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Activation of Human Monocyte-Derived Macrophages Cultured on Teflon: Response to Interferon-γ during Terminal Maturation in vitro
Immunobiol., vol. 177, pp. 186-198 (1988)
1 Medizinische
2 Department
Klinik I der Universitat Freiburg i. Brsg. Federal Republic of Germany, and of Nephrology, Prince Henry's Hospital, Melbourne, Australia
Activation of Human Monocyte-Derived Macrophages Cultured on Teflon: Response to Interferon-y during Terminal Maturation in vitro REINHARD ANDREESEN 1,', STEPHEN GADD 2, WOLFRAM BRUGGERt, GEORG W. LOHR
1
,
and ROBERT C. ATKINS2
Received January 13, 1988· Accepted in Revised Form February 29, 1988
Abstract Macrophages (M biology and function have been performed using immature mo precursor cells. However, the conclusions drawn may be questionable, as mo have to undergo terminal differentiation before they reach relevant tissue sites of inflammation and immune reaction. We have analyzed the ability of mo-derived, tefloncultured M to respond to activating stimuli with an increased tumor cytotoxic effector cell function using recombinant interferon-gamma (IFN-y), IFN-a2, granulocyte/macrophage colony stimulating factor (GM-CSF), interleukin(IL) 2, IL la, and bacterial lipopolysaccharides (LPS) as mediator molecules. It could be shown that the response of M to the most potent activator molecule, IFN -y, depends on the terminal differentiation from the mo stage to the mature M. Whereas adherent mo could be activated only moderately, M increased their cytotoxicity by a factor of up to 400. IFN -y activation positively correlated with the effector cell number, the time of incubation and the dosage used. Activation did not depend on the presence of LPS, and was lost within 24 to 48 h. LPS itself activated cells only in the microgram range. IFN-a2 activated M only at a two log higher concentration than IFN-y; GM-CSF was only slightly effective, whereas M incubation with IL 1a or IL 2 did not result in M activation. Thus, the ability of human M to become activated appears to be a function of cellular maturation and is acquired during the terminal step of M differentiation. Tefloncultured M could facilitate studies of the activation of human M and may be more suitable cells for adoptive immunotherapy in cancer patients than blood monocytes.
Introduction Human macrophages (M = macrophages; MPS = mononuclear phagocyte system; OD = optical density; RIA = radioimmunoassay; sin = supernatant; TF = transferrin; TM = teflon-cultured macrophages. 'R. A. is a recipient of a Heisenberg Award from the Deutsche Forschungsgemeinschaft.
M Maturation and Response to IFN . 187
of malignancies (1, 2). M are located throughout all tissues and body cavities, where they respond to chemoattracting and activating signals which modulate cell function and phenotype (3). Tissue M all derive from early progenitor cells in the bone marrow whose differentiation into blood monocytes (mo) creates a circulating precursor cell pool which undergoes terminal maturation and diversification upon leaving the capillary bed to migrate into the various tissues (4). With this last differentiation step, competence in various effector cell functions is achieved and, in addition, remarkable heterogeneity in terms of morphology and cell biology within the MPS is established (5-9). In vitro differentiation of blood mo to M on teflon membranes can serve as a model system to study several aspects of M maturation (9-12). Due to the hydrophobicity of the teflon material, M grow in suspension and can be recovered for experimentation at any stage of differentiation (10). Here, we present evidence that terminal maturation in vitro provides a strong priming signal for tumor cytotoxicity in that it is paralleled by the generation of responsiveness to IFN-y. Differentiated M could only be moderately activated by LPS and IFN-a2, even less by GM-CSF and not at all by IL 1a and IL2.
Materials and Methods Cells and cell cultures Mononuclear cells were isolated from buffy coat preparations of healthy blood donors by density gradient centrifugation over Ficoll-Hypaque. Five x 106 cells/ml were cultured in RPM I 1640 (supplemented with 20 [,1M 2-mercaptoethanol, 2 mM L-glutamine, 50 U/ml penicillin, and 5 [,Ig/ml streptomycin) plus 10 % fetal calf serum (FCS) for 60 min in plastic tissue culture flasks (Fa. Greiner, Niirtingen, F.R.G.) before the non-adherent cells were removed by washing twice in warm medium. The adherent mo, which were of more than 90 % purity as determined by morphology and immunophenotyping (9, 10), were cultured overnight in supp!. RPM I 1640 plus 5 % human AB-group serum pooled from different pretested donors. The cells could then be completely washed off the flasks after cooling them down to 4°C for 30 min and accounted for about 65 % of the circulating monocyte population. These mo were seeded into teflon bags at a concentration of 3 x 10 5 cells/ml supp!. RPMI 1640 plus 5 % AB-serum (10). At different stages of culture, M of more than 95 % purity were recovered from the bags by needle aspiration. The histiocytic lymphoma cell line U937 was maintained in log phase growth in supp!. RPMI 1640 plus 10 % FCS and passaged twice weekly.
Tumor cytostasis assay (11) Monocyte/macrophages were seeded in 96-well flat bottom plastic microtiter plates (Fa. Greiner) in a volume of 0.2 ml supp!. RPMI 1640 plus 10 % FCS at different concentrations and cultured with and without recombinant human IFN-y (IMMUNERON®, Biogen, Geneva, Switzerland), recombinant human IFN-a2 (ROFERON-A®, Hoffmann-LaRoche, Basel, Switzerland), recombinant human IL la (Hoffmann-LaRoche, Nutley, NJ, U.S.A.), recombinant human IL 2 (gift from Dr. Luckenbach, Sandoz AG, Vienna, Austria), recombinant human GM-CSF (Behringwerke AG, Marburg, F.R.G.), and LPS Salmonella abortus
188 . REINHARD ANDREE SEN et a!. equi (provided by Dr. C. Galanos, Max-Planck-Institut, Freiburg, F.R.G.). The units of the above-mentioned recombinant materials were calculated based on the information provided by the manufacturers. The microwells were then rinsed twice with medium before U937 target cells were added in various concentrations. Effector and target cells were cocultured for 48 h; then the microcultures were pipetted, O.l-ml aliquots transferred to a new microplate and pulsed for a further 6 h with 0.2 [lCi [3HJ-thymidine. To test for soluble cytotoxins released from control and activated M, they were cultured with fresh supp!. RPMI 1640 plus 10 % FCS for 48 h after which the supernatants (sin) were collected, filtered (0.4 [l) and stored at 4°C. The sin were added to 10" U937 cells at a final dilution of 1 :1, and the eHJ-thymidine incorporation was measured after another 48 h of culture. Data were expressed either as percentage of inhibition of [3HJ-thymidine uptake in test cultures as compared to tumor cell controls (% tumor growth), or an activation index (AI) was calculated by dividing the counts per minute (cpm) in tumor aliquots after co culture with normal M by that of tumor cells after coculture with activated M . Surface antigen expression analysis with the enzyme-linked immunosorbent assay (ELISA)
Monocytes/macrophages were seeded in supp!. RPM I 1640 in 96-well flat bottom microtiter plates (Greiner) at a density of lOs/well for mo and at 3 x 10 4/well for M and incubated for 30 min. Cells were then fixed at 4°C with 0.05 % glutaraldehyde for 10 min, triplicates were incubated with the following monoclonal antibodies (mAb) diluted in gelatine(0.2 %)-containing medium: anti-~2 microglobulin (Becton and Dickinson, Riidermark, F.R.G.), My4 (Coulter Immunology, Hialeah, FL, U.S.A.), OKT9 (Ortho Diagnostics, Raritan, Nj, U.S.A.), MAX.1, .2, .3,.11 (own laboratory, ref. 9, 13), Ki-1 (gift from Prof. Dr. Ax, Behringwerke, Marburg, F.R.G.), 13C2 (gift from Dr. M. Horton, Imperial Cancer Research Fund Laboratories, London, U.K., ref. 14), anti-CD16 CLB FeR gran 1 (Dr. P. Tetteroo, The Netherlands Red Cross Blood Transfusion Service, Amsterdam, ref. 15), HE10 and 12B1 (gift from Dr. F. Farrar, Villejuife, France, ref. 16). As controls, mAb of different immunoglobulin (Ig) types raised against cytoplasmic antigens of Salmonella enteritidis llRX (17) were used. Immunoperoxidase staining was done as described (18) using phenyl-diamine-dichloride as the substrate. Data are expressed as OD 4 8 6 corrected by the values measured in control wells without the first antibody, mean of triplicate values, S.D. > 15 'Yo. ELISA analysis of M secretory products
Cells were cultured at density described above in 96-well flat bottom microtiter plates (Greiner) in supp!. RPMI 1640 with 0.1 mg/mllactalbumin hydrolysate, 0.2 mg/ml fetuin, and 0.15 IU/ml bovine insulin (all from Sigma Chemicals, St. Louis, M, U.S.A.) for 24 h. The medium was then replaced with fresh medium, and the cells were cultured for a further 24 h. The supernatants were assayed. as followed: micro titer wells were coated overnight with 1 :1000 diluted rabbit antibodies raised against fibronectin, transferrin, and alpha-2-macroglobulin (all purchased from Dakopatts, Copenhagen, Denmark), respectively, and then incubated with undiluted and 1:50 diluted supernatants for 1 h. This was followed by incubation with the same antibodies coupled to horseradish peroxidase (Boehringer Mannheim GmbH, Mannheim, F.R.G.) for 30 min at room temperature. Concentrations were calculated from the values obtained with different dilutions of the standard compounds (all from Sigma Chemicals).
Results Mature human M
M Maturation and Response to IFN . 189 AI 250 X
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DAYS IN CULTURE Figure 1. Activation of human M at different stages of maturation from blood mo. Adherent mo (day 1) were seeded directly into 0.2-ml micro wells or recovered as differentiating TM from hydrophobic teflon cultures at the times indicated, seeded at 10 5 (for day 1 mol or 5 X 10 4 per well (for differentiating TM monolayer was then rinsed 3 times with serum-free medium before 104 U937 cells were added in medium plus 10 % FCS. CHJ-thymidine incorporation was measured into O.l-ml tumor cell aliquots after 48 h of cocultivation. Data are the mean of triplicate values and expressed as activation index (AI) which were calculated as described in «Materials and Methods», SD> 15 %.
ous cytotoxicity gradually increased upon culture from blood mo, as has been repeatedly reported (11, 25-28), but we also found their response to activation to be highly dependent on the stage of maturation. In Figure 1, the IFN-y-induced activation of teflon-cultured macrophages (TM
(TM
effector cells
control TM' (% U937 growth)
10,000 10,000 10,000 10,000 10,000 10,000
1,000 5,000 10,000 25,000 50,000 100,000
57.2 ± 4.5' 20.5 ± 3.4 17.3 ± 0.8 27.4 ± 1.9 38.0 ± 2.1 28.7 ± 4.2
activated TMb (activation index) 4.5 ± 0.5 d 20.8 ± 6.1 19.1 ± 2.2 84.2 ± 12.8 416.4±31.7 281.7 ± 26.4
, targets and effectors were cocultured for 48 h, the cultures pipetted and O.l-ml-aliquots transferred to a new microtiter plate were pulsed with CHJ-thymidine for 6 h. b mo-derived TM (day 8) were incubated with 500 U IFN-y per ml for 24 h (part A) and 8 h (part B), respectively. ( data are expressed as percentage of [3HJ-thymidine incorporation of the tumor cell control cultures and given as mean of triplicate values. d data are activation indices calculated in comparison to the activity of untreated TM
190 . REINHARD ANDREESEN
et al.
AI 200
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INCUBATION TIME
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72
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Figure 2. Activation of human mo-derived TM by IFN-y in dependence on the time of incubation. Five x 104 TM (day 12) were seeded per well and incubated with two different IFN -y concentrations for the times indicated. For further details see legend to Figure 1.
enhanced their activity upon stimulation with IFN-y at concentrations as high as 1,250 Ulml only by a factor of 5 to 8, their response gradually increased upon terminal differentiation in vitro. The spontaneous tumor growth inhibition of untreated TM
M Maturation and Response to IFN . 191
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Figure 3. Activation of mo-derived TM with different stimuli. Five x 10 TM/weli (day 10) were incubated with the indicated substances for 24 h in medium plus 10% FCS, the wells rinsed 3 times with serum-free medium and the TM monolayer then cocultured with 10 4 U937 cells. For further details see legend to Figure 1.
When the activating stimulus was withdrawn, TM!fJ lost the activated stage rapidly within 48 h, after which the cytotoxic activity fell below control levels (Fig. 4). While adherent mo mature into M!fJ they change in many aspects (Table 2): M!fJ terminal differentiation induced a tremendous increase in size, protein and DNA content on the single cell level, the latter being most likely the result of cell fusion. No uptake of eH]-thymidine can be demonstrated in mo/M!fJ cultures neither can mitotic figures be observed (10). Multinucleated cells appear, giving rise sometimes to giant syncytial
192 . REINHARD ANDREESEN et al. Table 2. Functional and phenotypic comparison of normal blood monocytes and tefloncultured mo-derived macrophages a
A multinucleated cells (%) cell diameter (flm) protein content (flg/106), DNA content (flg/106)' tartrate-resistent acid phosphatase (%) peroxidase (%)
blood mo
teflon Mb
<1
12-18 20-35 376 ± 115 12 ± 4 >90 <1
10-12 32 ± 15 5.7 ± 0.2 <1 80-90
B 24-h secretion' of: fibronectin (ng/106) transferrin (ng/10 6) alpha-2-macroglobulin (ng/106) interleukin 1d (cpm/1 06)' prostaglandin E 2d (ng/106)
<5 <5 <5 56,300 141
C antigen expression' (OD 486 ) beta-2-microglobulin HLA-DR (MAX.21) CD14 SC9 12B1 MAX.1 (gp64) MAX.2 (gp200) MAX.3 (gp68) MAX.11 (gp64) transferrin receptor (OKT9) CD16 l3C2 Ki-l (CD30) HE 10
1.699 1.439 1.498 0.155 0.274 negative negative negative negative negative negative negative negative negative
± 4,800 ± 19
204 ± 203 35 ± 16 792 ± 164 1,400 ± 280 25 ± 8
2.580 2.140 1.720 1.491 >2.600 1.943 0.498 1.202 1.841 2.330 0.897 1.182 0.514 0.819
, partially taken from ref. 9. b data pooled from experiments using TM between culture day 10 and 15. , cells were cultured in serum-free medium for 24 h. The sin were assayed for fibronectin, a2m and TF with the use of an ELISA, for prostaglandin E2 with a RIA and for IL 1 activity with the C3H/HeJ mouse thymocyte proliferation assay. d in the presence of 10 flg/ml LPS (Salmonella abortus equi). e cell were taken for cell-ELISA analysis either on day 1 (blood mo at lOS/well) or on day 15 harvested from the teflon bags (TM at 3 X 104/well). Data are expressed as OD 486 corrected by the unspecific binding in control wells with the second antibody, S.D. < 15 %.
cells with more than 40 nuclei. Characteristic changes in cytochemistry are documented. In addition, TM but not blood mo released very small amounts of transferrin (TF), more of alpha-2-macroglobulin (a2M, see also ref. 19) and fibronectin (see also ref. 20). In comparison to day 1 mo, mature mo-derived TM have an increased ability to secrete lysozyme and hematopoietic growth factors (ref. 9 and 11, to be published in more detail)
M Maturation and Response to IFN . 193
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92 h
Figure 4. Loss of M activity after prolonged incubation subsequent to incubation with IFN-y. Five x 104 TM/well (day 8) were incubated with two different concentrations of IFN -y for 3 h and the wells rinsed 3 times. U937 target cells were then added for 48 h either immediately «<0 h») or after the M had been further cultivated in fresh medium plus 10 % FCS for the time periods indicated «<20 h» to «96 h»). For further details see legend to Figure 1.
but decrease production of IL 1 activity and prostaglandins upon stimulation with LPS. On antigen analysis using the cell-ELISA technique, M increase the amount of beta-2-microglobulin, HLA-DR, and CD14 molecules and in addition express a number of antigens not present on blood mo: those of the MAX series (9), the receptor for TF (OKT9, ref. 12), the osteoclast-associated antigen 13C2 (14), the low affinity receptor for IgG (CD16, see also ref. 21), some still undefined myeloid antigens (HElO, 12B1, ref. 16) and the Hodgkin cell-associated antigen CD30 (Ki-1, ref. 22).
Discussion Human M become competent effector cells upon completion of their differentiation pathway. The terminal maturation step from circulating blood mo to mature M seems to be of special importance and is accompanied by dramatic changes in morphology, biochemistry, and cell biology (9, 10,23,24). With regard to the role of M as host defense cells against the spread of malignancies, it is of interest that spontaneous tumor cytotox-
194 . REINHARD ANDREESEN et al.
icity is developed during terminal differentiation (11, 25-28). From the results presented here, we conclude that terminal maturation also provides an essential priming signal for M to allow their response to IFN-y. IFN-y appears to be a major physiologic (though not sole) lymphokine that induces M activation for a variety of effector cell functions such as antimicrobial (29) and antitumor (30, 31) activity and the secretion of monokines (32, 33). However, activation is a complex phenomenon and has to proceed through various stages before the fully activated capacity is elicited (34, 35). Whereas in the murine system the responsive stage of M activation has been defined quite clearly, the activation of human mo/M is still incompletely understood. Controversial data have been published on the ability of LPS and IFN-y to activate human mo cytotoxicity (30, 31, 36-39). These conflicting findings, however, should be viewed within the context of different effector populations (elutriation vs. adherence purified cells), target cells, time course and type of cytotoxicity assay employed, and specific cell function should be taken into account. Our results, however, support other observations that adherent mo may indeed only be low responders to activating signals and require terminal differentiation before becoming competent effector cells (Fig. 1). A similar observation wa~ made by CAMERON and CHURCHILL in 1979 using a different culture system and crude lymphokine preparations (27). Unlike others (31), we can show that mature cells also respond to IFN -u, though at a two log higher concentration. On the contrary, when we tested GM-CSF (40) and IL2 (36), which also had been reported to activate human mo, we could demonstrate only marginal or no effects, respectively, on the cytotoxicity of mature TM. In contrast to murine M, the role of LPS in the cascade of events leading to a cytotoxic killer M in humans is uncertain (37-39). In our experiments, LPS had to be added to TM in microgram amounts to result in augmentation of cytotoxicity (Fig. 3) and had no effects at concentrations below this level. In addition, no synergistic effects on the tumor cytotoxicity of TM were observed when LPS and IFN-y were added to the cells either simultaneously or consecutively (not shown in detail). Thus, trace contaminations of the active reagents with endotoxin (according to the manufacturers, less than 0.1 ng/l0 6 units) most likely do not contribute to the generated tumor cytotoxicity. Also, TM washed and cultured for 48 h under strict endotoxin-free conditions did not lose their responsiveness to IFN-y (not shown in detail). The tumor cytotoxic capacity of activated mo-derived TM may not only be due to the responsiveness acquired during the terminal maturation process but may also be the result of a highly potent effector cell function attributed specifically to the fully differentiated M cell. In both respects, the in vitro culture could have mimicked those signals which M normally receive in their tissue environment. Especially the M membrane phenotype is remodelled during the terminal differentiation in vitro, result-
M Maturation and Response to IFN . 195
ing in the expression of a set of new antigens not presented on blood mo (Table 2). These membrane structures could be involved either in binding of the stimulus, in signal transduction or even in the effector phase of MO cytotoxicity. HAMILTON et al., for example, have related the expression of TF receptors to the responsive stage of M activation (41, 42). In our system, generation of MO response to IFN-y is indeed paralleled by the expression of TF receptors (Table 2 and ref. 12). Although no soluble cytotoxins could be detected in the supernatants of activated TM, other secretory poducts may play a role as accessory factors. Fibronectin might facilitate cell-cell adhesion (43) and thus interaction. Transferrin already had been implicated in binding and effector function of NK cells (44). Alpha-2-macroglobulin bound to cellular receptors might bridge M to tumor cell membranes by complexing to target cell proteases (45). In conclusion, M at various stages of culture in teflon bags provide an excellent system to study the events and stages of human M activation for tumor cytotoxicity. The results obtained indicate the importance of the terminal maturation within the M lineage. If our data reflect normal physiology in vivo, any impairment of this differentiation step wduld have serious consequences on M function in host defense and likewise in other regulatory circuits into which cells of the MPS are involved (e,.g., bone resorption, iron metabolism and hematopoiesis). In addition, in vitro matured M rather than blood mo may be suitable for use in adoptive immunotherapy of cancer. Acknowledgements We are indebted to GLADYS MACK and ANNEGRET REHM for their excellent technical aSSIstance. This work was supported by Deutsche Forschungsgemeinschaft and Boehringer Ingelheim Fonds.
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196 . REINHARD ANDREESEN et al. 6. DOUGHERTY, G. J., and W. H. McBRIDE. 1984. Macrophage heterogeneity. J. Clin. Lab. Immunol. 14: 1. 7. HANCOCK, W. W., H. ZOLA, and R. C. ATKINS. 1983. Antigenic heterogeneity of human mononuclear phagocytes: immunohistological analysis using monoclonal antibodies. Blood 62: 1271. 8. FRANKLIN, W. A., D. Y. MASON, K. PULFORD, B. FALINI, E. BLISS, K. C. GATTER, H. STEIN, L. C. CLARKE, and J. O. D. MCGEE. 1986. Immunohistological analysis of human mononuclear phagocytes and dendritic cells by using monoclonal antibodies. Lab. Invest. 54: 322. 9. ANDREESEN, R., K. J. BROSS, J. OSTERHOLZ, and F. EMMRICH. 1986. Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens. Blood 67: 1257. 10. ANDREE SEN, R., J. PICHT, and G. W. LOHR. 1983. Primary cultures of human blood-born macrophages grown on hydrophobic teflon membranes. J. Immunol. Meth. 56: 295. 11. ANDREE SEN, R., J. OSTERHOLZ, K. J. BROSS, A. SCHULZ, G. A. LUCKENBACH, and G. W. LOHR. 1983. Cytotoxic effector cell function at different stages of human monocytemacrophage maturation. Cancer Res. 43: 5931. 12. ANDREESEN, R., J. OSTERHOLZ, H. H. BODEMANN, K. J. BROSS, U. COSTABEL, and G. W. LOHR. 1984. Expression of transferrin receptors and intracellular transferrin during terminal differentiation of human monocytes. Blut 49: 195. 13. EMMRICH, F., and R. ANDREESEN. 1985. Monoclonal antibodies against differentiation antigens on human macrophages. Immunol. Lett. 9: 321. 14. HORTON, M. A., D. LEWIS, K. McNULTY, J. A. S. PRINGLE, and T. J. CHAMBERS. 1985. Monoclonal antibodies to osteoclastomas (giant cell bone tumors): definition of osteoclast-specific cellular antigens. Cancer Res. 45: 5663. 15. TETTERoo, P. A. T., C. E. VANDER SCHOOT, F. J. VISSER, M. J. E. Bos, and A. E. G. VON DEM BORNE. 1987. Three different types of Fcgamma receptors on human leukocytes defined by workshop antibodies: FcgammaRlow of neutrophils, FcgammaR low of KINK lymphocytes and FcgammaRII. In: McMICHAEL et al. (eds). Leucocyte Typing III, Oxford University Press, Oxford, UK, pp. 702-706. 16. FARACE, F., N. KIEFFER, B. CAILLOU, W. VAINCHENKER, T. TURsz, and M. C. DOKHELAR. 1986. A 93 KD Glycoprotein expressed on human cultured monocytes and dendritic reticulum cells defined by an anti K562 monoclonal antibody. Eur. J. Immunol. 16: 1521. 17. O'CONNOR, C. G., and L. K. ASHMAN. 1982. Application of the nitrocellulose transfer technique and alkaline phosphatase conjugated antiimmunoglobulin for determination of the specificity of monoclonal antibodies to protein mixtures. J. Immunol. Methods 54: 167. 18. ANDREESEN, R., A. MACKENSEN, J. OSTERHOLZ, and G. W. LOHR. 1988. Microculture assay for human macrophage maturation in vitro. Cell-ELISA analysis of differentiation antigen expression. lnt. Arch. Allerg. appl. lmmunol., in press. 19. HOVI, T., D. MOSHER, and A. VAHERI. 1977. Cultured human monocytes synthesize and secrete alpha-2-macroglobulin. J. Exp. Med. 145: 1580. 20. ALITALO, K., T. HOVI, and A. VAHERI. 1980. Fibronectin is produced by human macrophages. J. Exp. Med. 151: 602. 21. FLEIT, H. B., S. D. WRIGHT, and J. C. UNKELESS. 1982. Human neutrophil Fc-g receptor distribution and structure. Proc. Nat!. Acad. Sci. 79: 3275. 22. SCHWAB, U., H. STEIN,J. GERDES, H. LEMKE, H. KIRCHNER, M. SCHAADT, and V. DIEHL. 1982. Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin's disease and a subset of normal lymphoid cells. Nature 299: 65. 23. SUTTON, J. S., and L. WEISS. 1966. Transformation of monocytes in tissue culture into macrophages, epitheloid cells, and multinucleated giant cells. An electron microscopy study. J. Cell. BioI. 28: 303. 24. ZUCKERMANN, S. H., S. K. ACKERMANN, and S. D. DOUGLAS. 1979. Long-term human peripheral blood monocyte cultures: establishment, metabolism and morphology of primary human monocyte-macrophage cell cultures. Immunol. 38: 401.
M$ Maturation and Response to IFN . 197 25. UNSGAARD, G. 1979. Cytotoxicity to tumour cells induced in human monocytes cultured in vitro in the presence of different sera. Acta Path. Microbiol. Scand. Sect. 87: 141. 26. RINEHART, J. J., R. VESSELLA, P. LANGE, and M. E. KAPLAN. 1979. Characterization and comparison of human monocyte- and macrophage-induced tumor cell cytotoxicity. J. Lab. Clin. Med. 93: 361. 27. CAMERON, D. J., and W. H. CHURCHILL. 1979. Cytotoxicity of human macrophages for tumor cells. Enhancement by human lymphocyte mediators. J. Clin. Invest. 63: 977. 28. MANTOVANI, A., J. H. DEAN, T. R. JERRELS, and R. B. HERBERMANN. 1980. Augmentation of tumoricidal activity of human monocytes and macrophages by lymphokines. Int. J. Cancer 25: 691. 29. NATHAN, C. F., H. W. MURRAY, M. E. WIEBE, and B. Y. RUBIN. 1983. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J. Exp. Med. 158: 670. 30. HERBERMAN, R. B., J. R. ORTALDO, A. MANTOVANI, D. S. HOBBS, H. F. KUNG, and S. PRESTKA. 1982. Effect of human recombinant interferon on cytotoxic activity of natural killer cells and monocytes. Cell. Immunol. 67: 160. 31. JUNMING LE, W. PRENSKY, Y. K. YIP, ZONGLIANG CHANG, T. HOFFMAN, H. C. STEVENSON, I. BALAZS, J. R. SADLIK, and J. VILCEK. 1983. Activation of human monocyte cytotoxicity by natural and recombinant immune interferon. J. Immunol. 131: 2821. 32. HAMBURG, S. I., H. B. FLEIT, J. C. UNKELESS, and M. RABINOVITCH. 1980. Mononuclear phagocytes: responders to and producers of interferon. Ann. N.Y. Acad. Sci. 350: 72. 33. NATHAN, C. F. 1987. Secretory products of macrophages. J. Clin. Invest. 79: 319. 34. Ruco, L. P., and M. S. MELTZER. 1978. Macrophage activation for tumor cytotoxicity: Development of macrophage cytotoxic activity requires completion of a sequence of short-lived intermediary reactions. J. Immunol. 121: 2039. 35. MELTZER, M. S. 1981. Tumor cytotoxicity by lymphokine-activated macrophages: Development of macrophage tumoricidal activity requires a sequence of reactions. Lymphokines Vol. 3, Academic Press Inc., pp. 319-343. 36. MALKOVSKY, M., B. LOVELAND, M. NORTH, G. L. ASHERSON, and L. GAO. 1987. Recombinant interleukin-2 directly augments the cytotoxicity of human monocytes. Nature 325: 262. 37. WEINBERG, J. B., and A. F. HANEY. 1983. Spontaneous tumor cell killing by human blood monocytes and human peritoneal macrophages: Lack of alteration by endotoxin or quenchers of reactive oxygen species. J. N. C. 1. 70: 1005. 38. BIONDI, A., G. PERI, R. LORENZET, D. FUMAROLA, and A. MANTOVANI. 1983. Role of endotoxin in the expression of human monocyte cytotoxicity. J. Retic. Endoth. Soc. 33: 315. 39. SAIKI, 1., S. SONE, W. E. FOGLER, E. S. KLEINERMAN, G. LOPEZ-BERESTEIN, and 1. J. FIDLER. 1985. Synergism between human recombinant g-interferon and Muramyl Dipeptide encapsulated in liposomes for activation of antitumor properties of human monocytes. Cancer Res. 45: 6188. 40. GRABSTEIN, K. H., D. L. URDAL, R. J. TUSHINSKI, D. Y. MOCHIZUKI, V. L. PRICE, M. A. CANTRELL, S. GILLIS, and P. J. CONLON. 1986. Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor. Science 232: 506. 41. HAMILTON, T. A., J. E. WEIEL, and D. O. ADAMS. 1984. Expression of the transferrin receptor in murine peritoneal macrophages is modulated in the different stages of activation. J. Immunol. 132: 2285. 42. HAMILTON, T. A., P. W. GRAY, and D. O. ADAMS. 1984. Expression of the transferrin receptor on murine peritoneal macrophages is modulated by in vitro treatment with Interferon-gamma. Cell. Immunol. 89: 478. 43. ALI, I. U., and R. O. HYNES. 1978. Effects of LETS glycoprotein on cell motility. Cell 14: 439. 44. VODINELICH, 1., R. SUTHERLAND, C. SCHNEIDER, R. NEWMAN, and M. GREAVES. 1983. Receptor for transferrin may be a «target» structure for natural killer cells. Proc. Nat!' Acad. Sci. 80: 835.
198 . REINHARD ANDREESEN et al. 45. JOHNSON, W. J., S. V. PIZZO, M. J. IMBER, and D. O. ADAMS. 1982. Receptors for maleylated proteins regulate the secretion of neutral proteases by murine macrophages. Science 218: 574. Dr. REINHARD ANDREESEN, Medizinische Klinik, Hugstetter Str. 55, D-7800 Freiburg, Federal Republic of Germany
1 Medizinische
2 Department
Klinik I der Universitat Freiburg i. Brsg. Federal Republic of Germany, and of Nephrology, Prince Henry's Hospital, Melbourne, Australia
Activation of Human Monocyte-Derived Macrophages Cultured on Teflon: Response to Interferon-y during Terminal Maturation in vitro REINHARD ANDREESEN 1,', STEPHEN GADD 2, WOLFRAM BRUGGERt, GEORG W. LOHR
1
,
and ROBERT C. ATKINS2
Received January 13, 1988· Accepted in Revised Form February 29, 1988
Abstract Macrophages (M
Introduction Human macrophages (M
M
of malignancies (1, 2). M are located throughout all tissues and body cavities, where they respond to chemoattracting and activating signals which modulate cell function and phenotype (3). Tissue M all derive from early progenitor cells in the bone marrow whose differentiation into blood monocytes (mo) creates a circulating precursor cell pool which undergoes terminal maturation and diversification upon leaving the capillary bed to migrate into the various tissues (4). With this last differentiation step, competence in various effector cell functions is achieved and, in addition, remarkable heterogeneity in terms of morphology and cell biology within the MPS is established (5-9). In vitro differentiation of blood mo to M on teflon membranes can serve as a model system to study several aspects of M maturation (9-12). Due to the hydrophobicity of the teflon material, M grow in suspension and can be recovered for experimentation at any stage of differentiation (10). Here, we present evidence that terminal maturation in vitro provides a strong priming signal for tumor cytotoxicity in that it is paralleled by the generation of responsiveness to IFN-y. Differentiated M could only be moderately activated by LPS and IFN-a2, even less by GM-CSF and not at all by IL 1a and IL2.
Materials and Methods Cells and cell cultures Mononuclear cells were isolated from buffy coat preparations of healthy blood donors by density gradient centrifugation over Ficoll-Hypaque. Five x 106 cells/ml were cultured in RPM I 1640 (supplemented with 20 [,1M 2-mercaptoethanol, 2 mM L-glutamine, 50 U/ml penicillin, and 5 [,Ig/ml streptomycin) plus 10 % fetal calf serum (FCS) for 60 min in plastic tissue culture flasks (Fa. Greiner, Niirtingen, F.R.G.) before the non-adherent cells were removed by washing twice in warm medium. The adherent mo, which were of more than 90 % purity as determined by morphology and immunophenotyping (9, 10), were cultured overnight in supp!. RPM I 1640 plus 5 % human AB-group serum pooled from different pretested donors. The cells could then be completely washed off the flasks after cooling them down to 4°C for 30 min and accounted for about 65 % of the circulating monocyte population. These mo were seeded into teflon bags at a concentration of 3 x 10 5 cells/ml supp!. RPMI 1640 plus 5 % AB-serum (10). At different stages of culture, M
Tumor cytostasis assay (11) Monocyte/macrophages were seeded in 96-well flat bottom plastic microtiter plates (Fa. Greiner) in a volume of 0.2 ml supp!. RPMI 1640 plus 10 % FCS at different concentrations and cultured with and without recombinant human IFN-y (IMMUNERON®, Biogen, Geneva, Switzerland), recombinant human IFN-a2 (ROFERON-A®, Hoffmann-LaRoche, Basel, Switzerland), recombinant human IL la (Hoffmann-LaRoche, Nutley, NJ, U.S.A.), recombinant human IL 2 (gift from Dr. Luckenbach, Sandoz AG, Vienna, Austria), recombinant human GM-CSF (Behringwerke AG, Marburg, F.R.G.), and LPS Salmonella abortus
188 . REINHARD ANDREE SEN et a!. equi (provided by Dr. C. Galanos, Max-Planck-Institut, Freiburg, F.R.G.). The units of the above-mentioned recombinant materials were calculated based on the information provided by the manufacturers. The microwells were then rinsed twice with medium before U937 target cells were added in various concentrations. Effector and target cells were cocultured for 48 h; then the microcultures were pipetted, O.l-ml aliquots transferred to a new microplate and pulsed for a further 6 h with 0.2 [lCi [3HJ-thymidine. To test for soluble cytotoxins released from control and activated M
Monocytes/macrophages were seeded in supp!. RPM I 1640 in 96-well flat bottom microtiter plates (Greiner) at a density of lOs/well for mo and at 3 x 10 4/well for M
Cells were cultured at density described above in 96-well flat bottom microtiter plates (Greiner) in supp!. RPMI 1640 with 0.1 mg/mllactalbumin hydrolysate, 0.2 mg/ml fetuin, and 0.15 IU/ml bovine insulin (all from Sigma Chemicals, St. Louis, M
Results Mature human M
M Maturation and Response to IFN . 189 AI 250 X
W
200
Z
150
.... «
100
0 Z
•
12.5Ujml
ml125 Ujml
~ 1.250U/ml
0
>
....u «
50 0 DAYl
DAY
I,.
DAY7
DAY18
DAYS IN CULTURE Figure 1. Activation of human M at different stages of maturation from blood mo. Adherent mo (day 1) were seeded directly into 0.2-ml micro wells or recovered as differentiating TM from hydrophobic teflon cultures at the times indicated, seeded at 10 5 (for day 1 mol or 5 X 10 4 per well (for differentiating TM
ous cytotoxicity gradually increased upon culture from blood mo, as has been repeatedly reported (11, 25-28), but we also found their response to activation to be highly dependent on the stage of maturation. In Figure 1, the IFN-y-induced activation of teflon-cultured macrophages (TM
(TM
effector cells
control TM' (% U937 growth)
10,000 10,000 10,000 10,000 10,000 10,000
1,000 5,000 10,000 25,000 50,000 100,000
57.2 ± 4.5' 20.5 ± 3.4 17.3 ± 0.8 27.4 ± 1.9 38.0 ± 2.1 28.7 ± 4.2
activated TMb (activation index) 4.5 ± 0.5 d 20.8 ± 6.1 19.1 ± 2.2 84.2 ± 12.8 416.4±31.7 281.7 ± 26.4
, targets and effectors were cocultured for 48 h, the cultures pipetted and O.l-ml-aliquots transferred to a new microtiter plate were pulsed with CHJ-thymidine for 6 h. b mo-derived TM (day 8) were incubated with 500 U IFN-y per ml for 24 h (part A) and 8 h (part B), respectively. ( data are expressed as percentage of [3HJ-thymidine incorporation of the tumor cell control cultures and given as mean of triplicate values. d data are activation indices calculated in comparison to the activity of untreated TM
190 . REINHARD ANDREESEN
et al.
AI 200
100
50
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w
o
z
z
o 10
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19
sec min min
iT-----oi 1-1-.,--.,------.,------.,0.5 h
3
h
12 h
INCUBATION TIME
24. h
46
h
72
h
Figure 2. Activation of human mo-derived TM by IFN-y in dependence on the time of incubation. Five x 104 TM (day 12) were seeded per well and incubated with two different IFN -y concentrations for the times indicated. For further details see legend to Figure 1.
enhanced their activity upon stimulation with IFN-y at concentrations as high as 1,250 Ulml only by a factor of 5 to 8, their response gradually increased upon terminal differentiation in vitro. The spontaneous tumor growth inhibition of untreated TM
M Maturation and Response to IFN . 191
AI
200
IFN-gommo
200
IFN-oLpha
100
40 20 10 4 <{
2
xw
c
z z
Q 200
~ >
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u
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100
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20
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<{
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Figure 3. Activation of mo-derived TM with different stimuli. Five x 10 TM/weli (day 10) were incubated with the indicated substances for 24 h in medium plus 10% FCS, the wells rinsed 3 times with serum-free medium and the TM monolayer then cocultured with 10 4 U937 cells. For further details see legend to Figure 1.
When the activating stimulus was withdrawn, TM!fJ lost the activated stage rapidly within 48 h, after which the cytotoxic activity fell below control levels (Fig. 4). While adherent mo mature into M!fJ they change in many aspects (Table 2): M!fJ terminal differentiation induced a tremendous increase in size, protein and DNA content on the single cell level, the latter being most likely the result of cell fusion. No uptake of eH]-thymidine can be demonstrated in mo/M!fJ cultures neither can mitotic figures be observed (10). Multinucleated cells appear, giving rise sometimes to giant syncytial
192 . REINHARD ANDREESEN et al. Table 2. Functional and phenotypic comparison of normal blood monocytes and tefloncultured mo-derived macrophages a
A multinucleated cells (%) cell diameter (flm) protein content (flg/106), DNA content (flg/106)' tartrate-resistent acid phosphatase (%) peroxidase (%)
blood mo
teflon Mb
<1
12-18 20-35 376 ± 115 12 ± 4 >90 <1
10-12 32 ± 15 5.7 ± 0.2 <1 80-90
B 24-h secretion' of: fibronectin (ng/106) transferrin (ng/10 6) alpha-2-macroglobulin (ng/106) interleukin 1d (cpm/1 06)' prostaglandin E 2d (ng/106)
<5 <5 <5 56,300 141
C antigen expression' (OD 486 ) beta-2-microglobulin HLA-DR (MAX.21) CD14 SC9 12B1 MAX.1 (gp64) MAX.2 (gp200) MAX.3 (gp68) MAX.11 (gp64) transferrin receptor (OKT9) CD16 l3C2 Ki-l (CD30) HE 10
1.699 1.439 1.498 0.155 0.274 negative negative negative negative negative negative negative negative negative
± 4,800 ± 19
204 ± 203 35 ± 16 792 ± 164 1,400 ± 280 25 ± 8
2.580 2.140 1.720 1.491 >2.600 1.943 0.498 1.202 1.841 2.330 0.897 1.182 0.514 0.819
, partially taken from ref. 9. b data pooled from experiments using TM between culture day 10 and 15. , cells were cultured in serum-free medium for 24 h. The sin were assayed for fibronectin, a2m and TF with the use of an ELISA, for prostaglandin E2 with a RIA and for IL 1 activity with the C3H/HeJ mouse thymocyte proliferation assay. d in the presence of 10 flg/ml LPS (Salmonella abortus equi). e cell were taken for cell-ELISA analysis either on day 1 (blood mo at lOS/well) or on day 15 harvested from the teflon bags (TM at 3 X 104/well). Data are expressed as OD 486 corrected by the unspecific binding in control wells with the second antibody, S.D. < 15 %.
cells with more than 40 nuclei. Characteristic changes in cytochemistry are documented. In addition, TM but not blood mo released very small amounts of transferrin (TF), more of alpha-2-macroglobulin (a2M, see also ref. 19) and fibronectin (see also ref. 20). In comparison to day 1 mo, mature mo-derived TM have an increased ability to secrete lysozyme and hematopoietic growth factors (ref. 9 and 11, to be published in more detail)
M Maturation and Response to IFN . 193
AI
20
~ 125U ml rlFNg
ill
10
X
1,250Ujml r IFNg
5
lJ.J
0 ~
z
0
_m_
2
~
>
~
u
I --
0.5
0.2
o
20 44 72 TIME AFTER ACTIVATION
92 h
Figure 4. Loss of M activity after prolonged incubation subsequent to incubation with IFN-y. Five x 104 TM/well (day 8) were incubated with two different concentrations of IFN -y for 3 h and the wells rinsed 3 times. U937 target cells were then added for 48 h either immediately «<0 h») or after the M had been further cultivated in fresh medium plus 10 % FCS for the time periods indicated «<20 h» to «96 h»). For further details see legend to Figure 1.
but decrease production of IL 1 activity and prostaglandins upon stimulation with LPS. On antigen analysis using the cell-ELISA technique, M increase the amount of beta-2-microglobulin, HLA-DR, and CD14 molecules and in addition express a number of antigens not present on blood mo: those of the MAX series (9), the receptor for TF (OKT9, ref. 12), the osteoclast-associated antigen 13C2 (14), the low affinity receptor for IgG (CD16, see also ref. 21), some still undefined myeloid antigens (HElO, 12B1, ref. 16) and the Hodgkin cell-associated antigen CD30 (Ki-1, ref. 22).
Discussion Human M become competent effector cells upon completion of their differentiation pathway. The terminal maturation step from circulating blood mo to mature M seems to be of special importance and is accompanied by dramatic changes in morphology, biochemistry, and cell biology (9, 10,23,24). With regard to the role of M as host defense cells against the spread of malignancies, it is of interest that spontaneous tumor cytotox-
194 . REINHARD ANDREESEN et al.
icity is developed during terminal differentiation (11, 25-28). From the results presented here, we conclude that terminal maturation also provides an essential priming signal for M to allow their response to IFN-y. IFN-y appears to be a major physiologic (though not sole) lymphokine that induces M activation for a variety of effector cell functions such as antimicrobial (29) and antitumor (30, 31) activity and the secretion of monokines (32, 33). However, activation is a complex phenomenon and has to proceed through various stages before the fully activated capacity is elicited (34, 35). Whereas in the murine system the responsive stage of M activation has been defined quite clearly, the activation of human mo/M is still incompletely understood. Controversial data have been published on the ability of LPS and IFN-y to activate human mo cytotoxicity (30, 31, 36-39). These conflicting findings, however, should be viewed within the context of different effector populations (elutriation vs. adherence purified cells), target cells, time course and type of cytotoxicity assay employed, and specific cell function should be taken into account. Our results, however, support other observations that adherent mo may indeed only be low responders to activating signals and require terminal differentiation before becoming competent effector cells (Fig. 1). A similar observation wa~ made by CAMERON and CHURCHILL in 1979 using a different culture system and crude lymphokine preparations (27). Unlike others (31), we can show that mature cells also respond to IFN -u, though at a two log higher concentration. On the contrary, when we tested GM-CSF (40) and IL2 (36), which also had been reported to activate human mo, we could demonstrate only marginal or no effects, respectively, on the cytotoxicity of mature TM. In contrast to murine M, the role of LPS in the cascade of events leading to a cytotoxic killer M in humans is uncertain (37-39). In our experiments, LPS had to be added to TM in microgram amounts to result in augmentation of cytotoxicity (Fig. 3) and had no effects at concentrations below this level. In addition, no synergistic effects on the tumor cytotoxicity of TM were observed when LPS and IFN-y were added to the cells either simultaneously or consecutively (not shown in detail). Thus, trace contaminations of the active reagents with endotoxin (according to the manufacturers, less than 0.1 ng/l0 6 units) most likely do not contribute to the generated tumor cytotoxicity. Also, TM washed and cultured for 48 h under strict endotoxin-free conditions did not lose their responsiveness to IFN-y (not shown in detail). The tumor cytotoxic capacity of activated mo-derived TM may not only be due to the responsiveness acquired during the terminal maturation process but may also be the result of a highly potent effector cell function attributed specifically to the fully differentiated M cell. In both respects, the in vitro culture could have mimicked those signals which M normally receive in their tissue environment. Especially the M membrane phenotype is remodelled during the terminal differentiation in vitro, result-
M Maturation and Response to IFN . 195
ing in the expression of a set of new antigens not presented on blood mo (Table 2). These membrane structures could be involved either in binding of the stimulus, in signal transduction or even in the effector phase of MO cytotoxicity. HAMILTON et al., for example, have related the expression of TF receptors to the responsive stage of M activation (41, 42). In our system, generation of MO response to IFN-y is indeed paralleled by the expression of TF receptors (Table 2 and ref. 12). Although no soluble cytotoxins could be detected in the supernatants of activated TM, other secretory poducts may play a role as accessory factors. Fibronectin might facilitate cell-cell adhesion (43) and thus interaction. Transferrin already had been implicated in binding and effector function of NK cells (44). Alpha-2-macroglobulin bound to cellular receptors might bridge M to tumor cell membranes by complexing to target cell proteases (45). In conclusion, M at various stages of culture in teflon bags provide an excellent system to study the events and stages of human M activation for tumor cytotoxicity. The results obtained indicate the importance of the terminal maturation within the M lineage. If our data reflect normal physiology in vivo, any impairment of this differentiation step wduld have serious consequences on M function in host defense and likewise in other regulatory circuits into which cells of the MPS are involved (e,.g., bone resorption, iron metabolism and hematopoiesis). In addition, in vitro matured M rather than blood mo may be suitable for use in adoptive immunotherapy of cancer. Acknowledgements We are indebted to GLADYS MACK and ANNEGRET REHM for their excellent technical aSSIstance. This work was supported by Deutsche Forschungsgemeinschaft and Boehringer Ingelheim Fonds.
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