Journal of Immunological Methods, 127 (1990) 71-77 Elsevier
71
JIM 05463
Measurement of macrophage adherence and spreading with weak electric fields M. Kowolenko 1, C.R. Keese 2, D.A. Lawrence 3 and I. Giaever 2 I Bristol-Myers Co., Phar. Research and Development, P. 0. Box 4755, Syracuse, NIT, U.S.A., 2 School of Science, Rensselaer Polytechnic Institute, Troy, NY, U.S.A., and 3 Department of Microbiology, Albany Medical College, 47 New Scotland Aoe., Albany, NY, U.S.A. (Received 8 September 1989, revised received 13 October 1989, accepted 16 October 1989)
A new method to monitor macrophage attachment on protein-coated surfaces and spreading in response to activating agents is described. Murine macrophages were cultured on small gold electrodes coated with protein, and attachment and spreading were detected as electrical impedance changes. The rate of attachment of cells to fibronectin-coated electrodes was measured to be significantly greater than to other proteins tested. Activation agents used included interferon-v, lipopolysaccharide and heat killed Listeria monocytogenes. Addition of each agent to macrophages on electrodes resulted in characteristic patterns in the impedance time course with impedance changes as large as 40%. Key words: Macrophage activation; Cell attachment and spreading; Electrical measurement
Introduction Upon stimulation, macrophages undergo a series of events that lead to a number of biochemical and morphological changes. While many of these events are easily quantitated by conventional assay techniques, such as Ia antigen expression (Cowing et al., 1978; Warren and Vogel, 1985) or oxidative burst activity (Johnston et al., 1981; Sasada et al., 1983), two of the most difficult parameters to evaluate in a quantitative manner are macrophage adherence and spreading. Many investigators (Grinnell et al., 1977; Yamada et al.,
Correspondence to: I. Giaever, School of Science, Rensselaer Polytechnic Institute, Troy, NY, U.S.A. Abbreviations: HKLM, heat-killed Listeria monocytogenes; LPS, lipopolysaccharide; IFN-),, interferon-~/; BMDM, bone marrow-derived macrophages.
1981; Tarone et al., 1982) have described assays that measure attachment as a ratio of adherent to nonadherent cells or spreading as the mean diameter of cells observed in a given microscopic field; however, both of these methods are rather nonspecific and are subject to a wide degree of experimental variability. In this report, we describe a method that utilizes weak electric fields to monitor both macrophage attachment and spreading in response to known activating agents and to substrates coated with specific proteins. Bone marrow-derived macrophages (BMDM) were cultured on small evaporated gold electrodes immersed in tissue culture medium and subjected to an applied oscillating electric field. As previously reported (Giaever and Keese, 1984; Giaever and Keese, 1986), the impedance of this system varies with cell activities and can be used to monitor cell attachment and changes in cell morphology and cell density.
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72
Materials and methods
Mice 6-8-week-old CBA/J mice were obtained from Jackson Laboratory and maintained in the Albany Medical College Animal Care Facility, receiving Purina Lab chow and acid water ad libitum. Bone marrow-derived macrophages Femurs were removed from mice and the marrow collected by flushing the bone with balanced salt solution (BSS). Cells were aspirated several times until clumps were no longer visible, then counted using trypan blue exclusion to determine viability. Cells were placed in 150 cm2 flasks (Coming) at a final concentration of 7.5 x 10 4 cells/ml medium. Medium consisted of Dulbecco's Modified Eagle Medium (DMEM) (high glucose; Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), nonessential amino acids (MA Bioproducts), sodium pyruvate (MA Bioproducts), 25/~g/ml gentamicin (Elkin), and 10% L cell-conditioned media (a source of colony-stimulating factor 1; CSF-1). Cells were cultured for 5 days in 8% CO: at 37°C. On day 5, nonadherent cells were removed by washing each flask three times with BSS. To obtain adherent macrophages, chilled BSS was added, and each flask was placed on ice for 30 rain followed by scraping to remove the adherent cells. Cells were pelleted, resuspended in fresh medium and added to 60 mm tissue culture plates that contained electrodes. The final cell concentration was approximately 2.5 x 103 cells/ cm2 of available substrate; the final volume of medium in the dish was 3 ml.
fresh macrophage cultures to verify the results shown. Electric field measurements The electrode system was fabricated in 60 mm culture dishes as previously described (Giaever and Keese, 1984). Each finished dish contained one large (approx. 2 cm2) and four small (approx. 3 × 10 -4 cm2) electrodes. To perform electrical measurements the dish was placed in an incubator and 3 ml of medium was added over the electrodes. The large electrode and one of the small electrodes were connected to a phase-sensitive lock-in amplifier, and an AC signal of 1.0 V at 4000 Hz was supplied through a 1 Mf~ resistor (Fig. 1). Since the impedance of the dish with culture medium is at most 10 Kfl, the applied signal acts as a constant current source (--- 1 #A), and the measured voltage across the electrode is proportional to the impedance. The impedance of the dish is dominated by the boundary between the small electrode and the culture fluid, whereas the total additional impedance associated with the leads, the tissue culture solution, and the boundary between the large electrode and the culture fluid is negligible by comparison. In the work reported below, we treat the system as a simple RC series circuit in calculating resistance values from the measured impedance.
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Stimuli Macrophage-activating agents consisted of heat-killed Listeria monocytogenes (HKLM) prepared as described (Kowolenko et al., 1988), lipopolysaccharide (LPS, E. coli 055 :B5, Calbiochem), or recombinant interferon-y (IF/q-y, gift of R. Noelle, Dartmouth Medical College). Briefly, cells were added to electrode-containing culture plates, and impedance was monitored for 24-48 h. Cultures were then treated with either HKLM (3 x 1 0 7 cells/ml), LPS (10 ng/ml) or INF (100 U/ml), and impedance measurements continued for 3 days. All experiments were repeated with
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Fig. 1. Basic electrical setup used to monitor macrophage attachment and spreading. The small electrode is treated as a simple R C series circuit and the resistive portion of the measured impedance is calculated.
73
Protein-coated electrode To coat the small electrodes with specific proteins, small droplets (20 #1) of saline (0.15 M NaC1) containing 50 /~g/ml of protein were applied directly over each electrode. Following 15 rain incubation to assure adsorption of a complete monolayer of protein molecules, the droplets were removed by aspiration and the dish rinsed with saline. Complete culture medium was then added to the dish. Proteins used in these studies included rabbit IgG (Miles), gelatin (Grayslake)and human plasma fibronectin (Sigma). In addition, diluted whole fetal bovine serum was used as a control, giving the collection of adsorbed proteins norreally encountered by the cells in culture.
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Fig. 2. Attachment of macrophages to electrodes precoated with specific proteins (see text). The break in each curve is at the time of cell addition. Resistance in this and other figures is reported as the ratio of resistance measured at different times to that of the same electrode before cell addition. All electrodes have essentially the same resistance at time zero with only slight differenes due to minor variations in electrode size.
Results
Rate of cell attachment
Cell attachment studies have clearly demonstrated that certain proteins are involved in the process of ceil-surface binding in tissue culture (Yamada et al., 1981; Gieger et al., 1984; Maher and Singer, 1988). In the case of macrophages, workers have shown the presence of receptors specific for fibronectin (Molnar et al., 1987; Brown and Goodwin, 1988) and for the Fc portion of IgG molecules (Melewicz and Speigelberg, 1980; Vogel et al., 1983) on the macrophage surface; thus, we were interested in evaluating cell attachment to electrodes coated with these proteins, as well as gelatin and serum. The rate attachment of 3.0
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macrophages to precoated electrodes as determined by impedance measurements is shown in Fig. 2. In several independent tests, the fibronectin-coated electrode displayed a significantly greater initial increase in resistance due to cell attachment and spreading than the serum-coated control. Gelatin- and IgG-coated electrodes performed in a manner nearly identical to the serum-coated control, and the final resistance after several days for all four electrodes was essentially identical. Cell spreading in response to actioation agents Activation of macrophages results in distinct morphologic changes in the cell frequently de3.1]
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Fig. 3. Response of macrophages to HKLM. Cells were inoculated 1 day before the measurements shown were recorded. Electrical monitoring begins approximately 9 h before the addition of (a) H K L M (3 × 107 cells/ml) or (b) buffer only. (Arrow denotes addition of agents).
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Fig. 4. Response of macrophages to LPS and IFN-y. Cells were inoculated 1 day before the addition of activating agents. Agents were added at time zero on the graph, a: LPS (10 ng/ml); b: IFN-'t (100 U/ml).
scribed as spreading (Vasiliev and Gelfand, 1977). To determine if the electrode impedance measurements could detect these changes, three known macrophage activation agents were tested: IFN-y, lipopolysaccharide, and heat-killed Listeria monocytogenes. Cells exposed to these agents alone and in combination were electrically monitored for changes in electrode resistance. Upon addition of HKLM to an established cell layer, there was a precipitous drop in resistance followed by a significant rise when compared with a control system that received only buffer (Fig. 3). This increase in resistance reached a maximum about 10 h after the addition of HKLM. The high resistance is maintained for more than 12 h followed by rounding and detachment of cells and a large drop in pH. This is recorded as a decrease in
resistance and the loss of the small fluctuation associated with cell motility. The system was also capable of distinguishing differences in macrophage behavior induced with IFN-y and LPS, two agents that activate macrophages by different mechanisms (Adams and Hamilton, 1984, 1987). Both IFN-y and lipopolysaccharide caused increases in resistance (Fig. 4) but each with a different characteristic time course. The addition of LPS resulted in resistance patterns somewhat similar in character to those observed in the HKLM stimulated cultures showing an initial drop following by a sharp resistance increase; whereas, IFN-7 addition resulted in a more moderate increase in resistance A combination of addition of IFN-y followed approximately 1 day later by LPS resulted in enhanced activation
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Fig. 5. Response of macrophages to combinations of LPS and IFN-y. Cells were inoculated and grown 1 day before addition of the first agent. Approximately 24 h later, the second agent was added (arrow) and the resistance of the electrodes followed, a: IFN-'y followed by LPS; b: LPS followed by IFN-'t (concentrations as in Fig. 4).
75 by the LPS as determined by increased system impedance (Fig. 5a). Reversing the order of the agents in the same time sequence resulted in essentially no change in impedance upon IFN-2/ addition (Fig. 5b). The initial coating of electrodes with the various proteins outlined in Fig. 2 did not alter response patterns to the various stimuli, suggesting that the coated substrates tested in these experiments did not augment or impede macrophage activation in any detectable way (data not shown).
Discussion
The capability of macrophages to adhere to tissue culture surfaces has been utilized to evaluate the effects of both disease states (Arnaout et al., 1984) or toxicant exposure on cell function (Lopez-Cepero et al., 1986). The most common method of determining cell adherence involves incubation of a cell suspension with a tissue culture plate for a variety of times followed by washing the plates and counting cell number in the supernatant. The difference between the cells initially added and those recovered is related to adherence. Culturing bone marrow ceils in the presence of CSF-1, a macrophage-specific growth factor (Stanley et al., 1983), leads to the development of mature, functional macrophages (BMDM) that have characteristics similar to those of resident tissue macrophages. The BMDM, like the resident macrophage, display a wide variety of functions upon activation (MacKay and Russell, 1987) such as Ia antigen expression (Warren and Vogel, 1985) and tumor cell lysis (Keller and Keist, 1986). Upon stimulation with bacteria (Keller et al., 1987), IFN-7 (Adams and Hamilton, 1987), or lipopolysaccharide (Hamilton et al., 1986; Introna et al., 1986) macrophages undergo a series of events, the most obvious being an increase in cell spreading. Although the parameter of macrophage spreading has served as a rapid indication of activation, the ability to quantitate the degree of spreading has proved to be a tedious process, requiring methods such as microscopic cell diameter measurements (Vasiliev and Gelfand, 1977; Pizzey et al., 1984).
An objective and quantitative method to measure cell attachment and spreading is a needed tool in cell biology. In this paper we describe a new biophysical means to monitor these activities of macrophages in culture under a variety of experimental conditions. In this method ceils are cultured on small gold electrodes. As the cells are visually observed to spread on the electrodes, there is a concomitant increase in impedance. We have thoroughly studied this impedance change at different frequencies of the applied AC signal and have shown that the changes at 4000 Hz, used in this work, are the result of the spreading cells reducing the effective electrode area. Thus by measuring impedance, cell attachment and spreading can be continuously monitored. Electrode area and cell density affect the absolute impedance measured; however, in the work presented, we are interested in changes in the impedance and minor variations in electrode size or macrophage density can be neglected. This method necessarily looks at cells on the gold surface and not on conventional tissue culture substrates; although we have not observed differences in behavior, this limitation should be noted. Placement of cells in the electric fields does not induce cell activation nor affect cell viability. This is in agreement with previous findings concerning the detection of fibroblast motility and spreading that suggest these measurements have no effect on the cells (Cfiaever and Keese, 1984; Giaever and Keese, 1986). The adherence of cells to each other and to substrates depends on the interaction of various proteins such as fibronectin with its receptor on the cell surface. The ability to adsorb proteins on electrodes prior to the addition of cells allowed us to electrically measure the effects of protein substrates on the cell attachment behavior and activation. As would be predicted, fibronectin, when adsorbed to electrodes, resulted in more rapid attachment than did other proteins tested. While the cells also possess receptors for the Fc portion of IgG molecules, these proteins are not believed to be associated with adherence but rather function in clearance mechanisms (Waldman, 1989). Accordingly, the electric field measurements using adsorbed IgG molecules did not show any significant difference in the rate of attachment compared to controls. It should be noted that we
76 e m p l o y e d a d s o r b e d I g G in these studies a n d n o t a layer of I g G specifically b o u n d to a d s o r b e d antigen. It should b e p o s s i b l e to test a n d i d e n t i f y o t h e r molecules associated with a d h e r e n c e with this system, such as leukocyte a d h e r e n c e f a c t o r (Springer et al., 1987), b y c o a t i n g the electrodes with specific p r o t e i n s or isolated m e m b r a n e fragm e n t s c o n t a i n i n g c o m p l e m e n t a r y receptors. T h e three m a c r o p h a g e activators utilized in this study, Listeria monocytogenes, l i p o p o l y s a c c h a r i d e a n d I F N - 7 , a p p e a r to activate m a c r o p h a g e s through distinct receptor/ligand interactions ( A d a m s a n d H a m i l t o n , 1987). I F N - y has b e e n shown to b i n d to its r e c e p t o r a n d i n d u c e b i o c h e m ical changes within the cell, such as increased p r o t e i n kinase C activity a n d i n c r e a s e d levels of intracellular Ca 2+ (Weiel et al., 1986). L P S a n d H K L M are b o t h ' s e c o n d signal' agents, i n d u c i n g cytolytic activity of m a c r o p h a g e s a n d similar b i o chemical events, such as increased p r o t e i n synthesis ( H a m i l t o n et al., 1986). A n a l y s i s utilizing i m p e d a n c e m e a s u r e m e n t s d e m o n s t r a t e d t h a t the degree of s p r e a d i n g i n d u c e d b y either L P S or H K L M was similar. Interestingly, the electric field m e a s u r e m e n t d e t e c t e d differences in the degree of s p r e a d i n g b e t w e e n H K L M or LPS a c t i v a t e d cell a n d I F N - y a c t i v a t e d cells. T h e sequential a d d i t i o n of IFN-~, followed b y LPS resulted in e n h a n c e m e n t of s p r e a d i n g while LPS followed b y I F N - ' t showed n o increase a b o v e the m a x i m u m r e a c h e d b y LPS alone. This implies that the m a c r o p h a g e is c a p a b l e of a finite degree of s p r e a d i n g a n d that LPS alone delivers a sufficient signal to i n d u c e this m a x i m a l degree o f spreading. It w o u l d a p p e a r f r o m this d a t a t h a t j u s t as different a c t i v a t i o n agents induce characteristic b i o c h e m i c a l events within the cell, these s a m e agents induce specific m o r p h o l o g i c changes of the cell.
References Adams, D.O, and Hamilton, T.A. (1984) The cell biology of macrophage activation. Ann. Rev. Immunol. 2, 283. Adams, D.O. and Hamilton, T.A. (1987) Molecular transductial mechanisms by which interferon and other signals regulate macrophage development. Immunol. Rev. 97, 5. Arnaout, M.A., Spits, H., Terhorst, C., Pitt, J. and Todd, R.F.I. (1984) Deficiency of a leukocyte surface glycoprotein
(LFA-1) in two patients with mol deficiency. J. Clin. Invest. 74, 1291. Brown, E.J. and Goodwin, J.L. (1988) Fibronectin receptors of phagocytes. Characterization of the Arg-Gly-Asp binding proteins of human monocytes and polymorphonuclear leukocytes, J. Exp. Med. 167, 777. Cowing, C., Schwartz, B.D. and Dickler, H.B. (1978) Macrophage Ia antigens. J. Immunol. 120, 378. Giaever, I. and Keese, C.R. (1984) Monitoring fibroblast behavior in tissue culture with an applied electric field. Proc. Natl. Acad. Sci. U.S.A. 81, 3761. Giaever, I. and Keese, C.R. (1986) Use of electric fields to monitor the dynamical aspect of cell behavior in tissue culture. IEEE Trans. Biomed. Eng. 33, 242. Gieger, B., Avnur, Z., Rinnerthaler, G., Hinssen, H. and Small, V. (1984) Microfilament-organizing centers in aras of cell contact: Cytoskeletal interactions during cell atachment and locomotion, J. Cell Biol. 99, 83s. Grinnell, F., Hayes, D.G. and Minter, D. (1977) Cell adhesion and spreading factor. Partial purification and properties. Exp. Cell Res. 110, 175. Hamilton, T.A., Jansen, M.J., Somers, S.D. and Adams, D.O. (1986) Effect of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: Relationship to activation for macrophage tumoricidal function. J. Cell Physiol. 128, 9. Introna, M., Hamilton, T.A., Kaufman, R.E., Adams, D.O. and Bast, R.C. (1986) Functional activation of murine peritoneal macrophages with bacterial lipopolysaccharide alter expression of c-rnyc and c-fos oncogenes. J. Immunol. 137, 2711. Johnston, R.B., Chadwick, D.A. and Cohn, Z.A. (1981) Priming of macrophages for enhanced oxidative metabolism by exposure to proteolytic enzymes. J. Exp. Med. 153, 1678. Keller, R. and Keist, R. (1986) Induction and maintenance and reintroduction of tumoricidal activity in bone marrow derived mononuclear phagocytes by macrophage activating lymphokines (MAF). Cell. Imrnunol. 101, 659. Keller, R., Keist, R., Van Der Meide, P.H., Groscurth, P., Aguet, M. and Leist, T.P. (1987) Induction, maintenance and reinduction of tumoricidal activity in bone marrow derived mononuclear phagocytes by Corynebacterium parvum. Evidence for the involvement of a T cell and interferon-independent pathway of macrophage activation. J. Immunol. 138, 2366. Kowolenko, M., Tracy, L., Mudzinski, S. and Lawrence, D.A. (1988) Effect of lead on macrophage function, J. Leukocyte Biol. 43, 357. Lopez-Cepero, M., Friedman, M., Klein, T. and Friedman, H. (1986) Tetrahydrocannabinol-induced suppression of macrophage spreading and phagocytic activity in vitro. J. Leukocyte Biol. 39, 679, MacKay, R.J. and Russell, S.W. (1987) Protein phenotypes of mouse macrophages activated in vivo for tumor cell killing. J. Leukocyte Biol. 42, 213. Maher, P.A. and Singer, S.J. (1988) An integral membrane protein antigen associated with the membrane attachment
77 sites of actin microfilaments is identified as an integrin B chain. Mol. Cell Biol. 8, 564. Melewicz, F.M. and Speigelberg, H.L. (1980) Fc Receptors for IgG on a subpopulation of human peripheral blood monocytes. J. Immunol. 125, 1026. Molnar, J., Hoekstra, S., Ku, C.S. and Van Alten, P. (1987) Evidence for the recyling nature of the fibronectin receptor of macrophages. J. Cell Physiol. 131,374. Pizzey, J., Witkowski, J. and Jones, G. (1984) Monensin-induced inhibition of cell spreading in normal and dystrophic human fibroblasts. Proc. Natl. Aead. Sci. U.S.A. 81, 4960. Sasada, M., Pabst, M.J. and Johnston, R.B. (1983) Activation of mouse peritoneal macrophages by LPS alters the kinetic parameters of the superoxide producing NADPH oxidase. J. Biol. Chem. 258, 9631. Springer, T.A., Dustin, M.L., Kishimoto, T.K. and Marlin, S. (1987) The lymphocyte function associated LFA-1, CD2 and LFA-3 molecules: Cell adhesion receptors of the immune system. Ann. Rev. Immunol. 5, 223. Stanley, E.R., Guilbert, L.J., Tushinski, R.J. and Bartelmez, S.H. (1983) CSF-1-A lineage-specific hematopietic growth factor. J. Cell. Biochem. 21, 151. Tarone, G., Galetto, G., Prat, M. and Comoglio, P.M. (1982)
CeU surface molecules and fibronectin-mediated cell adhesion: Effect of proteolytic digestion of membrane proteins. J. Cell Biol. 94, 197. Vasiliev, J.M and Gelfand, I.M. (1977) Mechanism of morphogenesis in cell structures. Int. Rev. Cytol. 50, 159. Vogel, S.N., Finbloom, D.S., English, K.E., Rosenstreich, D.L. and Langreth, S.G. (1983) Interferon-induced enhancement of macrophage Fc receptor expression, fl-Interferon treatment of C3H/HeJ macrophages results in increased numbers and density of Fc repector. J. Immunol. 130, 1210. Waldman, H. (1989) Manipulation of T-cell responses with monoclonal antibodies. Ann. Rev. Immunol. 7, 407. Warren, M.K. and Vogel, S.N. (1985) Bone marrow-derived macrophages: Development and regulation of differentiation markers by colony stimulahing factor and interferons. J. Immunol. 134, 982. Weiel, J.E., Hamilton, T.A. and Adams, D.O. (1986) LPS induces altered phosphate labeling of proteins in murine peritoneal macrophages. J. Immuol. 136, 3012. Yamada, K.M., Kennedy, D.W., Grotendorst, G.R. and Moral, T. (1981) Glycolipids: Receptors for fibronectin. J. Cell Physiol. 109, 343.
71
JIM 05463
Measurement of macrophage adherence and spreading with weak electric fields M. Kowolenko 1, C.R. Keese 2, D.A. Lawrence 3 and I. Giaever 2 I Bristol-Myers Co., Phar. Research and Development, P. 0. Box 4755, Syracuse, NIT, U.S.A., 2 School of Science, Rensselaer Polytechnic Institute, Troy, NY, U.S.A., and 3 Department of Microbiology, Albany Medical College, 47 New Scotland Aoe., Albany, NY, U.S.A. (Received 8 September 1989, revised received 13 October 1989, accepted 16 October 1989)
A new method to monitor macrophage attachment on protein-coated surfaces and spreading in response to activating agents is described. Murine macrophages were cultured on small gold electrodes coated with protein, and attachment and spreading were detected as electrical impedance changes. The rate of attachment of cells to fibronectin-coated electrodes was measured to be significantly greater than to other proteins tested. Activation agents used included interferon-v, lipopolysaccharide and heat killed Listeria monocytogenes. Addition of each agent to macrophages on electrodes resulted in characteristic patterns in the impedance time course with impedance changes as large as 40%. Key words: Macrophage activation; Cell attachment and spreading; Electrical measurement
Introduction Upon stimulation, macrophages undergo a series of events that lead to a number of biochemical and morphological changes. While many of these events are easily quantitated by conventional assay techniques, such as Ia antigen expression (Cowing et al., 1978; Warren and Vogel, 1985) or oxidative burst activity (Johnston et al., 1981; Sasada et al., 1983), two of the most difficult parameters to evaluate in a quantitative manner are macrophage adherence and spreading. Many investigators (Grinnell et al., 1977; Yamada et al.,
Correspondence to: I. Giaever, School of Science, Rensselaer Polytechnic Institute, Troy, NY, U.S.A. Abbreviations: HKLM, heat-killed Listeria monocytogenes; LPS, lipopolysaccharide; IFN-),, interferon-~/; BMDM, bone marrow-derived macrophages.
1981; Tarone et al., 1982) have described assays that measure attachment as a ratio of adherent to nonadherent cells or spreading as the mean diameter of cells observed in a given microscopic field; however, both of these methods are rather nonspecific and are subject to a wide degree of experimental variability. In this report, we describe a method that utilizes weak electric fields to monitor both macrophage attachment and spreading in response to known activating agents and to substrates coated with specific proteins. Bone marrow-derived macrophages (BMDM) were cultured on small evaporated gold electrodes immersed in tissue culture medium and subjected to an applied oscillating electric field. As previously reported (Giaever and Keese, 1984; Giaever and Keese, 1986), the impedance of this system varies with cell activities and can be used to monitor cell attachment and changes in cell morphology and cell density.
0022-1759/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
72
Materials and methods
Mice 6-8-week-old CBA/J mice were obtained from Jackson Laboratory and maintained in the Albany Medical College Animal Care Facility, receiving Purina Lab chow and acid water ad libitum. Bone marrow-derived macrophages Femurs were removed from mice and the marrow collected by flushing the bone with balanced salt solution (BSS). Cells were aspirated several times until clumps were no longer visible, then counted using trypan blue exclusion to determine viability. Cells were placed in 150 cm2 flasks (Coming) at a final concentration of 7.5 x 10 4 cells/ml medium. Medium consisted of Dulbecco's Modified Eagle Medium (DMEM) (high glucose; Gibco) supplemented with 10% fetal bovine serum (FBS, Hyclone), nonessential amino acids (MA Bioproducts), sodium pyruvate (MA Bioproducts), 25/~g/ml gentamicin (Elkin), and 10% L cell-conditioned media (a source of colony-stimulating factor 1; CSF-1). Cells were cultured for 5 days in 8% CO: at 37°C. On day 5, nonadherent cells were removed by washing each flask three times with BSS. To obtain adherent macrophages, chilled BSS was added, and each flask was placed on ice for 30 rain followed by scraping to remove the adherent cells. Cells were pelleted, resuspended in fresh medium and added to 60 mm tissue culture plates that contained electrodes. The final cell concentration was approximately 2.5 x 103 cells/ cm2 of available substrate; the final volume of medium in the dish was 3 ml.
fresh macrophage cultures to verify the results shown. Electric field measurements The electrode system was fabricated in 60 mm culture dishes as previously described (Giaever and Keese, 1984). Each finished dish contained one large (approx. 2 cm2) and four small (approx. 3 × 10 -4 cm2) electrodes. To perform electrical measurements the dish was placed in an incubator and 3 ml of medium was added over the electrodes. The large electrode and one of the small electrodes were connected to a phase-sensitive lock-in amplifier, and an AC signal of 1.0 V at 4000 Hz was supplied through a 1 Mf~ resistor (Fig. 1). Since the impedance of the dish with culture medium is at most 10 Kfl, the applied signal acts as a constant current source (--- 1 #A), and the measured voltage across the electrode is proportional to the impedance. The impedance of the dish is dominated by the boundary between the small electrode and the culture fluid, whereas the total additional impedance associated with the leads, the tissue culture solution, and the boundary between the large electrode and the culture fluid is negligible by comparison. In the work reported below, we treat the system as a simple RC series circuit in calculating resistance values from the measured impedance.
TISSUE CULTURE MEDIUM (ELECTROLYTE) - CELLS
"
-
-
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GOLD ELECTflODE O04CM2)
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Stimuli Macrophage-activating agents consisted of heat-killed Listeria monocytogenes (HKLM) prepared as described (Kowolenko et al., 1988), lipopolysaccharide (LPS, E. coli 055 :B5, Calbiochem), or recombinant interferon-y (IF/q-y, gift of R. Noelle, Dartmouth Medical College). Briefly, cells were added to electrode-containing culture plates, and impedance was monitored for 24-48 h. Cultures were then treated with either HKLM (3 x 1 0 7 cells/ml), LPS (10 ng/ml) or INF (100 U/ml), and impedance measurements continued for 3 days. All experiments were repeated with
LAflUE GOLD COUNTER ELECTRODE (IOICM2)
I
I
1
Fig. 1. Basic electrical setup used to monitor macrophage attachment and spreading. The small electrode is treated as a simple R C series circuit and the resistive portion of the measured impedance is calculated.
73
Protein-coated electrode To coat the small electrodes with specific proteins, small droplets (20 #1) of saline (0.15 M NaC1) containing 50 /~g/ml of protein were applied directly over each electrode. Following 15 rain incubation to assure adsorption of a complete monolayer of protein molecules, the droplets were removed by aspiration and the dish rinsed with saline. Complete culture medium was then added to the dish. Proteins used in these studies included rabbit IgG (Miles), gelatin (Grayslake)and human plasma fibronectin (Sigma). In addition, diluted whole fetal bovine serum was used as a control, giving the collection of adsorbed proteins norreally encountered by the cells in culture.
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Fig. 2. Attachment of macrophages to electrodes precoated with specific proteins (see text). The break in each curve is at the time of cell addition. Resistance in this and other figures is reported as the ratio of resistance measured at different times to that of the same electrode before cell addition. All electrodes have essentially the same resistance at time zero with only slight differenes due to minor variations in electrode size.
Results
Rate of cell attachment
Cell attachment studies have clearly demonstrated that certain proteins are involved in the process of ceil-surface binding in tissue culture (Yamada et al., 1981; Gieger et al., 1984; Maher and Singer, 1988). In the case of macrophages, workers have shown the presence of receptors specific for fibronectin (Molnar et al., 1987; Brown and Goodwin, 1988) and for the Fc portion of IgG molecules (Melewicz and Speigelberg, 1980; Vogel et al., 1983) on the macrophage surface; thus, we were interested in evaluating cell attachment to electrodes coated with these proteins, as well as gelatin and serum. The rate attachment of 3.0
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macrophages to precoated electrodes as determined by impedance measurements is shown in Fig. 2. In several independent tests, the fibronectin-coated electrode displayed a significantly greater initial increase in resistance due to cell attachment and spreading than the serum-coated control. Gelatin- and IgG-coated electrodes performed in a manner nearly identical to the serum-coated control, and the final resistance after several days for all four electrodes was essentially identical. Cell spreading in response to actioation agents Activation of macrophages results in distinct morphologic changes in the cell frequently de3.1]
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Fig. 3. Response of macrophages to HKLM. Cells were inoculated 1 day before the measurements shown were recorded. Electrical monitoring begins approximately 9 h before the addition of (a) H K L M (3 × 107 cells/ml) or (b) buffer only. (Arrow denotes addition of agents).
74
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Fig. 4. Response of macrophages to LPS and IFN-y. Cells were inoculated 1 day before the addition of activating agents. Agents were added at time zero on the graph, a: LPS (10 ng/ml); b: IFN-'t (100 U/ml).
scribed as spreading (Vasiliev and Gelfand, 1977). To determine if the electrode impedance measurements could detect these changes, three known macrophage activation agents were tested: IFN-y, lipopolysaccharide, and heat-killed Listeria monocytogenes. Cells exposed to these agents alone and in combination were electrically monitored for changes in electrode resistance. Upon addition of HKLM to an established cell layer, there was a precipitous drop in resistance followed by a significant rise when compared with a control system that received only buffer (Fig. 3). This increase in resistance reached a maximum about 10 h after the addition of HKLM. The high resistance is maintained for more than 12 h followed by rounding and detachment of cells and a large drop in pH. This is recorded as a decrease in
resistance and the loss of the small fluctuation associated with cell motility. The system was also capable of distinguishing differences in macrophage behavior induced with IFN-y and LPS, two agents that activate macrophages by different mechanisms (Adams and Hamilton, 1984, 1987). Both IFN-y and lipopolysaccharide caused increases in resistance (Fig. 4) but each with a different characteristic time course. The addition of LPS resulted in resistance patterns somewhat similar in character to those observed in the HKLM stimulated cultures showing an initial drop following by a sharp resistance increase; whereas, IFN-7 addition resulted in a more moderate increase in resistance A combination of addition of IFN-y followed approximately 1 day later by LPS resulted in enhanced activation
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Fig. 5. Response of macrophages to combinations of LPS and IFN-y. Cells were inoculated and grown 1 day before addition of the first agent. Approximately 24 h later, the second agent was added (arrow) and the resistance of the electrodes followed, a: IFN-'y followed by LPS; b: LPS followed by IFN-'t (concentrations as in Fig. 4).
75 by the LPS as determined by increased system impedance (Fig. 5a). Reversing the order of the agents in the same time sequence resulted in essentially no change in impedance upon IFN-2/ addition (Fig. 5b). The initial coating of electrodes with the various proteins outlined in Fig. 2 did not alter response patterns to the various stimuli, suggesting that the coated substrates tested in these experiments did not augment or impede macrophage activation in any detectable way (data not shown).
Discussion
The capability of macrophages to adhere to tissue culture surfaces has been utilized to evaluate the effects of both disease states (Arnaout et al., 1984) or toxicant exposure on cell function (Lopez-Cepero et al., 1986). The most common method of determining cell adherence involves incubation of a cell suspension with a tissue culture plate for a variety of times followed by washing the plates and counting cell number in the supernatant. The difference between the cells initially added and those recovered is related to adherence. Culturing bone marrow ceils in the presence of CSF-1, a macrophage-specific growth factor (Stanley et al., 1983), leads to the development of mature, functional macrophages (BMDM) that have characteristics similar to those of resident tissue macrophages. The BMDM, like the resident macrophage, display a wide variety of functions upon activation (MacKay and Russell, 1987) such as Ia antigen expression (Warren and Vogel, 1985) and tumor cell lysis (Keller and Keist, 1986). Upon stimulation with bacteria (Keller et al., 1987), IFN-7 (Adams and Hamilton, 1987), or lipopolysaccharide (Hamilton et al., 1986; Introna et al., 1986) macrophages undergo a series of events, the most obvious being an increase in cell spreading. Although the parameter of macrophage spreading has served as a rapid indication of activation, the ability to quantitate the degree of spreading has proved to be a tedious process, requiring methods such as microscopic cell diameter measurements (Vasiliev and Gelfand, 1977; Pizzey et al., 1984).
An objective and quantitative method to measure cell attachment and spreading is a needed tool in cell biology. In this paper we describe a new biophysical means to monitor these activities of macrophages in culture under a variety of experimental conditions. In this method ceils are cultured on small gold electrodes. As the cells are visually observed to spread on the electrodes, there is a concomitant increase in impedance. We have thoroughly studied this impedance change at different frequencies of the applied AC signal and have shown that the changes at 4000 Hz, used in this work, are the result of the spreading cells reducing the effective electrode area. Thus by measuring impedance, cell attachment and spreading can be continuously monitored. Electrode area and cell density affect the absolute impedance measured; however, in the work presented, we are interested in changes in the impedance and minor variations in electrode size or macrophage density can be neglected. This method necessarily looks at cells on the gold surface and not on conventional tissue culture substrates; although we have not observed differences in behavior, this limitation should be noted. Placement of cells in the electric fields does not induce cell activation nor affect cell viability. This is in agreement with previous findings concerning the detection of fibroblast motility and spreading that suggest these measurements have no effect on the cells (Cfiaever and Keese, 1984; Giaever and Keese, 1986). The adherence of cells to each other and to substrates depends on the interaction of various proteins such as fibronectin with its receptor on the cell surface. The ability to adsorb proteins on electrodes prior to the addition of cells allowed us to electrically measure the effects of protein substrates on the cell attachment behavior and activation. As would be predicted, fibronectin, when adsorbed to electrodes, resulted in more rapid attachment than did other proteins tested. While the cells also possess receptors for the Fc portion of IgG molecules, these proteins are not believed to be associated with adherence but rather function in clearance mechanisms (Waldman, 1989). Accordingly, the electric field measurements using adsorbed IgG molecules did not show any significant difference in the rate of attachment compared to controls. It should be noted that we
76 e m p l o y e d a d s o r b e d I g G in these studies a n d n o t a layer of I g G specifically b o u n d to a d s o r b e d antigen. It should b e p o s s i b l e to test a n d i d e n t i f y o t h e r molecules associated with a d h e r e n c e with this system, such as leukocyte a d h e r e n c e f a c t o r (Springer et al., 1987), b y c o a t i n g the electrodes with specific p r o t e i n s or isolated m e m b r a n e fragm e n t s c o n t a i n i n g c o m p l e m e n t a r y receptors. T h e three m a c r o p h a g e activators utilized in this study, Listeria monocytogenes, l i p o p o l y s a c c h a r i d e a n d I F N - 7 , a p p e a r to activate m a c r o p h a g e s through distinct receptor/ligand interactions ( A d a m s a n d H a m i l t o n , 1987). I F N - y has b e e n shown to b i n d to its r e c e p t o r a n d i n d u c e b i o c h e m ical changes within the cell, such as increased p r o t e i n kinase C activity a n d i n c r e a s e d levels of intracellular Ca 2+ (Weiel et al., 1986). L P S a n d H K L M are b o t h ' s e c o n d signal' agents, i n d u c i n g cytolytic activity of m a c r o p h a g e s a n d similar b i o chemical events, such as increased p r o t e i n synthesis ( H a m i l t o n et al., 1986). A n a l y s i s utilizing i m p e d a n c e m e a s u r e m e n t s d e m o n s t r a t e d t h a t the degree of s p r e a d i n g i n d u c e d b y either L P S or H K L M was similar. Interestingly, the electric field m e a s u r e m e n t d e t e c t e d differences in the degree of s p r e a d i n g b e t w e e n H K L M or LPS a c t i v a t e d cell a n d I F N - y a c t i v a t e d cells. T h e sequential a d d i t i o n of IFN-~, followed b y LPS resulted in e n h a n c e m e n t of s p r e a d i n g while LPS followed b y I F N - ' t showed n o increase a b o v e the m a x i m u m r e a c h e d b y LPS alone. This implies that the m a c r o p h a g e is c a p a b l e of a finite degree of s p r e a d i n g a n d that LPS alone delivers a sufficient signal to i n d u c e this m a x i m a l degree o f spreading. It w o u l d a p p e a r f r o m this d a t a t h a t j u s t as different a c t i v a t i o n agents induce characteristic b i o c h e m i c a l events within the cell, these s a m e agents induce specific m o r p h o l o g i c changes of the cell.
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