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Disappearance and reappearance of resident macrophages: Importance in C. parvum-induced tumoricidal activity
CELLULAR
IMMUNOLOGY
90,
179-189 (1985)
Disappearance and Reappearance of Resident Macrophages: Importance in C. parvum-Induced Tumoricidal Activity STEPHEN HASKILL’
AND SUSANNE BECKER
Department of Obstetrics and Gynecology and Department of Microbiology and Immunology, Lineberger Cancer Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 Received June 4, 1984; accepted August 5. 1984 We have investigated the role of resident macrophages in the early tumoricidal response to C. parvum. The bacteria were labeled with FITC and resident cells were labeled in situ with blue fluorescent covaspheres to enable subsequent monitoring of cellular changes by flow cytometry. Macrophages disappeared within 5 hr of administration of bacteria. At 24 hr, fibrinous adhesions containing double labeled macrophages were observed at numerous sites on the peritoneum. Macrophages associated with large numbers of bacteria, levels of beads similar to control animals, and elevated plasminogen activator-like activity did not reappear in washings in significant numbers until 72 hr. Thus, the large bacteria-containing cells that account for the majority of the early tumoricidal activity are likely to be derived from resident macrophages. 0 1985 Academic Press,Inc.
INTRODUCTION Tumoricidal activity develops rapidly in response to intraperitoneal injection of C. pawum. We have described several features of this response which suggest the presence of two populations of macrophages as well as direct cooperation with bacteria containing granulocytes in the activation of one of these cells (l-3). A major population of cytolytic macrophages has been identified and isolated by both cell fractionation and cell sorting techniques. These cells sediment very rapidly and contain intracellular bacteria (1). In contrast to these tumoricidal cells, cytostasis was found to be associated with smaller cells that did not apparently contain intracellular bacteria (2). Granulocytes appear to play a direct role in the activation process. When isolated from the peritoneum 5 hr after stimulation, granulocytes transferred to normal mice elicited a cytolytic macrophage response comparable to that which developed with intact bacteria at a lOOO-fold higher dose of C. parvum. We also observed that phagocytosis of granulocytes containing bacteria was a prominent feature of the macrophages detected immediately prior to the period of maximum cytolytic activity (3). These results suggested that resident macrophages might be responsible for the cytolytic activity present at the peak of the tumoricidal response. In addition, there ’ Address reprints requests and correspondence to Dr. Stephen Haskill, Lineberger Cancer Research Center 237-H, University of North Carolina, Chapel Hill, N.C. 27514. 179 0008-8749/85 $3.00 Copyri&t Q 1985 by Academic Pres. Inc. All rights of reproduction in any form reserved.
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appeared to be a need to study the interactions between bacteria, granulocytes, and the different macrophage populations in the early stagesof the activation process. We have used a dual fluorescent probe approach in combination with flow cytometry to carry out this study. Resident macrophages were labeled in situ with blue fluorescent spheres prior to administration of FITC conjugated C. parvum. Multiparameter analysis of the two fluorescent markers was carried out simultaneously with electronic cell volume determinations. The results demonstrate the importance of resident macrophages in the response to C. parvum and the close association of these cells with granulocytes during the first hours of the response. MATERIALS
AND METHODS
Animals AB6Fi mice, 6 to 8 weeks old were obtained from the Trudeau Institute (Saranac Lake, N.Y.). All animals were used within 3 weeks of arrival in our vivarium. In Situ Labeling Techniques Conjugation of C. parvum with fluorescein isothiocyanate (FITC). Three milliliters of bacteria (Burroughs Wellcome Co., Research Triangle Park, N.C.) containing 21 mg of protein was centrifuged to a pellet and resuspended in 5 ml of the FITC reagent (Molecular Probes, Junction City, Or.). This was made up of FITC at 0.1 mg/ml in 0.1 M borate buffer pH 9.1. The reaction was carried out at 25°C for 30 min. The labeled bacteria were then washed 4 times in PBS pH 7.4 prior to use. Labeled bacteria were stored for several weeks at 4°C. We have previously reported that FITC-labeled bacteria are as stimulatory as nonconjugated C. parvum (1). For flow cytometric analysis the bacteria were stimulated with 365 nm light in order to simultaneously excite both the covaspheres and bacteria. FITC-stained material can be efficiently excited in the uv when two conditions are met. First the conjugation level is high as it was in the present study, thus enhancing the uv excitation spectrum (4). Second, when FITC is in lysozomes, as is to be expected from phagocytized bacteria, the pH falls to 4.5-5.0 thus shifting the excitation spectra toward the uv (5). Labeling of resident macrophages. Blue fluorescent covaspheres (type CX) were obtained as a gift from Dr. David Wood of Covalent Technology Corporation, Ann Arbor, Mich. Excitation maximum for these spheres was 365 nm and emission maximum was 420 nm. It was assumed that these did not have bacterial contamination. All dilutions were made in LPS-free solutions of saline. Endotoxin determinations carried out with the Microbiological Associates (Walkersville, Md.) OCG 1000 assay kit indicated that individual animals received less than 8 pg of LPS. Approximately lo8 spheres were injected per mouse. New syringes and needles were used for each animal. Three days after the beads were given, the appropriate FITC conjugated bacteria or saline vehicle was injected. Instrumentation An Ortho (Ortho Instruments, Westwood, Mass.) ICP-22 flow cytometer modified to include a third photomultiplier tube and a prototype electronic cell volume (ECV) flow chamber was used for all measurements as described previously (6). Cell volume measurements determined according to the Coulter principle were carried
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out in a new 100~pm orifice flow cell and associated electronics provided by Ortho Instruments. Approximate size standardizations were carried out with lo-pm polystyrene spheres. Excitation and Barrier Filters All of the studies described here were carried out using the 365 peak of the mercury arc-lamp with an UG-1 exciter filter. Blue emission reflected from a 500 nm dichroic mirror was read through a 430 f 10 nm interference filter supplied by Pomfret Research Optics (Stamford, Conn.). Green emission was detected with a 585 nm dichroic mirror and a 535 + 35 nm interference hlter (supplied by Ortho Instruments). Data Computation For cytogram (two parameter) analysis, a Radio Shack (Ft. Worth, Tex.) TRS-80 Model 1 microcomputer was interfaced through a parallel input bus with a 3 channel analog-digital converter developed in the Biomedical Microelectronics Laboratory at the University of North Carolina at Chapel Hill. Appropriate software was created which permitted storage of both the parameter used for gating as well as cytogram storage of two other parameters. From these data, histogram analysis on selected portions of a cytogram, histogram subtraction, and calculation of the weighted mean of a histogram could be accomplished. Hard copy was derived from a Qume daisy wheel printer also supplied from Radio Shack Corporation. For the 3 parameter volume, blue and green fluorescence studies, as list mode was not available, we gated on one parameter (for instance, blue fluorescence) and stored data as a 2 parameter cytogram of green fluorescence and volume. Single parameter histograms were run on the gating parameter (volume gated for macrophage size cells) prior to choosing upper or lower limits. Fluorescent Assay for Plasminogen Activator-Like Activity The general techniques have been taken from Do&are and Smith (7), and as modified by ourselves (6, 8). Briefly, 1 mg of the CBZ-Gly-Gly-Arg-MNA substrate (Enzyme Systems Products, P.O. Box 2033, Livermore, Calif.) was dissolved in 20 ~1 of dimethyl formamide to which 1.O ml of 0.1 M MES buffer pH 6.5 was added. In a separate tube, 1.0 mg of NSA was dissolved with 20 ~1 of dimethyl formamide, and 5 ml of MES buffer pH 6.5 was added. Next, 0.3 ml of the NSA mixture was added to 1.0 ml of the substrate solution. This was adequate reagent to stain 5 X IO6cells. The reaction mixture was stable for at least 30 min at room temperature. It was found that a final concentration of 0.0 1% Triton X- 100 markedly enhanced the reaction. The cells were washed once in MES buffer without detergent prior to analysis. Cells were maintained at 4°C prior to and during analysis. The MNANSA reaction product can be excited at 365 nm and has an emission maximum near 540 nm (6). Cell Cultures HeLaS3 (H-S3) were used as target cells in the macrophage cytotoxicity assays. Cells were grown in MEM (Gibco, Grand Island, N.Y.) supplemented with Lglutamine, 20 &ml gentamycin at 10% FBS (KC Biological Co., Kansas City, MO.).
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Macrophage Harvest Cells were obtained by washing the peritoneal cavity with lo-20 ml warm PBS containing 50 units/ml of heparin and were washed in MEM. The percentage of macrophages was determined by differential staining using Wright’s stain. The macrophages were diluted to a concentration of 2.0 X lo6 per ml to be used in the cytotoxicity assay. Target Cell Preparation for Cytotoxicity Assays H-S3 cells were seeded into a 25-cm flask 2 to 3 days before use. Before the assay, medium was poured off and fresh medium containing 5 mCi/ml of [3H]thymidine (Schwartz/Mann, Spring Valley, N.Y.) was added. The cells were then incubated 24 to 30 hr. Radiolabeled cells were dispersed from tissue culture flasks 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 IO4 viable radiolabeled cells per ml. Target cell viability determined by trypan blue exclusion was always greater than 95% after dispersion. One X lo4 target cells per well were used in each macrophage cytotoxicity assay. Adherent Macrophage Cytotoxicity Assay The macrophage cytotoxicity assay was performed in 96-well microtiter plates employing [3H]thymidine prelabeled targets. Macrophages were added to microtiter plate wells in a volume of 0.1 ml at a concentration of 20 X lo5 macrophages per ml. Cells were allowed to adhere for 1 to 2 hr and wells were washed vigorously to leave only adherent cells (>85% macrophages). The assay plates were incubated 18 to 20 hr at 37°C in a humidified CO* incubator. After incubation the percentage specific 3H was calculated by removing 0. I ml of supernatant from each well and counting the amount of activity in a scintillation counter (Packard Instruments Co., Rockville, Md.). Maximum release was determined by placing 1 X lo4 cells directly into aquasol and counting. Spontaneous releasewas determined by incubating target cells in media for the duration of the assayand ranged between 7 and 15% for these assays. The % specific 3H release for each sample was calculated as follows: % Specific release =
% Releasewith macrophages - % Spontaneous release x 100. % Maximum release - % Spontaneous release
Collagenase Treatment of Fibrin Adhesions Rapid disaggregation of the fibrin cellular adhesions was accomplished by a brief 10 min incubation in 0.1% collagenase (Sigma Type 1) in PBS. The cells were washed in RPMI-1649 without FCS prior to analysis. This resulted in a complete digestion of the material into a single cell suspension. We have previously used this same method of digestion to isolate tumor and inflammatory cells from gelled material present in ovarian cancer efhisions (9).
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RESULTS Evolution of the Macrophage Response to C. parvum In order to investigate if resident macrophages phagocytized C. pawum and interacted with granulocytes early in the response, we labeled the phagocytic peritoneal cells in situ with blue fluorescent covaspheres. Control experiments reported in Fig. 1 indicated that the total number of macrophages containing spheres declined slowly over the 7 days of study with an indicated half-life of 18.5 days (mean of 2 experiments). In order to selectively follow the different cells which had ingested C. parvum, the bacteria were conjugated with FITC. Within 5 hr of administration, ~2% macrophages were detected in the washout material. The only fluorescent bacteria present appeared to be present in the inflammatory granulocytes. This disappearance reaction appeared to be of the classical coagulation dependent type (10, 11) as it was prevented by administration of 50 units of heparin at the time of bacterial stimulation. (Resident type cells were 51% of control levels compared to 1.5% without heparin.) An influx of peroxidase positive monocyte-like cells was detected at 24 hr. These cells did not contain spheres and only 6% contained detectable levels of bacteria. Thereafter, the numbers of peroxidase positive cells declined as the total number of macrophages increased by Day 3 of the response. Three and 4 days after stimulation, significant numbers of large macrophagescontaining both blue spheresand bacteria were identified. Quantitation by flow cytometry of the number of blue spheres in C. pawum as compared to control donors demonstrated that there was little if any loss of the marker during the time period studied. In 3 experiments, the cells had a mean fluorescence of 102 + 18% of the uninjected controls (see also Fig. 4). No attempt was made to quantitate the level of green fluorescence in the sphere positive group; visual observation indicated that in agreement with our previous observations, over 90%
HOURS POST SPHERE ADMINISTRATION FIG. 1. Time course of disappearance and reappearance of resident macrophages as well as influx of inflammatory macrophages. Values represent the mean of two separate experiments with three animals per time point. Total macrophage numbers were determined on Giemsa-stainedcytocentrifuge preparations.
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of these cells were positive for bacteria at Days 3 and 4 in two different time course studies. Influence of Sphere Administration on General Inflammatory Characteristics In order to have as little influence on resident macrophage function as possible, sphere administration was carried out under LPS-free conditions. Endotoxin determinations indicated individual animals received less than 8 pg of LPS. Influx of inflammatory granulocytes and nonspecific activation of macrophages did not appear in control mice not injected with bacteria but receiving the other agents. Less than 2 X lo5 granulocytes were observed at any time point in the sphere control group as shown by differential counts on giemsa stained preparations (Table 1). Tumoricidal activity assayed against HeLa cells was elevated in the group of animals receiving both C. parvum and spheres (Table 2). Sphere administration by itself did not significantly elevate background cytotoxicity over that in control mice injected only with saline. Flow Cytometric Analysis of Bacteria and Sphere Containing Cells at Dl@erent Time Points Labeling of cells with spheres and/or fluorescent bacteria permitted us to monitor changes in cell size of resident and inflammatory macrophages. This parameter is related both to macrophage maturation as well as to the sedimentation velocity separation characteristics previously used by our group in identifying the cytotoxic cells at Day 4 (1). Macrophage size in the control sphere injected animals was constant throughout the study. These cells were compared daily with the lo-pm bead standards. At 5 hr, the only labeled cells were granulocytes which contained solely the FITC-labeled bacteria (Fig. 2). Similar types of analyses were carried out to determine cell volume profiles for both sphere positive and negative cells at each of the time points (Fig. 2). Cells containing both spheres and bacteria were similar in size (twice the volume of residents) at the 3 time points. The volume of macrophages containing only bacteria was restricted to smaller cells with a peak size being obtained at 48 hr.
TABLE 1 In Viva Labeling with Spheres Does Not Induce Inflammation Total granulocytes per animal (X10e5)’ Time W
Control
Spheres
C. parvum + spheres
5 24 48 72 96
0.6 ND ND ND 0.5
0.5 1.8 1.0 0.5 0.2
61 45 16 10 10
o Mean of three animals per time point.
CYTOMETRIC
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ANALYSIS OF MACROPHAGE ACTIVATION TABLE 2
Tumoricidal Activity of Various Control and C. purvum Stimulated Peritoneal Macrophages” % Spec. isotope releaseb
Control Saline + spheres C. parvum C. parvum + saline C. parvum + spheres
5.3 * 9.3 + 46.6 k 46.5 f 64.7 f
1.1 2.8 8.2 9.2 14.4
4.0 f 5.0 f 13.0 k 18.0 + 27.7 k
1.8 2.8 3.2 4.1 10.2
4.3 f 2.3 f 5.7 + 10.0 f 9.7 f
2.1 1.5 1.1 1.4 1.8
’ Efkctor to target cell ratio. bMean and standard deviation of three experiments,
Fibrinous Adhesions in the Peritoneum Contain Resident Macrophages At 24 hr, fibrinous adhesions were detected at several points in the abdomen. Single cell preparations of these areas were readily accomplished by gentle digestion of the material in collagenase. Differential counts of the cells indicated that the cells were either macrophages (30%) or granulocytes (70’S), both of which contained bacteria. Flow cytometric analysis based on intensity of fluorescence due to FITC also indicated that two populations of cells were present (Fig. 3). The one with the lowest green fluorescence had the same cell volume as expected for granulocytes. The other cells contained both spheresand large amounts of bacteria (>95% positive for both markers). The cell volume profile indicated the presence of cells with the same size as resident macrophages as well as much larger cells. These data indicate a close association of both granulocytes and resident macrophages in fibrin-like
FIG. 2. Volume analysis of two distinct macrophage populations present at different times after C. 0) or positive for the blue fluorescent spheres (0 - - - 0). Whereas most of the sphere containing cells contained bacteria (>90%), the nonsphere,bacteria containing macrophagesre.presemeda much smaller proportion of the inflammatory population (6-249s). The data at 5 hr are representative of the cell volume of gratmlocytes (PMN’s). Insufficient macrophages were detected to analyze at this time point. The volume values for control animals receiving only spheres were standardized with IO-pm spheres.
purvum stimulation. Ceg volumes were determined on cells negative (0 -
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.
AND BECKER
-..
b102030405060708090tooID120 CELL VOLUME
FIG. 3. Cell volume analysis of bacteria and sphere containing cells present in the fibrin adhesions 24 hr after C. purvwm stimulation. Cell volume values were determined on the lowest 5% of green tluorescence (Low Cp) and the highest 90% (High Cp). Blue fluorescence and volume values were stored for both ranges of green fluorescence. Control donors receiving only spheres 4 days previously were used for comparison. The high Cp group was composed of a minor population of cells the size of resident macrophages as well as cells that were markedly enlarged.
adhesions which is not surprising in view of our knowledge that bacteria containing granulocytes are very efficient at activating macrophages (3).
Reappearing Resident Macrophages Have Enhanced Plasminogen Activator-Like Activity Release of the macrophages from fibrin gels probably requires the development of fibrinolytic activity which can be derived directly from macrophages. Plasminogen activator-like activity can be determined with the active center modelled substrate Gly-Gly-Arg-MNA in combination with the NSA coupling reaction (7). Plow
0
IO 20 30 40 50 60
PLASMINOGEN
7;
80 9b KiO Ilb Ii0
ACTIVATOR
FIG. 4. Cell associatedplasminogen activator-like activity determined with the synthetic substrate CBZGly-Gly-Arg-MNA. Green fluorescence was gated on cell volumes the size of macrophages and blue fluorescence..Normal resident macrophages (8 - - - *) were grated only on cell size. Mean fluorescence intensity for both the plasminogen activator-like activity (P.Act.) and blue spheres are.included. Control (0 - - - 0) and C. parvum (0 0) donors.
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cytometric analysis of this reaction in combination with the quantitation of blue spheres was carried out to assessthe level of cell-associated fibrinolytic activity in the reemergent resident cells. The data shown in Fig. 4 clearly indicate the enhanced level of enzyme activity in these cells. The presence of spheres in the control mice caused only a modest absolute increase in activity. DISCUSSION We have demonstrated the disappearance and subsequent reappearance of resident macrophages in the response to C. parvum through the use of fluorescent in situ labeling of both cells and bacteria. Our data demonstrate that resident macrophages and granulocytes containing intracellular bacteria are placed in close contact as a result of the rapid coagulation response to the bacterial inoculation. This close proximity of granulocytes and macrophages appears to be important for induction of macrophage tumoricidal activity in this model system. We have previously shown that granulocytes containing C. purvum induce a typical profile of macrophage activation on transfer to normal mice (12) and large peroxidase negative cytolytic cells appearing 3-5 days after inoculation are characterized by the presence of intracellular bacteria ( 1). In spite of the absence of detectable endotoxin, the introduction of spheres is not completely without influence on the resident macrophage; however, these effects appear to be minor and of small consequence to the overall concept of resident cell involvement in the cytolytic response. Various characteristics of the resident cell were analyzed to evaluate any effects on their general behavior. The half-life of resident macrophages in mice appears to be about 25 days based on blood monocyte turnover times and distribution data of van Furth et al. ( 13). The values for turnover of sphere-taggedcells we observed (13 and 24 days in two separate experiments) are in agreement with expected times. Analysis of the cell volume data also showed no change during this period. The background level of plasminogen-activator activity was modestly enhanced by the presence of the spheres. This is not surprising in view of the reports of Gordon et al. (14) and Vassalli and Reich (15) that latex sphere ingestion heightens neutral protease secretion. While sphere administration alone did not lead to tumoricidal activity, we did observe an increase in cytolytic activity in the C. purvum stimulated group. Aggregation of peritoneal macrophages during the disappearance reaction has recently been elegantly studied by a combination scanning and thin section electron microscopy approach ( 16). Bacterial toxins and adjuvant induced a rapid adherance of the macrophage, followed by large numbers of granulocytes adhering to the mesothelial surface. The fibrin investments containing predominantly granulocytes and macrophages did not break down until 72 hr. These observations parallel our own results and further support the concept of coagulation, the close association of macrophages with granulocytes, and the role of induced fibrinolysis in releasing the trapped cells. We do not know if the resident macrophages are directly responsible for procoagulant activity. Resident macrophages have been shown to rapidly disappear from the peritoneal fluid following stimulation with bacterial toxins. Abundant evidence suggeststhat this is dependent upon the initiation of coagulation as it can be directly triggered by thrombin (17) and inhibited by anticoagulants such as heparin and warfarin (10). Nelson (10) has reported that macrophage adherance
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seemsto precede the actual appearance of fibrin on the cells. Chapman et al. (11) have recently reported that in BCG immune mice, the disappearance reaction can be initiated by endotoxin only in the endotoxin-responsive C3H/HeN strain. Furthermore, procoagulant activity was detected in vitro only with endotoxin stimulation of the C3H/HeN macrophages suggesting a direct role of these cells in the disappearance reaction. A plausible explanation for the different stagesin macrophage activation induced by C. purvum, based upon the present data and previous publications from us (l3), includes induction of a coagulation response which brings into close proximity bacteria, granulocytes, and resident macrophages. Migration of some of these cells is likely to occur through the mesothelial stoma into the lymphatics (16) thus stimulating cellular immunity at distant sites. At the sametime, resident macrophages, still retaining bacteria and trapped in the fibrin gels, are induced to develop a series of neutral proteases needed to degrade the fibrin and thus produce the neutral protease cytolytic factor, known to have anti-tumor activity in this model (18, 19). While resident-derived macrophages account for the early cytotoxic response, inflammatory macrophagesbecome activated and account for the sustained cytotoxic response induced by C. parvum. The present study has not addressed the question of the role of intracellular bacteria in activation of these inflammatory macrophages. A significant proportion of the small cells contains bacteria at the early time points studied here. However, it seems likely that as cytotoxicity at Day 4 is associated only with the largest macrophages, whereas smaller ones are cytostatic (2), that different steps in activation, presumably involving in situ lymphokine production, account for activation of these cells to the cytotoxic state. A similar involvement of coagulation induced disappearance and activation by intracellular bacteria may also occur upon repeated stimulation by bacteria. We have recently used similar labeling techniques to demonstrate that large macrophages “in residence” at the time of a third stimulation with BCG similarly disappear and reappear 48-72 hr later (S. Haskill, and D. 0. Adams, manuscript in preparation). These large macrophages are directly cytotoxic, whereas the smaller inflammatory cells require the addition of LPS for maximum tumoricidal activity. ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grant CA29589. The authors wish to thank Suzanne Morris for her interest and help in the early aspects of this work, Dr. Yancey Gillespie for carrying out the endotoxin determinations, and Willia Bell for handling the animal immunizations. We also thank Linda McAlister for assistancein preparing the manuscript.
REFERENCES 1. Chap-es,S., and Haskill, S., Cell. Immunol. 70, 65, 1982. 2. Chapes, S. K., and Haskill, S., Cell. Immunol. 76, 49, 1983. 3. Chap+ S. K., and Haskill, S., Cell. Immunol. 75, 367, 1983. 4. Schauenstein,K., Bock, G., and Wick, G., In “Immunofluorescence and Related Staining Techniques” (W. Knapp, K. Holubar, and G. Wick, Eds.), p. 81. Elsevier/North-Holland Biomedical Press, New York, 1978. 5. Murphy, R. F., Powers, S., Verderame, M., Cantor, C. R., and Pollack, R., Cytometry 2, 402, 1982. 6. Haskill, S., Becker, S., Johnson, T., Marro, D., Nelson, K., and Propst, R. H., Cytometry 3, 359, 1983. 7. Dolbeare, F. A., and Smith, R. E., Clin. Chem. 23, 1485, 1977.
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8. Haskill, S., and Becker, S., .I. Reticuloendothel. Sot. 32, 213, 1982. 9. Haskill, S., Becker, S., Fowler, W., and Walton, L., Bit. .Z.Cancer 45, 728, 1982. IO. Nelson, D. S., Zmmunology 9, 219, 1965. 1I. Chapman, H. A., Vavrin, Z., and Hibbs, J. B., .Z.Zmmunol. 130,261, 1983. 12. Chapes, S. K., and Haskill, S., Cancer Res. 44, 3 1, 1984. 13. van Furth, R., Diesselhoffden Dulk, M. M. C., Raebum, J. A., van Zwet, T. L., Crofion, R., and Vlusse van Oud Alblas, A., In “Mononuclear Phagocytes, Functional Aspects” (R. van Furth, Ed.), p. 279. Martinus Nijhoff, The Hague, 1980. 14. Gordon, S., Unkeless, J. C., and Cohn, Z. A., J. Exp. Med. 140, 995, 1974. 15. Vassalli, J. D., and Reich, E., J. Exp. Med. 145, 429, 1977. 16. Leak, L. V., Lab. Invest. 48, 479, 1983. 17. Jokay, I., and Karczag, E., Experientia 29, 334, 1972. 18. Adams, D. O., Kao, K. J., Farb, R., and Pizzo, S. V., J. Zmmunol. 124, 293, 1980. 19. Adams, D. O., and Marino, P. A., In “Contemporary Hematology/Oncology.” Plenum, New York (in press).
IMMUNOLOGY
90,
179-189 (1985)
Disappearance and Reappearance of Resident Macrophages: Importance in C. parvum-Induced Tumoricidal Activity STEPHEN HASKILL’
AND SUSANNE BECKER
Department of Obstetrics and Gynecology and Department of Microbiology and Immunology, Lineberger Cancer Research Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27514 Received June 4, 1984; accepted August 5. 1984 We have investigated the role of resident macrophages in the early tumoricidal response to C. parvum. The bacteria were labeled with FITC and resident cells were labeled in situ with blue fluorescent covaspheres to enable subsequent monitoring of cellular changes by flow cytometry. Macrophages disappeared within 5 hr of administration of bacteria. At 24 hr, fibrinous adhesions containing double labeled macrophages were observed at numerous sites on the peritoneum. Macrophages associated with large numbers of bacteria, levels of beads similar to control animals, and elevated plasminogen activator-like activity did not reappear in washings in significant numbers until 72 hr. Thus, the large bacteria-containing cells that account for the majority of the early tumoricidal activity are likely to be derived from resident macrophages. 0 1985 Academic Press,Inc.
INTRODUCTION Tumoricidal activity develops rapidly in response to intraperitoneal injection of C. pawum. We have described several features of this response which suggest the presence of two populations of macrophages as well as direct cooperation with bacteria containing granulocytes in the activation of one of these cells (l-3). A major population of cytolytic macrophages has been identified and isolated by both cell fractionation and cell sorting techniques. These cells sediment very rapidly and contain intracellular bacteria (1). In contrast to these tumoricidal cells, cytostasis was found to be associated with smaller cells that did not apparently contain intracellular bacteria (2). Granulocytes appear to play a direct role in the activation process. When isolated from the peritoneum 5 hr after stimulation, granulocytes transferred to normal mice elicited a cytolytic macrophage response comparable to that which developed with intact bacteria at a lOOO-fold higher dose of C. parvum. We also observed that phagocytosis of granulocytes containing bacteria was a prominent feature of the macrophages detected immediately prior to the period of maximum cytolytic activity (3). These results suggested that resident macrophages might be responsible for the cytolytic activity present at the peak of the tumoricidal response. In addition, there ’ Address reprints requests and correspondence to Dr. Stephen Haskill, Lineberger Cancer Research Center 237-H, University of North Carolina, Chapel Hill, N.C. 27514. 179 0008-8749/85 $3.00 Copyri&t Q 1985 by Academic Pres. Inc. All rights of reproduction in any form reserved.
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appeared to be a need to study the interactions between bacteria, granulocytes, and the different macrophage populations in the early stagesof the activation process. We have used a dual fluorescent probe approach in combination with flow cytometry to carry out this study. Resident macrophages were labeled in situ with blue fluorescent spheres prior to administration of FITC conjugated C. parvum. Multiparameter analysis of the two fluorescent markers was carried out simultaneously with electronic cell volume determinations. The results demonstrate the importance of resident macrophages in the response to C. parvum and the close association of these cells with granulocytes during the first hours of the response. MATERIALS
AND METHODS
Animals AB6Fi mice, 6 to 8 weeks old were obtained from the Trudeau Institute (Saranac Lake, N.Y.). All animals were used within 3 weeks of arrival in our vivarium. In Situ Labeling Techniques Conjugation of C. parvum with fluorescein isothiocyanate (FITC). Three milliliters of bacteria (Burroughs Wellcome Co., Research Triangle Park, N.C.) containing 21 mg of protein was centrifuged to a pellet and resuspended in 5 ml of the FITC reagent (Molecular Probes, Junction City, Or.). This was made up of FITC at 0.1 mg/ml in 0.1 M borate buffer pH 9.1. The reaction was carried out at 25°C for 30 min. The labeled bacteria were then washed 4 times in PBS pH 7.4 prior to use. Labeled bacteria were stored for several weeks at 4°C. We have previously reported that FITC-labeled bacteria are as stimulatory as nonconjugated C. parvum (1). For flow cytometric analysis the bacteria were stimulated with 365 nm light in order to simultaneously excite both the covaspheres and bacteria. FITC-stained material can be efficiently excited in the uv when two conditions are met. First the conjugation level is high as it was in the present study, thus enhancing the uv excitation spectrum (4). Second, when FITC is in lysozomes, as is to be expected from phagocytized bacteria, the pH falls to 4.5-5.0 thus shifting the excitation spectra toward the uv (5). Labeling of resident macrophages. Blue fluorescent covaspheres (type CX) were obtained as a gift from Dr. David Wood of Covalent Technology Corporation, Ann Arbor, Mich. Excitation maximum for these spheres was 365 nm and emission maximum was 420 nm. It was assumed that these did not have bacterial contamination. All dilutions were made in LPS-free solutions of saline. Endotoxin determinations carried out with the Microbiological Associates (Walkersville, Md.) OCG 1000 assay kit indicated that individual animals received less than 8 pg of LPS. Approximately lo8 spheres were injected per mouse. New syringes and needles were used for each animal. Three days after the beads were given, the appropriate FITC conjugated bacteria or saline vehicle was injected. Instrumentation An Ortho (Ortho Instruments, Westwood, Mass.) ICP-22 flow cytometer modified to include a third photomultiplier tube and a prototype electronic cell volume (ECV) flow chamber was used for all measurements as described previously (6). Cell volume measurements determined according to the Coulter principle were carried
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out in a new 100~pm orifice flow cell and associated electronics provided by Ortho Instruments. Approximate size standardizations were carried out with lo-pm polystyrene spheres. Excitation and Barrier Filters All of the studies described here were carried out using the 365 peak of the mercury arc-lamp with an UG-1 exciter filter. Blue emission reflected from a 500 nm dichroic mirror was read through a 430 f 10 nm interference filter supplied by Pomfret Research Optics (Stamford, Conn.). Green emission was detected with a 585 nm dichroic mirror and a 535 + 35 nm interference hlter (supplied by Ortho Instruments). Data Computation For cytogram (two parameter) analysis, a Radio Shack (Ft. Worth, Tex.) TRS-80 Model 1 microcomputer was interfaced through a parallel input bus with a 3 channel analog-digital converter developed in the Biomedical Microelectronics Laboratory at the University of North Carolina at Chapel Hill. Appropriate software was created which permitted storage of both the parameter used for gating as well as cytogram storage of two other parameters. From these data, histogram analysis on selected portions of a cytogram, histogram subtraction, and calculation of the weighted mean of a histogram could be accomplished. Hard copy was derived from a Qume daisy wheel printer also supplied from Radio Shack Corporation. For the 3 parameter volume, blue and green fluorescence studies, as list mode was not available, we gated on one parameter (for instance, blue fluorescence) and stored data as a 2 parameter cytogram of green fluorescence and volume. Single parameter histograms were run on the gating parameter (volume gated for macrophage size cells) prior to choosing upper or lower limits. Fluorescent Assay for Plasminogen Activator-Like Activity The general techniques have been taken from Do&are and Smith (7), and as modified by ourselves (6, 8). Briefly, 1 mg of the CBZ-Gly-Gly-Arg-MNA substrate (Enzyme Systems Products, P.O. Box 2033, Livermore, Calif.) was dissolved in 20 ~1 of dimethyl formamide to which 1.O ml of 0.1 M MES buffer pH 6.5 was added. In a separate tube, 1.0 mg of NSA was dissolved with 20 ~1 of dimethyl formamide, and 5 ml of MES buffer pH 6.5 was added. Next, 0.3 ml of the NSA mixture was added to 1.0 ml of the substrate solution. This was adequate reagent to stain 5 X IO6cells. The reaction mixture was stable for at least 30 min at room temperature. It was found that a final concentration of 0.0 1% Triton X- 100 markedly enhanced the reaction. The cells were washed once in MES buffer without detergent prior to analysis. Cells were maintained at 4°C prior to and during analysis. The MNANSA reaction product can be excited at 365 nm and has an emission maximum near 540 nm (6). Cell Cultures HeLaS3 (H-S3) were used as target cells in the macrophage cytotoxicity assays. Cells were grown in MEM (Gibco, Grand Island, N.Y.) supplemented with Lglutamine, 20 &ml gentamycin at 10% FBS (KC Biological Co., Kansas City, MO.).
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Macrophage Harvest Cells were obtained by washing the peritoneal cavity with lo-20 ml warm PBS containing 50 units/ml of heparin and were washed in MEM. The percentage of macrophages was determined by differential staining using Wright’s stain. The macrophages were diluted to a concentration of 2.0 X lo6 per ml to be used in the cytotoxicity assay. Target Cell Preparation for Cytotoxicity Assays H-S3 cells were seeded into a 25-cm flask 2 to 3 days before use. Before the assay, medium was poured off and fresh medium containing 5 mCi/ml of [3H]thymidine (Schwartz/Mann, Spring Valley, N.Y.) was added. The cells were then incubated 24 to 30 hr. Radiolabeled cells were dispersed from tissue culture flasks 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 IO4 viable radiolabeled cells per ml. Target cell viability determined by trypan blue exclusion was always greater than 95% after dispersion. One X lo4 target cells per well were used in each macrophage cytotoxicity assay. Adherent Macrophage Cytotoxicity Assay The macrophage cytotoxicity assay was performed in 96-well microtiter plates employing [3H]thymidine prelabeled targets. Macrophages were added to microtiter plate wells in a volume of 0.1 ml at a concentration of 20 X lo5 macrophages per ml. Cells were allowed to adhere for 1 to 2 hr and wells were washed vigorously to leave only adherent cells (>85% macrophages). The assay plates were incubated 18 to 20 hr at 37°C in a humidified CO* incubator. After incubation the percentage specific 3H was calculated by removing 0. I ml of supernatant from each well and counting the amount of activity in a scintillation counter (Packard Instruments Co., Rockville, Md.). Maximum release was determined by placing 1 X lo4 cells directly into aquasol and counting. Spontaneous releasewas determined by incubating target cells in media for the duration of the assayand ranged between 7 and 15% for these assays. The % specific 3H release for each sample was calculated as follows: % Specific release =
% Releasewith macrophages - % Spontaneous release x 100. % Maximum release - % Spontaneous release
Collagenase Treatment of Fibrin Adhesions Rapid disaggregation of the fibrin cellular adhesions was accomplished by a brief 10 min incubation in 0.1% collagenase (Sigma Type 1) in PBS. The cells were washed in RPMI-1649 without FCS prior to analysis. This resulted in a complete digestion of the material into a single cell suspension. We have previously used this same method of digestion to isolate tumor and inflammatory cells from gelled material present in ovarian cancer efhisions (9).
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RESULTS Evolution of the Macrophage Response to C. parvum In order to investigate if resident macrophages phagocytized C. pawum and interacted with granulocytes early in the response, we labeled the phagocytic peritoneal cells in situ with blue fluorescent covaspheres. Control experiments reported in Fig. 1 indicated that the total number of macrophages containing spheres declined slowly over the 7 days of study with an indicated half-life of 18.5 days (mean of 2 experiments). In order to selectively follow the different cells which had ingested C. parvum, the bacteria were conjugated with FITC. Within 5 hr of administration, ~2% macrophages were detected in the washout material. The only fluorescent bacteria present appeared to be present in the inflammatory granulocytes. This disappearance reaction appeared to be of the classical coagulation dependent type (10, 11) as it was prevented by administration of 50 units of heparin at the time of bacterial stimulation. (Resident type cells were 51% of control levels compared to 1.5% without heparin.) An influx of peroxidase positive monocyte-like cells was detected at 24 hr. These cells did not contain spheres and only 6% contained detectable levels of bacteria. Thereafter, the numbers of peroxidase positive cells declined as the total number of macrophages increased by Day 3 of the response. Three and 4 days after stimulation, significant numbers of large macrophagescontaining both blue spheresand bacteria were identified. Quantitation by flow cytometry of the number of blue spheres in C. pawum as compared to control donors demonstrated that there was little if any loss of the marker during the time period studied. In 3 experiments, the cells had a mean fluorescence of 102 + 18% of the uninjected controls (see also Fig. 4). No attempt was made to quantitate the level of green fluorescence in the sphere positive group; visual observation indicated that in agreement with our previous observations, over 90%
HOURS POST SPHERE ADMINISTRATION FIG. 1. Time course of disappearance and reappearance of resident macrophages as well as influx of inflammatory macrophages. Values represent the mean of two separate experiments with three animals per time point. Total macrophage numbers were determined on Giemsa-stainedcytocentrifuge preparations.
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of these cells were positive for bacteria at Days 3 and 4 in two different time course studies. Influence of Sphere Administration on General Inflammatory Characteristics In order to have as little influence on resident macrophage function as possible, sphere administration was carried out under LPS-free conditions. Endotoxin determinations indicated individual animals received less than 8 pg of LPS. Influx of inflammatory granulocytes and nonspecific activation of macrophages did not appear in control mice not injected with bacteria but receiving the other agents. Less than 2 X lo5 granulocytes were observed at any time point in the sphere control group as shown by differential counts on giemsa stained preparations (Table 1). Tumoricidal activity assayed against HeLa cells was elevated in the group of animals receiving both C. parvum and spheres (Table 2). Sphere administration by itself did not significantly elevate background cytotoxicity over that in control mice injected only with saline. Flow Cytometric Analysis of Bacteria and Sphere Containing Cells at Dl@erent Time Points Labeling of cells with spheres and/or fluorescent bacteria permitted us to monitor changes in cell size of resident and inflammatory macrophages. This parameter is related both to macrophage maturation as well as to the sedimentation velocity separation characteristics previously used by our group in identifying the cytotoxic cells at Day 4 (1). Macrophage size in the control sphere injected animals was constant throughout the study. These cells were compared daily with the lo-pm bead standards. At 5 hr, the only labeled cells were granulocytes which contained solely the FITC-labeled bacteria (Fig. 2). Similar types of analyses were carried out to determine cell volume profiles for both sphere positive and negative cells at each of the time points (Fig. 2). Cells containing both spheres and bacteria were similar in size (twice the volume of residents) at the 3 time points. The volume of macrophages containing only bacteria was restricted to smaller cells with a peak size being obtained at 48 hr.
TABLE 1 In Viva Labeling with Spheres Does Not Induce Inflammation Total granulocytes per animal (X10e5)’ Time W
Control
Spheres
C. parvum + spheres
5 24 48 72 96
0.6 ND ND ND 0.5
0.5 1.8 1.0 0.5 0.2
61 45 16 10 10
o Mean of three animals per time point.
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Tumoricidal Activity of Various Control and C. purvum Stimulated Peritoneal Macrophages” % Spec. isotope releaseb
Control Saline + spheres C. parvum C. parvum + saline C. parvum + spheres
5.3 * 9.3 + 46.6 k 46.5 f 64.7 f
1.1 2.8 8.2 9.2 14.4
4.0 f 5.0 f 13.0 k 18.0 + 27.7 k
1.8 2.8 3.2 4.1 10.2
4.3 f 2.3 f 5.7 + 10.0 f 9.7 f
2.1 1.5 1.1 1.4 1.8
’ Efkctor to target cell ratio. bMean and standard deviation of three experiments,
Fibrinous Adhesions in the Peritoneum Contain Resident Macrophages At 24 hr, fibrinous adhesions were detected at several points in the abdomen. Single cell preparations of these areas were readily accomplished by gentle digestion of the material in collagenase. Differential counts of the cells indicated that the cells were either macrophages (30%) or granulocytes (70’S), both of which contained bacteria. Flow cytometric analysis based on intensity of fluorescence due to FITC also indicated that two populations of cells were present (Fig. 3). The one with the lowest green fluorescence had the same cell volume as expected for granulocytes. The other cells contained both spheresand large amounts of bacteria (>95% positive for both markers). The cell volume profile indicated the presence of cells with the same size as resident macrophages as well as much larger cells. These data indicate a close association of both granulocytes and resident macrophages in fibrin-like
FIG. 2. Volume analysis of two distinct macrophage populations present at different times after C. 0) or positive for the blue fluorescent spheres (0 - - - 0). Whereas most of the sphere containing cells contained bacteria (>90%), the nonsphere,bacteria containing macrophagesre.presemeda much smaller proportion of the inflammatory population (6-249s). The data at 5 hr are representative of the cell volume of gratmlocytes (PMN’s). Insufficient macrophages were detected to analyze at this time point. The volume values for control animals receiving only spheres were standardized with IO-pm spheres.
purvum stimulation. Ceg volumes were determined on cells negative (0 -
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-..
b102030405060708090tooID120 CELL VOLUME
FIG. 3. Cell volume analysis of bacteria and sphere containing cells present in the fibrin adhesions 24 hr after C. purvwm stimulation. Cell volume values were determined on the lowest 5% of green tluorescence (Low Cp) and the highest 90% (High Cp). Blue fluorescence and volume values were stored for both ranges of green fluorescence. Control donors receiving only spheres 4 days previously were used for comparison. The high Cp group was composed of a minor population of cells the size of resident macrophages as well as cells that were markedly enlarged.
adhesions which is not surprising in view of our knowledge that bacteria containing granulocytes are very efficient at activating macrophages (3).
Reappearing Resident Macrophages Have Enhanced Plasminogen Activator-Like Activity Release of the macrophages from fibrin gels probably requires the development of fibrinolytic activity which can be derived directly from macrophages. Plasminogen activator-like activity can be determined with the active center modelled substrate Gly-Gly-Arg-MNA in combination with the NSA coupling reaction (7). Plow
0
IO 20 30 40 50 60
PLASMINOGEN
7;
80 9b KiO Ilb Ii0
ACTIVATOR
FIG. 4. Cell associatedplasminogen activator-like activity determined with the synthetic substrate CBZGly-Gly-Arg-MNA. Green fluorescence was gated on cell volumes the size of macrophages and blue fluorescence..Normal resident macrophages (8 - - - *) were grated only on cell size. Mean fluorescence intensity for both the plasminogen activator-like activity (P.Act.) and blue spheres are.included. Control (0 - - - 0) and C. parvum (0 0) donors.
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cytometric analysis of this reaction in combination with the quantitation of blue spheres was carried out to assessthe level of cell-associated fibrinolytic activity in the reemergent resident cells. The data shown in Fig. 4 clearly indicate the enhanced level of enzyme activity in these cells. The presence of spheres in the control mice caused only a modest absolute increase in activity. DISCUSSION We have demonstrated the disappearance and subsequent reappearance of resident macrophages in the response to C. parvum through the use of fluorescent in situ labeling of both cells and bacteria. Our data demonstrate that resident macrophages and granulocytes containing intracellular bacteria are placed in close contact as a result of the rapid coagulation response to the bacterial inoculation. This close proximity of granulocytes and macrophages appears to be important for induction of macrophage tumoricidal activity in this model system. We have previously shown that granulocytes containing C. purvum induce a typical profile of macrophage activation on transfer to normal mice (12) and large peroxidase negative cytolytic cells appearing 3-5 days after inoculation are characterized by the presence of intracellular bacteria ( 1). In spite of the absence of detectable endotoxin, the introduction of spheres is not completely without influence on the resident macrophage; however, these effects appear to be minor and of small consequence to the overall concept of resident cell involvement in the cytolytic response. Various characteristics of the resident cell were analyzed to evaluate any effects on their general behavior. The half-life of resident macrophages in mice appears to be about 25 days based on blood monocyte turnover times and distribution data of van Furth et al. ( 13). The values for turnover of sphere-taggedcells we observed (13 and 24 days in two separate experiments) are in agreement with expected times. Analysis of the cell volume data also showed no change during this period. The background level of plasminogen-activator activity was modestly enhanced by the presence of the spheres. This is not surprising in view of the reports of Gordon et al. (14) and Vassalli and Reich (15) that latex sphere ingestion heightens neutral protease secretion. While sphere administration alone did not lead to tumoricidal activity, we did observe an increase in cytolytic activity in the C. purvum stimulated group. Aggregation of peritoneal macrophages during the disappearance reaction has recently been elegantly studied by a combination scanning and thin section electron microscopy approach ( 16). Bacterial toxins and adjuvant induced a rapid adherance of the macrophage, followed by large numbers of granulocytes adhering to the mesothelial surface. The fibrin investments containing predominantly granulocytes and macrophages did not break down until 72 hr. These observations parallel our own results and further support the concept of coagulation, the close association of macrophages with granulocytes, and the role of induced fibrinolysis in releasing the trapped cells. We do not know if the resident macrophages are directly responsible for procoagulant activity. Resident macrophages have been shown to rapidly disappear from the peritoneal fluid following stimulation with bacterial toxins. Abundant evidence suggeststhat this is dependent upon the initiation of coagulation as it can be directly triggered by thrombin (17) and inhibited by anticoagulants such as heparin and warfarin (10). Nelson (10) has reported that macrophage adherance
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seemsto precede the actual appearance of fibrin on the cells. Chapman et al. (11) have recently reported that in BCG immune mice, the disappearance reaction can be initiated by endotoxin only in the endotoxin-responsive C3H/HeN strain. Furthermore, procoagulant activity was detected in vitro only with endotoxin stimulation of the C3H/HeN macrophages suggesting a direct role of these cells in the disappearance reaction. A plausible explanation for the different stagesin macrophage activation induced by C. purvum, based upon the present data and previous publications from us (l3), includes induction of a coagulation response which brings into close proximity bacteria, granulocytes, and resident macrophages. Migration of some of these cells is likely to occur through the mesothelial stoma into the lymphatics (16) thus stimulating cellular immunity at distant sites. At the sametime, resident macrophages, still retaining bacteria and trapped in the fibrin gels, are induced to develop a series of neutral proteases needed to degrade the fibrin and thus produce the neutral protease cytolytic factor, known to have anti-tumor activity in this model (18, 19). While resident-derived macrophages account for the early cytotoxic response, inflammatory macrophagesbecome activated and account for the sustained cytotoxic response induced by C. parvum. The present study has not addressed the question of the role of intracellular bacteria in activation of these inflammatory macrophages. A significant proportion of the small cells contains bacteria at the early time points studied here. However, it seems likely that as cytotoxicity at Day 4 is associated only with the largest macrophages, whereas smaller ones are cytostatic (2), that different steps in activation, presumably involving in situ lymphokine production, account for activation of these cells to the cytotoxic state. A similar involvement of coagulation induced disappearance and activation by intracellular bacteria may also occur upon repeated stimulation by bacteria. We have recently used similar labeling techniques to demonstrate that large macrophages “in residence” at the time of a third stimulation with BCG similarly disappear and reappear 48-72 hr later (S. Haskill, and D. 0. Adams, manuscript in preparation). These large macrophages are directly cytotoxic, whereas the smaller inflammatory cells require the addition of LPS for maximum tumoricidal activity. ACKNOWLEDGMENTS This work was supported by the National Institutes of Health Grant CA29589. The authors wish to thank Suzanne Morris for her interest and help in the early aspects of this work, Dr. Yancey Gillespie for carrying out the endotoxin determinations, and Willia Bell for handling the animal immunizations. We also thank Linda McAlister for assistancein preparing the manuscript.
REFERENCES 1. Chap-es,S., and Haskill, S., Cell. Immunol. 70, 65, 1982. 2. Chapes, S. K., and Haskill, S., Cell. Immunol. 76, 49, 1983. 3. Chap+ S. K., and Haskill, S., Cell. Immunol. 75, 367, 1983. 4. Schauenstein,K., Bock, G., and Wick, G., In “Immunofluorescence and Related Staining Techniques” (W. Knapp, K. Holubar, and G. Wick, Eds.), p. 81. Elsevier/North-Holland Biomedical Press, New York, 1978. 5. Murphy, R. F., Powers, S., Verderame, M., Cantor, C. R., and Pollack, R., Cytometry 2, 402, 1982. 6. Haskill, S., Becker, S., Johnson, T., Marro, D., Nelson, K., and Propst, R. H., Cytometry 3, 359, 1983. 7. Dolbeare, F. A., and Smith, R. E., Clin. Chem. 23, 1485, 1977.
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8. Haskill, S., and Becker, S., .I. Reticuloendothel. Sot. 32, 213, 1982. 9. Haskill, S., Becker, S., Fowler, W., and Walton, L., Bit. .Z.Cancer 45, 728, 1982. IO. Nelson, D. S., Zmmunology 9, 219, 1965. 1I. Chapman, H. A., Vavrin, Z., and Hibbs, J. B., .Z.Zmmunol. 130,261, 1983. 12. Chapes, S. K., and Haskill, S., Cancer Res. 44, 3 1, 1984. 13. van Furth, R., Diesselhoffden Dulk, M. M. C., Raebum, J. A., van Zwet, T. L., Crofion, R., and Vlusse van Oud Alblas, A., In “Mononuclear Phagocytes, Functional Aspects” (R. van Furth, Ed.), p. 279. Martinus Nijhoff, The Hague, 1980. 14. Gordon, S., Unkeless, J. C., and Cohn, Z. A., J. Exp. Med. 140, 995, 1974. 15. Vassalli, J. D., and Reich, E., J. Exp. Med. 145, 429, 1977. 16. Leak, L. V., Lab. Invest. 48, 479, 1983. 17. Jokay, I., and Karczag, E., Experientia 29, 334, 1972. 18. Adams, D. O., Kao, K. J., Farb, R., and Pizzo, S. V., J. Zmmunol. 124, 293, 1980. 19. Adams, D. O., and Marino, P. A., In “Contemporary Hematology/Oncology.” Plenum, New York (in press).