(X 106) + standard deviation of 13 separate experiments. In each experiment groups of 5 mice were infected i.p. with approximately lo4 Listeriu 2 days prior to harvest.
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
Control
’ Infected
AND RESISTANT
Control
167
MICE
I Infected
5
1
3 Time
(hrs)
FOG.5. Bactericidal activity of spleen cells from C57BL/lO (Q and BALB/c (Cl) mice. Mice were uninfected or infected i.v. with IO’ Listeria 2 days prior to assay.Bactericidal activity was measured at 1 and 3 hr after incubation of bacteria with cells. Spontaneous release of label at 1 hr (209 f 40) and 3 hr (236 f 6).
neutrophils contributed experiments.
only minimaly
to the bactericidal activity measured in these
DISCUSSION Peritoneal cells from intraperitoneally, but not intravenously, Listeria-infected C57BL/lO mice rapidly developed far greater bactericidal activity than the same number of cells from susceptible BALB/c and CBA mice. The induction of enhanced bactericidal activity of cells from C57BL/lO mice was sensitive to irradiation of the donor mice at the time of infection, as is the in vivo resistance to Listeria of these mice (4, 14), suggesting a role for recently divided cells in C57BL/lO and their unavailability in BALB/c mice. That expression of the enhanced bactericidal activity TABLE 4 Bactericidal and Phagocytic Activity of Neutrophil-enriched
Cell Populations”
No. of bacteriaphagocytosedper cellb Cell type
w of total population
Nil
1-5
5-10
>lO
Proteosepeptone
Macrophage Neutrophil
29 52
48 a4
15 12
6 3
31
Listeria
Macrophage Neutrophil
39 36
37 76
13 16
9 a
40 0
Control
Macrophage Neutrophil
75
33
13
15
39
-
-
-
-
Treatment
0
a Groups of 5 mice were injected i.p. with IO6 Lisferia or 1 ml proteosepeptone 6 hr prior not injected. b 200 cells were counted and the results expressed as a percentage.
1
CPM releasedC 953k
a7
1029 f 134 767 k
69
to assay.Control mice were
‘Counts per minute releasedinto supematant following a 2-hr incubation of cells with labeled Listeria.
168
WOOD
ET
AL.
was not radiosensitive was shown by the fact that irradiation of the donors just before harvesting the cells did not affect their activity. We found no evidence that differences between C57BL/lO and BALB/c mice in either the total number of peritoneal cells recovered, the percentage of macrophages in these populations or their ability to phagocytose Listeria would account for the enhanced bactericidal activity of cells from C57BL/lO mice. The finding that i.p. injection of proteose peptone also induced a higher degree of bactericidal activity in resistant mice compared to susceptible mice suggests that this phenomenon is not the result of a specific response to Listeria infection. However as with Listeria infection, the bactericidal differences observed in these mouse strains following injection of proteose peptone were not due to differences in the total numbers of macrophages recruited into their peritoneal cavities. Therefore the enhanced bactericidal activity of 2 day infected C57BL/ 10 compared with BALB/c cells is most likely due to selective recruitment to the site of infection and/or activation of recently divided bone marrow-derived monocytes/macrophages with enhanced bactericidal activity. C57BL/lO mice have twice as many monocyte precursors (macrophage colony forming cells) as BALB/c and these are rapidly depleted from the bone marrow following Listeria infection ( 15). These results may be compred with those of Gervais et al. (6), who found an enhanced inflammatory reponse in C57BL/lO (Lrx) mice compared with A/J(L?) mice 3 days post infection. This difference was genetically linked in back-cross and recombinant inbred mice with differences in Listeria resistance, the in vitro chemotactic response of peritoneal cells and the Hc gene which determines the level of the Cs component of complement. They believed that there was also a second gene acting to render the A/J mice susceptible. Czuprynski et al. (16) have confirmed that the inflammatory response of A/J mice is less efficient than that of C57BL/6 mice and have partially restored the response by supplying Cs. They found that while there were higher numbers of peritoneal cells in their i.p. infected resistant mice compared with infected susceptible mice, there was little difference in the bactericidal activity, cell for cell, between the strains. Since BALB/c and CBA mice are both Cs sufficient strains, the Hc gene cannot account for the difference in resistance we observe between them and C57BL/ 10 mice. Furthermore, they appear to differ from C57BL/lO mice, not in the numbers of cells recruited to the site of infection, but in the bactericidal efficiency of these cells. Indeed no difference was observed in the in vitro chemotactic response of the two strains. While higher numbers of cells were attracted to the peritoneal cavity of BALB/c mice following i.p. infection, this was possibly due to the attainment of higher bacterial numbers in this strain than in C57BL/lO. When spleen cell bactericidal activity was compared, resistant C57BL/lO showed greater efficiency than BALB/c, even before infection. Intravenous infection somewhat enhanced the activity of both strains, but the degree of enhancement was not as great as that seen in the peritoneal cavity of C57BL/lO mice infected i.p. It is possible that the spleen, which like the bone marrow is a hemopoietic organ, contains many less mature monocyte/macrophages which our studies in the peritoneal cavity suggest are strongly bactericidal. Spleens of uninfected C57BL/lO mice contain three times the number of colony forming cells (macrophage precursors) found in BALB/c mice ( 15). If a correspondingly high number of young, efficiently bactericidal monocytes exist
BACTERICIDAL
ACTIVITY
OF SUSCEPTIBLE
AND RESISTANT
MICE
169
in the C57BL/lO spleens, this could account for the difference in innate bactericidal ability in the spleens of the two mouse strains. The finding that neutrophils played only a minor role in the phagocytosis and killing of Listeria in vitro disagrees with the findings of Czuprynski et al. (17), who reported that inflammatory peritoneal neutrophils killed Listeria in vitro even better than did inflammatory macrophages. These differences may be associated with the different bactericidal systems used, in particular the use of opsonins in their assay. Macrophages do not require antibody for the uptake and lysis of Listeria (18) and serum has been found to be toxic for Listeria ( 19). Therefore we excluded it from our assay system. However, we cannot discount the possibility that neutrophils have a greater requirement for opsonins that macrophages for the phagocytosis and lysis of Listeria. Finally the bactericidal assay used in this paper measures the actual lysis of bacteria by cells in contrast to other assays which quantitate the survival of bacteria following incubation with cells. The advantage of this system is that the problems of growth of extracellular bacteria are avoided thus making this assay far more sensitive. ACKNOWLEDGMENTS We thank Wayne Davies for his assistance in establishing the bactericidal assay.The work supported by the National Health and Medical Research Council of Australia.
REFERENCES 1. Cheers, C., McKenzie, I. F. C., Pavlov, H., Ward, C., and York, J., Infect. Zmmun. 19, 763, 1978. 2. Cheers, C., McKenzie, I. F. C., Mandel, T. E., and Chan, Y. Y., In “Genetic Control of Natural Resistance to Infection and Malignancy.” (E. Skamene, P. A. L., Kongshavn, and M. Landy, Eds.), p. 141. Academic Press, New York, 1980. 3. Skamene, E., and Kongshavn, P. A. L., Infect. Zmmun. 25, 345, 1979. 4. Sadarangani, C., Skamene, E., and Kongshavn, P. A. L., Infect. Zmmun. 28,381, 1980. 5. Stevenson, M. M., Kongshavn, P. A. L., and Skamene, E., J. Zmmunol. 127,402, 1981. 6. Gervais, F., Stevenson, M., and Skamene, E., J. Zmmunol. 132, 1, 1984. 7. Cheers, C., and McKenzie, I. F. C., Infect. Zmmun. 19, 755, 1978. 8. Wood, P., and Cheers, C., Immunology 54, 113, 1985. 9. Davies, W. A., J. Reticuloendoth. Sot. 32, 461, 1982. 10. Boyden, S., J. Exp. Med. 115,453, 1962. 11. Miles, A. A., and Misra, S. S., J. Hyg. 28, 732, 1938. 12. Boyle, W., Transplantation 6, 761, 1968. 13. Zigmond, S. H., and Hirsch, J. G., J. Exp. Med. 137, 387, 1973. 14. Cheers, C., and Macgeorge, J. In “NK Cells and Other Natural Effector Cells.” (R. B. Herberman, Ed.), p. 152 1. Academic Press, New York, 1982. 15. Young, A. M., and Cheers, C., Cell. Zmmunol. 97,227, 1986. 16. Czuprynski, C. J., Canons, B. P., Henson, P. M., and Campbell, P. A., Immunology 55, 5 11, 1985. 17. Czuprynski, C. J., Henson, P. M., and Campbell, P. A., J. Leuk. Biol. 35, 193, 1984. 18. Cooper, J. M., Johnson, R. B., and Rowley, D., Aust. J. Exp. Med. Sci. 61, 63, 1983. 19. Davies, W. A., J. Reticuloendoth. Sot. 34, 13 1, 1983.