Role of Corynebacterium parvum in the activation of peritoneal macrophages

CELLULAR

IMMUNOLOGY

70, 65-75 (1982)

Role of Corynebacterium of Peritoneal I. Association

between intracellular STEPHEN

parvum in the Activation Macrophages C. parvum and Cytotoxic

Macrophages’

K. CHAPES~ AND STEPHEN HASKILL

The Cancer Research Cenier and the Department of Obstetrics and Gynecology, University of North Carolina, Chapel Hill, North Carolina 27514 Received February

2. 1982: accepted March 25, 1982

We have investigated the association between intracellular C. parvum (CP) and macrophage (Mg) cytotoxicity. Mouse peritoneal M+s were activated by ip administration of CP and were subjected to a combination of fractionation techniques to study this. Velocity sedimentation demonstrated that only the largest cells were cytotoxic. These same cells contained CP and suggested an association between the two variables. Further separation of the largest M& using a BSA equilibrium buoyant density gradient demonstrated that cytotoxicity was due to M& and further substantiated the strong correlation between intracellular CP and cytotoxicity. Various fluorochrome tagged CP preparations were also used to activate M& and to isolate CP-containing M& using fluorescence-activated cell sorting. When velocity-enriched M& were sorted on the basis of the presence or absence of fluorescent CP, only the M&J fractions which contained CP were cytotoxic. The results indicate that most cytolytic macrophages present at the peak of the responsecontain CP. Thus, a convenient probe with which to follow macrophage activation at the single cell level was provided.

INTRODUCTION The many and diverse contributions of the macrophage (M4)3 to the immune response is becoming well appreciated. Macrophages have been found not only to be nonspecific scavenger cells, but are also important accessory cells in the development of humoral and cellular immunity. Furthermore, they can control the immune response by acting as suppressor cells. These various roles have been well studied and documented recently ( l-6). Furthermore, current studies point out the importance of recognizing the heterogeneity within the M$ population (7-12). Use of immunostimulants to induce and activate M@ has been common in M4 studies. Agents such as viruses, C. PCI~VU~?, and BCG are most commonly injected ’ This investigation was supported in part by National Cancer Institute Grant 1 PO1 CA 29589 01 and American Cancer Society Grant IM 84 E. * SKC is supported by National Institutes of Health, National Service Award CA 09156 from the National Cancer Institute. 3 Abbreviations used: CP, C. parvum; H-S3, HeLa S-3 cells; FBS, fetal bovine serum; PEC, peritoneal exudate cells; BSA, bovine serum albumin; VSG, velocity sedimentation gradient; FITC, fluorescein isothiocyanate; TRITC, tetramethyl rhodamine isothiocyanate; FACS, fluorescence-activated cell sorting. 65 OOOS-8749/82/090065-11%02.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ip and have been the activating stimuli in many of the studies finding functional heterogeneity between M$Jsubpopulations (4,8-10). The differences in the cytolytic activity of Mr$s for tumor cells has been especially evident in these investigations. The fate and role of the agents which induce cytotoxic M& are poorly understood. Early reports hinted that persistence of the stimulus was important in inducing tumoricidal M& (13, 14), but few studies have documented the fate of the agent in the peritoneal cavity and how it might contribute to the generation of M4 heterogeneity. This investigation reports a strong correlation between the intracellular presence of bacteria in CP-activated murine M+ and the cytolytic activity of those M& for tumor cells. MATERIALS

AND METHODS

Animals. B6AFi female mice, 6 to 8 weeks old, were obtained from Jackson Laboratories (Bar Harbor, Me.). Immunization of animals. Eight- to lZweek-old animals were immunized with 0.2 ml ( 1400 pg) fixed Corynebacteria parvum (C. parvum, Burroughs Wellcome, Co., Research Triangle Park, N.C.) 4 days before use. Cell cultures. HeLaS3 (H-S3) cells were obtained from Dr. Barry Goz, University of North Carolina, Chapel Hill, N.C. and were used as target cells in M4 cytotoxicity assays. Cells were grown in 25-cm’ tissue culture flask (3013 Falcon Plastics, Oxnard, Calif.) in RPMI-1640 medium (GIBCO, Grand Island, N.Y.) supplemented with L-glutamine, 20 pg/ml gentamycin, and 10% FBS (KC Biological Co., Kansas City, MO.). Culture media was not screened for LPS. This HS3 line has been in culture since 1976 and has been described previously (31). Peritoneal exudate macrophuges. Peritoneal exudate cells (PEC) were harvested from mice killed by cervical dislocation. Cells were obtained by washing the peritoneal cavity with 10 ml ice-cold medium (w/o FBS) and were washed 2X. The PEC were counted in a hemocytometer and were diluted to a concentration of 2.0 X lo5 cells per ml if they were to be used in cytotoxicity assays. Target cell preparation. H-S3 cells were seeded into 25-cm2 flask 2 to 3 days before use. Before the assay, medium was poured off and fresh medium containing 5 mCi/ml of [3H]thymidine (Schwarz/Mann, Spring Valley, N.Y.) was added. The cells were then incubated 24 to 30 hr. Radiolabeled cells were dispersed from tissue culture flask by washing the cells from the plastic with strong jets of media from a Pasteur pipet. Cells were washed, counted, and adjusted to a concentration of 5 X lo4 viable radiolabelled cells per ml. Target cell viability was always greater than 95% after dispersion. One X lo4 target cells per well were used in each M9 cytotoxicity assay. Velocity sedimentation gradients (VSG). The VSG was first described by Miller and Phillips ( 15) and was modified as described by Haskill (16). Cell recovery was always greater than 80%. BSA equilibrium buoyant density. BSA was obtained from Sigma (#A-4503) mixed to a concentration of 15% in deionized H20 and dialyzed against Hz0 for 2 days. Water was changed two to three times daily. BSA was lyophilized and reconstituted to isotonicity in PBS to 14 and 28% concentrations. The continuous gradient was constructed according to the method of Shortman et al. (17) in a

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67

standard mixing chamber with VSG fractionated cells added to the 28% BSA side. All materials and cells were kept at 4°C throughout. The gradient was formed into a 15ml tube and centrifuged at 2OOOgfor 20 min to achieve equilibrium. Onemilliliter fractions were collected, washed 2X, and counted. The percentage of macrophages in each fraction was determined by staining cells from each fraction with Wright’s stain and identifying cells with M4 morphology. Each fraction was adjusted to give 2O:l M4 to target cell ratio for cytotoxicity assays. Adherent macrophage cytotoxicity assay. The M4 cytotoxicity assay was performed in 96-well microtiter plates as described earlier ( 18) employing [ ‘Hlthymidine prelabeled targets. Macrophages were added to microtiter plate wells in a volume of 0.1 ml at a concentration of 20 X lo5 M& per ml. Cells were allowed to adhere for 1 to 2 hr and wells were washed vigorously to leave only adherent cells. Following washing, target cells were added to wells in a volume of 0.2 ml at a concentration of 5 X lo4 cells per ml. The assay plates were incubated 18 to 20 hr at 37°C in a humidified CO1 incubator. After incubation the percentage specific 3H release was calculated by removing 0.1 ml of supernatant from each well and counting the amount of activity in a scintillation counter (Packard Instr. Co., Rockville, Md.). Maximum release was determined by placing 1 X lo4 cells directly into aquasol and counting. Spontaneous release was determined by incubating target cells in media for the duration of the assay and ranged between 20 and 30% for our assays. The percentage specific 3H release for each sample was calculated as follows: % Release with M@ - % Spont. Release x loo % Specific Release = ’ % Max. Release - % Spont. Release Conjugation of C. parvum withfluorescein isothiocyanate (FITC) or tetramethyl rhodamine isothiocyanate (TRZTC). FITC: 3.0 ml of CP (7 mg/ml) was centrifuged in a 12 X 75 mm tube at 8OOgfor 15 min to pellet bacteria. The FITC (isomer #l, #F-7250, Sigma Chemical Co., St. Louis, MO.) was dissolved at a concentration of 5 mg/ml in Na,CO,. Two-tenths milliliter of this suspension was added to the CP pellet. The pellet was vortexed and incubated in the dark for 1 hr, with occasional mixing. After conjugation, 3 to 5 ml of cold PBS was added to the tube and bacteria were thoroughly suspended. The tube was centrifuged at 800g for 15 min and the FITC-CP were washed and centrifuged twice. The FITCCP suspension was resuspended in 2.7 ml of PBS and injected into animals as described above. TRITC: Bacteria were treated similarly as above. Five milligrams of TRITC (Research Organics, Inc., Cleveland, Ohio) in a 12 X 75 mm tube was first suspended in five drops of iV,N-dimethylformamide to solubilize the powder. One milliliter of 0.02 M Na2C03 buffer pH 9.5 was then added. The precipitate was removed by centrifuging the tube for 5 min at 800g and the supernatant was added to the bacteria as described for FITC conjugation; the remaining steps for TRITC conjugation remained the same as for FITC conjugation. Cell sorting. Macrophages labeled with TRITC, FITC, or left to be separated according to autofluorescence were sorted on an Ortho Cytofluorograf Systems 50 (Ortho Diagnostics, Westwood, Mass.). The cytofluorograf was equipped with a Lexel ion laser (Model 95) and was operated under sterile conditions. Excitation for FITC-CP-containing M@ and for autofluorescent M$J separations was 488 nm. The 512 nm line was used when CP was conjugated with TRITC. For sorting

68

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experiments using 5 12 nm excitation, a 520-nm-long pass filter, a 530-nm series D filter, a Corning 3-68 filter, and a 580~rim-long pass filter were used. Approximately 1 X lo7 PEC (95% M@) were sorted in each experiment. Recovery (the percentage of cells recovered that were initially added to the cell sorter) ranged from 22 to 25%. After sorting, M@ were prepared for cytotoxicity experiments as described above. RESULTS Intracellular

Localization of CP in Murine Peritoneal M&

Four days following stimulation, C’. parvum-activated murine peritoneal M&s exhibit considerable heterogeneity in both the percentage of peroxidase-positive cells, size, and the presence of intracellular bacteria. In particular, the C. parvum appeared to be associated predominantly with the larger M~#J(Fig. 1). This suggested that a fractionation procedure based on size would permit at least a preliminary assessment of the relationship between cytotoxic activity and the presence of the activating agent. Velocity Sedimentation of CP Activated M&: Cellular Analysis To see if we could isolate C. parvum-containing M&, CP-stimulated M@ were fractionated using lg VSG. Figure 2 shows the cell profile of the collected fractions. The cells isolated from the high-velocity fractions were predominantly M&. Furthermore, many of the M& in these fractions contained CP. In contrast, the lowvelocity (~5.8 mm/hr) fractions contained very few or no CP-containing M&. Thus, we found that we could isolate Mr$s, using velocity sedimentation, that were different in the amount of intracellular CP. The MC@from each fraction were then tested for cytotoxicity against H-S3 tumor cells. Our findings indicated that M& harvested from high-velocity fractions were the most cytolytic (Table 1). For example, in experiment #l, Mb in a fraction with a velocity of 10.6 mm/hr showed 44.1% specific cytotoxicity and 61% of these M$s contained CP. In contrast, M@ harvested from the lower velocity fractions (~6.5 mm/hr) demonstrated little specific cytotoxicity and did not contain visually apparent CP. Since specific release may not reflect the relative importance of a particular fraction of a heterogeneous cell population (i.e., an effector cell population present in high frequency but with a low specific release may contribute more to tumor cell lysis than an infrequent cell of high specific release), we also calculated the total lytic units of each M$ fraction and plotted these data along with the total number of CP-containing M&. Figure 3 is a representative plot of this analysis using the data from Table 1. It illustrates the close association that exists between the number of CP-containing M+s in each fraction and the total lytic units in each fraction. Subfractionation of the Most Active Fractions of CP-Containing Mr#~s In a further attempt to characterize the association of CP and M4 cytotoxicity and to determine whether it was possible to obtain pure populations of CP-containing and CP-free M&, we carried out a second fractionation step. C. parvumstimulated M& were first separated on a lg VSG as in Fig. 2. The M@JS sedimenting at or above 8.7 mm/hr were isolated by equilibrium buoyant density gradient

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C. parvum

d lz

70

CHAPES AND HASKILL

ns

?

25-

i

VELOCITY

(mm/hr)

FIG. 2. Cell distribution in fractions harvested from lg velocity sedimentation gradients. Total cell profile is scaled on the left ordinate and percentage MI@ and CP-containing M& are scaled on the right ordinate; both are plotted vs velocity.

centrifugation. All except the most dense fractions were found to be highly purified for M&s and cytotoxicity was found throughout the density gradient (see Table 2). However, when the total lytic units per fraction was plotted vs the total number of M+-containing CP, there was a strong correlation between the number of CP M&s in a fraction and the total cytolytic activity in the fraction (r = 0.98) indicating that cytolytic activity is associated with large (high-velocity Mr#~s),CP-containing M& which are quite heterogeneous in density. Fluorescence-Activated Cell Sorting of CP-Containing M& A further type of experiment was carried out to confirm the association between cytolytic activity and the intracellular content of CP. To achieve this, M@ enriched TABLE 1 Assessment of Macrophage Cytotoxicity, and Intracellular C. parvum following lg Velocity Sedimentation Gradient Separation Percentage C. parvum-containing M+sb

Percentage specific ‘H release” FR. vel.’ 10.6 8.1 7.5 6.8 6.2 5.0 4.3

EXP 1 44.1 * 17.8 + 8.0 + 2.0 + 1.9 f 2.3 f 0.7 f

1.2d 1.9 2.6 2.0 0.3 1.6 0.4

FR. vel*

EXP 2

FR. vel.

EXP 1

FR. vel.

EXP 2

13.9 9.5 8.1 7.2 6.3 5.5 4.2

51.2 + 3.9 38.0 + 5.1 1.6 + 0.8 -3.8 IIZ0.6 2.1 k 1.7 0.3 + 2.0 -4.2 f 1.3

10.6 8.1 7.5 6.8 6.2 5.0 4.3

61 31 9 0 2 0 0

13.9 9.5 8.1 7.2 6.3 5.5 4.2

92 74 15 6 0 0 0

a Normal MI$ cytotoxicity was 0.1 f 1.0 and 4.2 + 3.8 for experiments 1 and 2, respectively. Spontaneous release was less than 25%. b Percentage CP-containing M&s was determined by scoring cytocentrifuge preparations under a light microscope after staining with Wright’s stain. ’ Macrophages obtained from lg VSG fractions as described under Materials and Methods. d Numbers represent mean + standard deviation of triplicates. ET ratio = 20 M&s: 1 target cell.

CYTOTOXICITY

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71

C parvum

[mm /hr)

FIG. 3. Correlation between macrophage cytotoxicity and intracellular C. parvum. Total lytic units per fraction are calculated as in Table 2 and are expressed on the left ordinate. Total number of CPcontaining M&s is scaled on the right. Both are plotted vs velocity.

as in Fig. 2 (>8.1 mm/hr) were isolated from mice injected with CP conjugated with either FITC or TRITC and were sorted on an Ortho cytofluorograf. The first experiments utilized FITC-conjugated CP-stimulated mouse M&. This experiment sorted for FITC-positive (bacteria-containing) or FITC-negative (nonbacteria-containing) M+. These results are illustrated in Table 3 (Experiment 1). TABLE 2 Association of Intracellular C. Purvum with Cytolytic Activity after lg Velocity Sedimentation and BSA Equilibrium Buoyant Density Gradient Centrifugation

Fraction

Density

Normal M$s Unfractioned CP M& Pooled fractionated (FRS 1-6) BSA 1’ 2 3 4 5 6

1.040 1.048 1.054 1.062 1.069

Percentage specific ‘H Release H-S3

Number of CPcontaining M+s

4.2 + 1.5’

50

21.5 + 1.3

55

15.4 + 1.0

92

16.7 k 20.5 + 17.4 + 12.7 + 15.0 + 12.1 +

2.3 2.2 1.8 2.1 1.5 1.4

99 98 100 100 99 57

6.5 X 9.1 X 1.3 X 7.3 x 6.1 X 4.1 x

lo5 (3.3)” 10’ (4.6) lo6 (6.5) IO5 (3.7) lo5 (3.1) lo5 (1.4)

Total Lytic units per fraction’

54 86 105 66 52 36

’ Total lytic units are calculated by multiplying the number of equivalents per fraction X the number of possible assays per fraction. Equivalents are obtained for each fraction by constructing dose-response curves with ‘H specific release values from different M&target ratios from each experiment. 6 Numbers represent mean + standard deviation. ‘Collected fractions from a BSA continuous gradient prepared as described under Materials and Methods. d Numbers in parentheses are the number of possible assays per fraction.

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g 120z 2 IOOIA. i

00-

U-J

-

;

60-

u I-3

40-

E d

20- 0’

I I I 3 2 4 6 0 TOTAL NO. CP CONTAINING

I I 1 IO 12 14 M‘#‘S (x10’)

FIG. 4. Comparison of the total number of CP-containing M+s per fraction vs the total lytic units per fraction. The total lytic units of fractions from the experiment illustrated in Table 2 are plotted against the total number of CP-containing M&s per fraction to show a strong correlation between the two variables.

Normal M& showed low cytotoxicity and CP Mq5sshowed good cytolytic activity, illustrating that the conjugation procedure did not affect the stimulatory ability of CP. Furthermore, the FITC-negative cells only showed 5.8% cytotoxicity which is background level for this experiment. In contrast, FITC-positive Mqjs showed TABLE 3 Cytotoxicity of Fluorescence-Positive or -Negative Macrophage Populations Separated by a Fluorescence-Activated Cell Sorter Percentage specific ‘H release of H-S3

Exp 1’ FITC-conjugated C. parvum Normal M&s Whole C. parvum Ws lg separated C. parvum M&s’ Negative M$& Positive M&b

Exp 2’ TRITC-conjugated C. parvum 1.8

3.4 * 3.3

25.2 + 5.1

16.6 + 19.6

55.3 -+ 3.3

34.0 f 1.8 5.8 2 1.8 (0)" 28.5 + 6.4 (81)

63.8 + 29.1 + 59.0 +

41.8 -t 2.9

1.3 * 2.P

14.0 r

Exp 3’ Green autofluorescence

5.1

1.0 (0)

15.6 + 1.2 (1%)

0.5 (89)

39.9 k 6.2 (68%)

’ Effector (M+) to target ratio = 20 M$s to 1 target. b M&s separated by velocity sedimentation (velocities 2 8.1 mm/hr) were used for sorting. ’ Excitation at 488 nm for Exp 1 and Exp 3 and at 512 for Exp 2. d Numbers represent mean k standard deviation of triplicates. c Pooled Mb population from which fluorescent-positiveor -negative M&s were separated (see Materials and Methods). ‘An Ortho Cytofluorograf Systems 50, equipped with a Lexel ion laser, was used to perform all sorting experiments. 8 Fluorescent-negative or -positive M$s tested for cytotoxicity after cell sorting. ’ Numbers in parentheses are the percentage CP-containing M&s.

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28.5% specific cytotoxicity. Thus, this experiment indicated that bacteria-containing M&r are indeed strongly associated with cytotoxicity, whereas the noncytolytic M$s are in the subpopulation that appear(s) not to contain bacteria. During our experiments, however, we observed that some of the CP-activated M& autofluoresce (green) under 488 nm excitation. Therefore, we were concerned that some autofluorescent (potentially non-CP-containing) M&J might be included in the positive fraction in our initial sorts. Visual assessmentsof the positive fraction usually showed about 15% of the M&, apparently not containing CP. To see if green autofluorescing, apparently nonbacteria-containing M&G contribute to the cytotoxic activity of the positive fraction, CP was conjugated with TRITC and M& equivalent to those used in experiment 1 were sorted for TRITC-positive and TRITC-negative cells using an excitation wavelength of 5 12 nm. There was little red autofluorescence of the CP-activated M& using excitation of 512 nm (data not shown). When TRITC-negative M&s were tested for cytotoxicity of H-S3 target cells (see Table 3, Experiment 2), results similar to those found when we sorted for FITC-CP-containing M& were obtained. As before, the majority of cytolytic activity resided in the TRITC-positive fraction. In contrast to the results in Experiment 1, however, we found higher than background levels of cytotoxicity in the TRITC-negative fractions. These results suggest that indeed, in Experiment 1, some autofluorescing non-CP-containing M&G were included in the FITC-positive subpopulation; but that most cytolytic activity was still associated with CP-containing Mbs. In an attempt to see if autofluorescent M&s were cytotoxic, we sorted M@ stimulated by unconjugated CP on the basis of autofluorescence (488 nm excitation). We found that most cytolytic activity was associated with the positive fraction, of which >68% contained bacteria (Table 3, Experiment 3). In addition, there was a small amount of cytolytic activity in the negative subpopulation and this was apparently not accounted for by CP-containing M+s (1% CP M&). However, this activity was far less than that found in the autofluorescent M$J fraction. DISCUSSION We have demonstrated that there is a strong association between M4 cytotoxicity and the intracellular localization of CP 4 days following ip stimulation. The evidence for this rests upon three different types of cell fractionation experiments. When M& were separated by velocity sedimentation and scored for the presence of CP, the most rapidly sedimenting (largest) M&s contained the most bacteria and had the highest cytotoxic activity; the slower sedimenting M& contained little or no CP and were low in cytolytic activity, indicating a strong association between size, cytolytic activity, and intracellular bacteria. Further separation of the highvelocity M&s by density (BSA equilibrium buoyant density gradient centrifugation) yielded M&rich fractions containing varying amounts of cytotoxicity and intracellular CP. In spite of the wide range in density which characterized the cytolytic state, when the total lytic units of each of these fractions was plotted against the total number of CP-containing M&s in each fraction, a strong correlation between intracellular CP and cytotoxicity was again found. This association was further strengthened in experiments utilizing FACS anal-

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ysis. Fluorescein isothiocyanate-conjugated CP or TRITC-CP-containing M&s were highly cytolytic whereas the CP-free populations were significantly less cytolytic. Thus, from this series of experiments, utilizing various methods to isolate M&, the results indicate that cytotoxicity is highly associated with intracellular CP. The close association of intracellular CP and cytotoxicity 4 days after immunization suggests that CP may be acting as a direct activator of M& in this system. This does not preclude the possibility that M@ may be activated by other mechanisms, especially T cells. Past studies have shown that T cells or their products can activate M$JS(19-21). However, the generation of T-cell activity is usually not apparent until 6 to 7 days postcontact with antigen (22, 23). Additionally, tumor rejection by normal and nude mice following CP administration in vivo also suggests T-independent as well as T-cell-dependent mechanisms of M4 activation (24). Thus, the evidence provided here suggests that direct processing of the CP by M& may be important in the early development of the cytolytic state. Previous observations have pointed out that the most cytotoxic M& are the largest or most dense cells (4, 8, 10, 25-27). While we found that the cytolytic CP-containing M&s were the largest M&, they were highly heterogeneous with regard to density. The association between increasing size, cytolytic activity, and bacterial content suggests that these features are dependent on one another. Cytolytic activation by CP would thus appear to result from a response to the ingested bacteria which leads to cell enlargement and an increase in a number of biochemical markers (acid phosphatase, leucineamino-peptidase) associated with M$J stimulation (manuscript in preparation). Others have described how M& might exist in a continuum of activation states (28, 29). The physical state (i.e., degraded or intact) of the antigen may be reflected by the degree of autofluorescent cytolytic M&s just as the disperse density characteristics of these same cells probably reflect varying stages of M4 lysosomal content. Increasing the efficiency of cell sorting the various M4 subpopulations, or sorting at different times postimmunization may be reasonable ways to address these possibilities. This investigation clearly illustrates the heterogeneity of the M4 population following stimulation with a biological response modifier. Other studies (4, 7, 9, 26, 27) have found functional heterogeneity between MI#Jsubpopulations, activated with CP, fractionated by size or density, but to our knowledge, no study has associated the presence of CP with cytotoxicity or other functions. Chapes and Tompkins (8, 18) have previously demonstrated that infectious vaccinia virus is virtually eliminated from vaccinia-activated Mr$ populations before they become cytotoxic. However, no data on the presence or absence of noninfectious, but still immunogenic, virus particles in M&s are available. In contrast, Smialowicz and Schwab (30) have demonstrated that Fischer rat M& become cytotoxic only if M&s ingest nonbiodegradable group A streptococcal cell walls. These same M@ do not become cytotoxic if biodegradable group D streptococcal cell walls are phagocytosed. In conclusion, we feel that our observations may prove to be important for future advances in immunotherapy since the most active tumorcidal M& can be identified by the presence of intracellular bacteria. We also feel that this system allows us to further investigate the role of bacteria in the direct activation of M+s. We are now using quantitative flow cytometry to study the association between intracellular CP and the development and loss of various enzyme and antigenic markers known to be influenced during M4 activation.

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ACKNOWLEDGMENTS We thank Dr. John Camhier and Ms. Wendy Havrian of Duke University for assistance with cell sorting. We also express our appreciation to Mrs. Nancy Raybourne and Mrs. Willia Bell for outstanding laboratory assistance. Our thanks also to Dr. Susanne Becker for reviewing this manuscript.

REFERENCES 1. Rosenthal, A. S., and Shevach, E. M., J. Exp. Med. 138, 1194, 1973. 2. Chapes, S. K., Fan, S. S., Cummins, J. M., and Tompkins, W. A. F., J. Reticuloendothel. Sot. 29, 341, 1981. 3. Niederhuber, J. E., Immunol. Rev. 40, 28, 1978. 4. Lee, K. C., and Berry, D., J. Immunof. 118, 1530, 1977. 5. Gillespie, G. Y., and Russell, S. W., J. Reticuloendothel. Sot. 27, 535, 1980. 6. Oehler, J. R., Cambell, D. A., and Herberman, R. B., Cell Zmmutwl. 28, 355, 1977. 7. Moore, K., and McBride, W. H., Znt. J. Cuncer 26, 609, 1980. 8. Chapes, S. K., and Tompkins, W. A. F., J. Reticuloendothel. Sot. 30, 517, 1981. 9. Miller, G. A., Campbell, M. W., and Hudson, J. L., J. Reticuloendothel. Sot. 27, 167, 1980. 10. Lee, K. C., Wong, M., and McIntyre, D., J. Zmmunol. 126, 2474, 1981. 11. Weinberg, D. S., Fishman, M., and Veit, B. C., Cell. Zmmunol. 38, 94, 1978. 12. Fishman, M., and Weinberg, D. S., Cell. Immunol. 45, 437, 1979. 13. Wilkinson, P. E., In “Recent Results in Cancer Research” (G. Mathe, Ed.), pp. 41-49. SpringerVerlag, Berlin, 1976. 14. Hibbs, J. B., Trunsplantution 19, 81, 1975. 15. Miller, R. G., and Phillips, R. A., J. Cell Physiol. 73, 191, 1969. 16. Haskill, S., In “Manual of Macrophage Methodology” (H. B. Herscowitz, et al., Eds.), pp. 43-49. Marcel Dekker, New York, 1981. 17. Shortman, K., Haskill, J. S., Szenberg, A., and Legge, D. G., Nature (London) 216, 1227, 1967. 18. Chapes, S. K., and Tompkins, W. A. F., J. Immunol. 123, 303, 1979. 19. Godal, T., Rees, R. J. W., and Lamvik, J. O., Clin. Exp. Immunol. 8, 625, 1970. 20. Piessens, W. F., Churchill, W. H., and David, F. R., J. Zmmunol. 114, 293, 1975. 21. Kripke, M. L., Budman, M. B., and Fidler, I. J., Cell. Immunol. 30, 341, 1977. 22. Perrin, L. H., Zinkernagel, R. M., and Oldstone, M. B. A., J. Exp. Med. 146, 949, 1977. 23. Dunlop, M. B. C., and Blanden, R. V., Cell. Zmmunof. 28, 190, 1977. 24. Woodruff, M. F. A., and Warner, N. L., J. Nut. Cancer Inst. 58, 111, 1977. 25. Serio, C., Gandour, D. M., and Walker, W. S., J. Reticuloendothel. Sot. 25, 97, 1979. 26. Lee, K. C., Kay, J., and Wong, M., Cell. Immunol. 42, 28, 1979. 27. Campbell, M. W., Sholley, M. M., and Miller, G. A., Cell. Immunol. 42, 28, 1980. 28. Russell, S. W., Doe, W. F., and McIntosh, A. T., J. Exp. Med. 146, 1511, 1977. 29. Hibbs, J. B., Taintor, R. R., Chapman, H. A., and Weinberg, J. B., Science 197, 279, 1977. 30. Smialowicz, R. J., and Schwab, J. H., Infect. Immun. 17, 599, 1977. 31. Goz, B., and Walker, K. P., Cancer Rex 36, 4480, 1976.