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The role of divalent cations in interactions between lymphokines and macrophages
CELLULAR
IMMUNOLOGY
53, 236-245 (1980)
The Role of Divalent Cations in Interactions Lymphokines and Macrophages1g2 JAMES
between
D. JOHNSON, W. LEE HAND, AND NEVA L. KING-THOMPSON
Veterans Administration Medicine,
Medical Center (Atlanta), Decatur, Georgia 30033, and the Department Emory University School of Medicine, Atlanta, Georgia 30303
of
Received May 16, I979
The divalent cation requirements of lymphokine-mediated alterations in macrophage function (activation and inhibition of migration) were examined. Normal rabbit alveolar macrophages exposed to incubation supernatants of antigen-stimulated sensitized lymphocytes (lymphokine) were activated, manifested by increased adherence and enhanced bactericidal activity, as compared with control cells. This lymphokine-mediated activation was dependent upon the presence of extracellular Mg2+ (but not Ca2+). Our data from both current and previous studies suggest that Mg*+ influx is necessary for initiation or support of the macrophage activation process. The divalent cation requirements for lymphokine (MIF)induced inhibition of macrophage migration differed from that of the activation phenomenon. Specifically, both Ca2+and Mg2+ were required for expression of MIF activity. Adsorption experiments indicate that these cations are needed for binding of MIF to the macrophage surface.
INTRODUCTION Activation of macrophages, a process by which these cells develop an increased capacity to inactivate microorganisms, is responsible for acquired resistance to many intracellular organisms (1). Macrophage activation in viva is a consequence of the cell-mediated immune response. Biologically active products (lymphokines) released by sensitized lymphocytes after contact with specific antigen have a profound effect upon mononuclear phagocytes. Among those lymphokines with effects upon macrophages are factors which inhibit cell migration and stimulate macrophage activation (reviewed in Ref. (2)). It is this latter property, the activation of macrophages, whereby these cells become the antimicrobial effector of cell-mediated immunity (CMI).3 In previous studies we found that lymphokines exert their effects upon alveolar macrophages (3, 4). The molecular mechanisms of lymphokine-mediated altera* This work was supported by funds from the Medical Research Service of the Veterans Administration. * Requests for reprints should be sent to Dr. W. Lee Hand at the Veterans Administration Medical Center (Atlanta), 1670 Clairmont Road, Decatur, Ga. 30033. 3 Abbreviations used: CMI, cell-mediated immunity; LRT, lower respiratory tract; LN, lymph node; PBS, phosphate-buffered saline; TC199, tissue culture medium 199; NRS, pooled, normal heat-inactivated rabbit serum; MEM, modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; MIF, migration inhibitory factor. 236 0008~8749/80/10023610$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
Mg2+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
237
tions in macrophages are still uncertain. However, we recently demonstrated that activation of alveolar macrophages can be induced by exposure to the divalent cation ionophore A23187 (5). This ionophore-mediated activation required the presence of extracellular magnesium (Mg2+) and was presumably dependent upon the influx of Mg2+. The possibility that lymphokine-mediated alterations in macrophage function (including activation) are also dependent upon the presence of specific divalent cations is the basis of the present study. Specifically, we have examined the Mg2+ and Ca2+requirements of both the activation process and the inhibition of migration induced in alveolar macrophages by lymphokines. MATERIALS
AND METHODS
Bacteria A clinical isolate ofListeria monocytogenes was stored at -70°C and then grown overnight in trypticase soy broth (BBL, Division of Becton, Dickinson, and Co., Cockeysville, Md.) for the studies described below. Lower Respiratory Tract (LRT) Infection New Zealand male rabbits were exposed to aerosols of L. monocytogenes (T&R airborne infection apparatus, Tri-R Instruments, Rockville Center, N.Y .) as previously described in detail (6). Bacterial Antigen Overnight cultures of Listeria were washed, resuspended in phosphate-buffered saline (PBS), and disrupted in an ultrasonic apparatus (Lab-Line Instrument, Inc., Melrose Park, Ill.). Supernatants were cleared by centrifugation, dialyzed against PBS, sterilized by membrane filtration, and stored at -20°C (3). Collection of Alveolar Macrophages and Lymph Nodes (LN) Animals were anesthetized with sodium thiopental prior to exsanguination. Cells were collected from the LRT of normal animals by lavage with sterile saline (0.15 M) containing heparin (10 units/ml), penicillin (100 U/ml), and streptomycin (100 pg/ml) (Grand Island Biological Co., Grand Island, N.Y.) (3, 6, 7). Thoracic LN were aseptically removed from infected animals. Incubation of LN Lymphocytes for Lymphokine Production LN cells (lymphocytes) were obtained by teasing nodes from infected animals on stainless steel sieves (100 mesh) (W. S. Tyler, Inc., Mentor, Ohio). Cells were washed twice in tissue culture medium 199 (BBL) containing gentamicin, 25 pg/ml (TC199), but without serum, and then resuspended in the same medium at a concentration of 1 to 2 x lo7 viable cells/ml. Sets of culture tubes containing 2.5 ml of cell suspensions were set up with or without Listeria sonicate antigen (60 pg protein/ml) and placed in a CO2 incubator for 24 hr. After incubation, the supernatants were collected by centrifugation and antigen was added to the control supernatants (4).
238 Fractionation
JOHNSON,
of Migration
HAND,
Inhibitory
AND KING-THOMPSON
Factor
Some lymphocyte culture supernatants were then fractionated on Sephadex G-75 as previously described (3). Volumes (150 ml) of antigen-stimulated and control supernatants were dialyzed against PBS and concentrated to a volume of 10 ml. These concentrated supernatants were then fractionated on a calibrated 2 x 90-cm column of Sephadex G-75 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) MIF-containing fractions of antigen-stimulated supernatants (corresponding to a molecular weight of approximately 65,000) and the corresponding control supernatant fractions were collected, concentrated, and used in the macrophage migration assay as described below. Macrophage
Monolayers
Cells from the LRT of normal animals were washed and suspended in TC199 containing 15%normal heat-inactivated rabbit serum (NRS), placed in 35-mm plastic tissue culture dishes (3 to 5 x lo6 cells per dish), and incubated for 4 hr at 37°C in 95% air-5% CO, (4). Assay of Macrophage
Function
Lymphocyte culture supernatants were dialyzed against saline and then against Ca2+-and Mg2+-“free” modified Eagle’s medium (MEM) at 4°C over a 72-hr period. NRS, depleted of divalent cations by exposure to a chelating resin (Chelex 100, Bio-Rad, Richmond, Calif.), was added to these supernatants in a concentration of 15%. Ca2+ (1.8 x 10m3M) and/or Mg2+ (0.8 x lop3 M) were added to these supernatants as indicated below. Divalent cation levels in these preparations were measured with an atomic absorption spectrophotometer (Perkin-Elmer). Ca2+ and Mg2+ concentrations in the divalent cation-“free” salt solutions, media, and supernatants were approximately 1 x 1O-5M. (a) Macropkage adherence and bactericidal activity. After removal of nonadherent cells, monolayers of normal alveolar macrophages were covered with antigenstimulated or control supernatants (with or without Ca2+and Mg2+) and incubated for 72 hr prior to studies of macrophage function. At the conclusion of this incubation period, determinations of adherence to tissue culture dishes and of bactericidal activity were performed as previously described (4). Adherence to culture dishes (cell count) of macrophages incubated in antigen-stimulated supernatants was compared with the corresponding cell count in dishes containing control supernatants and expressed as a percentage: % cell adherence = No. of macrophages on dishes with stimulated supernatants x IO0 No. of macrophages on dishes with control supernatants * Survival of L. monocytogenes phagocytized (during a 30-min period) by macrophage monolayers was determined by comparing the number of viable intracellular bacteria remaining after an additional 2-hr incubation with that
Mg*+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
239
immediately.after the phagocytic period (0 time): % intracellular bacterial survival (2 hr) viable intracellular bacteria at 2 hr x 100. viable intracellular bacteria at 0 time (b) Inhibition of macrophage migration. Whole or fractionated stimulated and control supernatants (in MEM-15% NRS with or without Ca2+ and Mg2+) were added to Sykes-Moore chambers containing capillary tubes filled with normal alveolar macrophages (3). The chambers were incubated at 37°C in 95% air-5% COz. Migration of cells from the capillaries was measured at 24 hr and expressed as percentage inhibition of macrophage migration: % inhibition of migration = 100 -
area of migration in antigen-stimulated supernatant x 100 . area of migration in control supernatant ( 1
In some experiments, the macrophages were washed with 1 mM EDTA, to remove divalent cations bound to cell surface, prior to the migration assay. Macrophage viability, migratory capacity, and response to lymphokine were unaffected by this exposure to EDTA. The role of divalent cations in the binding of MIF to the surface of alveolar macrophages was examined in the following manner. Macrophages were incubated at 4°C for 2 hr in MIF-containing (antigen-stimulated) or control supernatants with or without Ca2+and/or Mg2+, at a concentration of 5 X lo6 cells/ml of supernatant. The cells were then removed by centrifugation, and the supernatants were supplemented with Ca2+ and/or Mg2+, if needed, to achieve physiologic concentrations of each cation. Supernatants were then utilized in studies of alveolar macrophage migration. Statistical Analysis Differences between experimental groups were evaluated with Student’s t test. RESULTS Characterization of LRT Cells This cell population has been previously described in detail (3). Cells (40 to 50 x 106) were obtained by LRT lavage of normal animals. Numbers of cells obtained from animals with L. monocytogenes infection of the LRT were increased two to six times above normal. A mean of 85-90% of LRT cells were macrophages. Viability of these cells, as determined by trypan blue exclusion, was routinely ~95%. Assays of Macrophage Function (a) Macrophage adherence and bactericidal activity. LN lymphocytes, obtained from animals 12 to 14 days after initiation of L. monocytogenes respiratory tract infection, were incubated in the presence or absence of Listeria antigen. Supernatants from antigen-stimulated or control lymphocyte incubations were then
240
JOHNSON, HAND, AND KING-THOMPSON TABLE 1 Function of Alveolar Macrophages after Exposure to Supernatants from Antigen-Stimulated or Control Lymphocyte Incubations Experimental group
Percentage intracellular bacterial survival (2 hr)”
Percentage cell adherence”
Control Antigen stimulated
100
54.0 k 6.1” (13)
191.9 k 31.6’
28.0 ? 3.4” (13)
(20) ” ’ ” ”
Calculated as described under Materials and Methods. Mean + SEM. Number of experiments in parentheses. Significantly different from control (P < 0.01). Significantly different from control (P < 0.005).
placed on monolayers of normal alveolar macrophages. Cells exposed to antigen-stimulated supernatants for 72 hr showed evidence of activation. That is, they exhibited increases in adherence to tissue culture dishes and bactericidal activity against L. monocytogenes as compared with control cells (Table 1). Furthermore, these activated cells manifested enhanced agglutinability or clumping (data not shown). TABLE 2 Bactericidal Activity of Alveolar Macrophages after Exposure to Lymphocyte Incubation Supernatants Divalent cations (M)
Percentage intracellular bacterial survival (2 hr)”
Experimental group
Mg’+
Ca’+
Control Ca’+ and M g2+
0.8 x 10m3
1.8 x IO-”
54.0 t 6.1” (13)
ca2+ 6‘free’ 1
0.8 x lo-”
1 x 10-S
55.8 t 9.5 (5)
Mg’+ “free”
1 x 10-s
1.8 x 10m3
61.0 k 8.4 (7)
0.8 x lO-3
I.8 x lO-3
28.0 k 3.4’ (13)
Ca’+ “free”
0.8 x lO-3
1
20.2 2 5.4” (5)
Mg’+ “free”
1
1.8 x 10m3
Antigen stimulated CaZf and Mg’+
x 10-s
x 10-b
67.5 + 12.8
(8) a Calculated as described under Materials and Methods. b Mean + SEM. Number of experiments in parentheses c Significantly different from control (P < 0.005). d Significantly different from control (P < 0.001).
Mg2+ AND Caz+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
241
TABLE 3 Divalent Cations and Lymphokine-Mediated Inhibition of Alveolar Macrophage Migration
Experimental group
Percentage inhibition of migration by lymphokine”
Ca’+ and Mg’+
32.1 + 3.0 (26)* P < 0.00001’
Ca*+ “free”
29.3 + 2.9 (26) P < 0.00001
Mg’+ “free”
24.5 + 2.6 (26) P < 0.00001
Ca2+and Mg2’ “free”
6.7 c 4.8 (19) NS
fl Calculated as described under Materials and Methods. * Mean 2 SEM. Number of experiments in parentheses. c Comparisons of macrophage migration in lymphokine preparations versus control supematants are denoted by P values.
The requirement for specific extracellular divalent cations in this lymphokinemediated activation of alveolar macrophages was then examined. Macrophages were exposed to antigen-stimulated or control supernatants with or without the addition of Caz+and/or Mg2+. Lymphokine (antig en-stimulated supernatants) in the presence of physiological concentrations of Ca*+ and Mg*+, or with physiological Mg*+ alone (Ca*+ “ free”), stimulated macrophage killing of Listeria (Table 2). In contrast, exposure of cells to antigen-stimulated supernatants containing physiological Ca*+, but without Mg*+, produced no augmentation of macrophage bactericidal capability. Macrophages incubated in Ca2+- or Mg*+-“free” control supernatants exhibited bactericidal activity identical to that of control incubations with physiological concentrations of Ca*+ and Mg’+. In addition, the increased adherence of macrophages incubated in antigenstimulated supernatants was also dependent upon the presence of Mg*+. The adherence of macrophages incubated in antigen-stimulated supernatants with supernatants physiological Mg*+ was greater than that in Mgz+-“free” (184.3 & 32.3%, P < 0.05). In contrast, the adherence of macrophages incubated in antigen-stimulated supernatants containing physiological Ca*+ was not significantly different from that of cells in Ca*+-“free” supernatants. (b) Inhibition of macrophage migration. Incubation supernatants of LN lymphocytes from animals with Listeria LRT infections were used in assays of alveolar macrophage migration. Antigen-stimulated supernatants (lymphokine) caused marked inhibition of cell migration at 24 hr as compared to control supernatants. Control assays comparing migration in chambers containing MEM-15% NRS alone versus MEM-15% NRS plus Listeria sonicate antigen revealed no evidence of antigen toxicity. Migration of macrophages was inhibited by lymphokine preparations containing physiological concentrations of Ca*+ and Mg*+, physiological Mg*+ alone (Ca*+ “free”), or physiological Caz+ alone (Mg*+ “free”) (Table 3). In the absence of both Ca*+ and Mg*+ in the extracellular
242
JOHNSON, HAND, AND KING-THOMPSON TABLE 4
Divalent Cations and Fractionated Lymphokine (MIF)-Mediated Inhibition of Alveolar Macrophages Percentage inhibition of migration by MIF”
Experimental group Ca2+and Mg*+
22.6 2 2.3 (5)* P = 0.003’
Ca*+ “free”
5.8 f 3.7 (5) NS
Mg*+ “free”
14.4 2 5.8 (5) NS
Ca2+ and Mg*+ “free”
7.8 + 3.7 (5) NS
a Calculated as described under Materials and Methods. * Mean f SEM. Number of experiments in parentheses. c Comparisons of macrophage migration in fractionated lymphokine preparations (MIF) versus control supernatants are denoted by P values.
medium, inhibition of macrophage migration was erratic and poorly reproducible in antigen-stimulated supernatants. MIF-containing and control Sephadex fractions were also utilized in the macrophage migration assay. Divalent cation requirements for inhibition of migration (EDTA-washed macrophages) were similar to those observed in studies with unfractionated supernatants. Maximal MIF activity occurred only in the presence of both Mg2+ and Ca2+. Once again, no MIF activity was seen in the absence of both cations (Table 4). The possibility that the divalent cation dependence of MIF activity reflects the necessity of Ca2+and Mg2+for binding of the lymphokine to the macrophage surface was then examined. Antigen-stimulated supernatants preincubated with alveolar macrophages in the presence of both Ca2+ and Mg2+ lost their ability to subsequently inhibit macrophage migration, presumably because MIF was removed from solution by adsorption to cells. MIF activity was partially lost during preincubation in the presence of either Ca2+or Mg 2+. MIF activity was unaffected when both Ca2+ and Mg2+ were absent during preincubation (Fig. 1). DISCUSSION It is well known that CM1 produces marked changes in macrophages (1). These alterations in macrophage function are believed to be mediated largely by lymphokines, products of antigen-stimulated sensitized lymphocytes. The effects of lymphokines upon macrophages include inhibition of macrophage migration and initiation of the activation process (2). Despite earlier doubts (9) it is now clear that lymphokines will elicit activation and inhibition of migration in alveolar as well as systemic macrophages (3, 4). The means whereby lymphokines alter macrophage function is uncertain. However, features of the activation process in other cells suggested a potential mechanism for macrophage activation. Specifically, the initiating event in
MgZ+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE &j
Lymphokine
q
Preincubated
Co” Mg”
Co” no Mg*’
INTERACTIONS
243
Lymphokine
no Co*’ Mg”
FIG. 1. Inhibition of alveolar macrophage migration by lymphokine. MIF-containing (antigenstimulated) and control lymphocyte supematants, with or without Ca*+ and/or Mg’+ as indicated, were preincubated in the presence of alveolar macrophages (0). After removal of cells, the supematants were supplemented with divalent cations, as needed to achieve physiological concentrations of Ca2+and Mg*+, and used in assays of alveolar macrophage migration. These results are compared with simultaneous studies utilizing portions of the same supematants, which had not been preincubated with macrophages (a).
of certain cells was shown to be the influx or intracellular redistribution activation of divalent cations (IO- 17). As a result of these findings, we recently examined the effect on alveolar macrophages of A23187, an ionophore which selectively binds divalent cations and passively transports them across biological membranes (18, 19). That study showed that exposure to A23187 initiated activation in alveolar macrophages. This ionophore-mediated activation was dependent upon the presence of extracellular Mgz+ and was apparently associated with Mg2+ influx (5). With this background information, we have examined the role of divalent cations in lymphokine-mediated alterations of alveolar macrophage function. As in the ionophore activation model, the presence of extracellular Mg2+ is required for lymphokine-mediated activation. In contrast extracellular Ca2+is not essential for activation. Thus, Ca2+ influx, which has been described as the initiating event in activation of various cells (12, 14, 16, 17) is not the stimulus for lymphokine-induced macrophage activation. Although the need for Mg 2+ in activation of alveolar macrophages is clear, the means by which this divalent cation augments cellular function is uncertain. There are several possible mechanisms for Mg2+ initiation or support of lymphokineinduced stimulation of cellular processes. First, Mg2+ may be essential for binding ofthe macrophage activating factor(s) to the macrophage plasma membrane. In that case no interaction between lymphokine and macrophage would occur in the absence of Mg2+. Second, increases in cyclic GMP levels have been associated with proliferation or activation in fibroblasts and lymphocytes (20-26). Divalent cations, including Mg2+, are utilized in several steps of cyclic GMP synthesis and control (reviewed in Ref. (27)). Third, Mg2+ stimulation of fibroblast proliferation has been ascribed to the necessity of this cation for transphosphorylation reactions (15,28, 30). The concentration of intracellular free Mg2+ may serve as a limiting factor in cellular protein synthesis, and increasing the availability of Mg2+ may thus activate cellular metabolism. In regard to the potential interactions of Mg2+, cyclic GMP,
244
JOHNSON,
HAND, AND KING-THOMPSON
and transphosphorylation, it is of interest that cyclic GMP-dependent protein kinase is uniquely dependent upon high concentrations of Mg2+ for optimal function (27). We previously postulated (5) that at least A23 187-induced macrophage activation might be initiated by an exchange of two H+ for each Mg*+ transported across the plasma cell membrane (18, 19, 31). This H+ efflux could lead to an increase in intracellular cytoplasmic pH, which has been reported to initiate activation of sea urchin eggs (32). However, we found no change in intracellular pH of alveolar macrophages after exposure to A23187. Furthermore, the intracellular pH of normal alveolar macrophages is similar to that of alveolar macrophages from animals with Listeria LRT infection (R. W. Corwin, J. D. Johnson, and W. L. Hand, unpublished observations). These findings may reflect the ready permeability of the alveolar macrophage plasma cell membrane to H+, CO*, and HCO; (33). Divalent cation requirements for lymphokine (MIF)-mediated inhibition of alveolar macrophage migration are somewhat different from that of lymphokinemediated activation. Thus, the presence of both Ca*+ and Mg*+ is required for MIF activity. Other investigators have noted that divalent cations are needed for MIF activity in assays utilizing guinea pig peritoneal macrophages, but the specificity of the cation requirement (i.e., Ca’+, MgZ+, or both) was not examined (34, 35). Our adsorption experiments strongly suggest that both Ca*+ and Mg*+ are necessary for optimal binding of MIF to alveolar macrophages. Obviously, divalent cations might also play additional roles in the MIF-macrophage interaction. Lastly, the observation that the two lymphokine-mediated macrophage functions evaluated in the present study have different divalent cation requirements might suggest that these functions are controlled by separate lymphocyte mediators. This is of interest since MIF, which causes the inhibition of alveolar macrophage migration, is thought by some investigators to also produce macrophage activation (2,36,37). However, it must be remembered that migration and bactericidal activity are very different cellular functions and are not altered at identical times after lymphokine exposure. Additional study is needed to determine whether the lymphokine activities known as migration inhibitory factor and macrophage activating factor are actually distinct entities or a single factor with multiple effects upon macrophages. REFERENCES 1. Mackaness, G. B., Sem. Hematol. 7, 172, 1970. 2. David, J. R., and Remold, H. G., In “Immunobiology ofthe Macrophage” (D. S. Nelson, Ed.), pp. 401-426. Academic Press, New York, 1976. 3. Cantey, J. R., and Hand, W. L., .I. Clin. Invest. 54, 1125, 1974. 4. Johnson, J. D., Hand, W. L., King, N. L., and Hughes, C. G., J. Immunol. 115, 80, 1975. 5. Hand, W. L., King, N. L., Johnson, J. D., and Lowe, D. A., Nature (London) 265, 543, 1977. 6. Hand, W. I,., and Cantey, J. R., J. Clin. Invest. 53, 354, 1974. 7. Myrvik, Q. N., Leake, E. S., and Fariss, B., J. Immunol. 86, 128, 1961. 8. Brennan, J. K., Mansky, J., Roberts, G., and Lichtman, M. A., In Vitro 11, 354, 1975. 9. Mackaness, G. B., Ann. N.Y. Acad. Sci. 221, 312, 1974. 10. Alford, R. H., J. Zmmunol. 104, 698, 1970. 11. Steinhardt, R. A., Epel, D., and Carroll, E. J., Jr., Nature (London) 252, 41, 1974. 12. Maino, V. C., Green, N. M., and Crumpton, M. J., Nature (London) 251, 324, 1974. 13. Luckasen, J. R., White, J. G., and Kersey, J. H., Proc. Nat. Acad. Sci. USA 71, 5088, 1974. 14. Freedman, M. H., Raff, M. C., and Gomperts, B., Nature (London) 255, 378, 1975.
Mgz+ AND Caz+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
245
15. Rubin, H., Proc. Nat. Acad. Sci. USA 72, 3551, 1975. 16. Berridge, M. .I., J. Cyclic Nucleotide Res. 1, 305, 1975. 17. Jensen, P., Winger, L., Rasmussen, H., and Nowell, P., Biochim. Biophys. Acta 4%, 374, 1977. 18. Reed, P. W., and Lardy, H. A., In “The Role of Membranes in Metabolic Regulation” (M. A. Mehlman and R. W. Hanson, Eds.), pp. 111-131. Academic Press, New York, 1972. 19. Reed, P. W., and Lardy, H. A., .I. Biol. Chem. 247, 6970, 1972. 20. Hadden, J. W., Hadden, E. M., Haddox, M. K., and Goldberg, N. D., Proc. Nat. Acad. Sci. USA 69, 3024, 1972. 21. Leifert, W. E., and Rudland, P. S., Nature (London) 248, 138, 1974. 22. Weinstein, Y., Chambers, D. A., Boume, H. R., and Melmon, K. L., Nature (London) 2.51, 352, 1974.
23. Whitfield, J. E., McManus, J. P., Boynton, A. L., Gillan, D. J., and Isaacs, R. J. ,J. Cell. Physiol. 84, 445, 1975. 24. Diamantstein,.T., and Ulmer, A., Immunology 28, 113, 1975. 25. Watson, J., J. Exp. Med. 141, 97, 1975. 26. Weinstein, Y., Segal, S., and Melmon, K. L., J. Immunol. 115, 112, 1975. 27. Goldberg, N. D., and Haddox, M. K., Annu. Rev. Biochem. 46, 823, 1977. 28. Rubin, H., and Koide, T., Proc. Nat. Acad. Sci. USA 73, 168, 1976. 29. Kamine, J., and Rubin, H., Nature (London) 263, 143, 1976. 30. Sanui, H., and Rubin, H., J. Cell. Physiol. 92, 23, 1977. 31. Pfeiffer, D. R., and Lardy, H. A., Biochemistry 15, 935, 1976. 32. Johnson, J. D., Epel, D., and Paul, M., Nature (London) 262,661, 1976. 33. Laman, D., Theodore, J., and Robin, E. D., C/in. Res. 24, 386A (Abstr.), 1976. 34. Pick, E., and Manheimer, S., Cell. Immunol. 11, 30, 1974. 35. Block, L. H., Aloni, B., Biemesderfer, D., Kashgarian, M., and Bitensky, M. W., .I. Immunol. 121, 1416, 1978. 36. Nathan, C. F., Remold, H. G., and David, J. R., J. Exp. Med. 137, 275, 1973. 37. Fowles, R. E., Fajardo, I. M., Leibowitch, J. L., and David, J. R., J. Exp. Med. 138, 952, 1973.
IMMUNOLOGY
53, 236-245 (1980)
The Role of Divalent Cations in Interactions Lymphokines and Macrophages1g2 JAMES
between
D. JOHNSON, W. LEE HAND, AND NEVA L. KING-THOMPSON
Veterans Administration Medicine,
Medical Center (Atlanta), Decatur, Georgia 30033, and the Department Emory University School of Medicine, Atlanta, Georgia 30303
of
Received May 16, I979
The divalent cation requirements of lymphokine-mediated alterations in macrophage function (activation and inhibition of migration) were examined. Normal rabbit alveolar macrophages exposed to incubation supernatants of antigen-stimulated sensitized lymphocytes (lymphokine) were activated, manifested by increased adherence and enhanced bactericidal activity, as compared with control cells. This lymphokine-mediated activation was dependent upon the presence of extracellular Mg2+ (but not Ca2+). Our data from both current and previous studies suggest that Mg*+ influx is necessary for initiation or support of the macrophage activation process. The divalent cation requirements for lymphokine (MIF)induced inhibition of macrophage migration differed from that of the activation phenomenon. Specifically, both Ca2+and Mg2+ were required for expression of MIF activity. Adsorption experiments indicate that these cations are needed for binding of MIF to the macrophage surface.
INTRODUCTION Activation of macrophages, a process by which these cells develop an increased capacity to inactivate microorganisms, is responsible for acquired resistance to many intracellular organisms (1). Macrophage activation in viva is a consequence of the cell-mediated immune response. Biologically active products (lymphokines) released by sensitized lymphocytes after contact with specific antigen have a profound effect upon mononuclear phagocytes. Among those lymphokines with effects upon macrophages are factors which inhibit cell migration and stimulate macrophage activation (reviewed in Ref. (2)). It is this latter property, the activation of macrophages, whereby these cells become the antimicrobial effector of cell-mediated immunity (CMI).3 In previous studies we found that lymphokines exert their effects upon alveolar macrophages (3, 4). The molecular mechanisms of lymphokine-mediated altera* This work was supported by funds from the Medical Research Service of the Veterans Administration. * Requests for reprints should be sent to Dr. W. Lee Hand at the Veterans Administration Medical Center (Atlanta), 1670 Clairmont Road, Decatur, Ga. 30033. 3 Abbreviations used: CMI, cell-mediated immunity; LRT, lower respiratory tract; LN, lymph node; PBS, phosphate-buffered saline; TC199, tissue culture medium 199; NRS, pooled, normal heat-inactivated rabbit serum; MEM, modified Eagle’s medium; EDTA, ethylenediaminetetraacetic acid; MIF, migration inhibitory factor. 236 0008~8749/80/10023610$02.00/O Copyright 0 1980 by Academic Press, Inc. All rights of reproduction in any form reserved
Mg2+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
237
tions in macrophages are still uncertain. However, we recently demonstrated that activation of alveolar macrophages can be induced by exposure to the divalent cation ionophore A23187 (5). This ionophore-mediated activation required the presence of extracellular magnesium (Mg2+) and was presumably dependent upon the influx of Mg2+. The possibility that lymphokine-mediated alterations in macrophage function (including activation) are also dependent upon the presence of specific divalent cations is the basis of the present study. Specifically, we have examined the Mg2+ and Ca2+requirements of both the activation process and the inhibition of migration induced in alveolar macrophages by lymphokines. MATERIALS
AND METHODS
Bacteria A clinical isolate ofListeria monocytogenes was stored at -70°C and then grown overnight in trypticase soy broth (BBL, Division of Becton, Dickinson, and Co., Cockeysville, Md.) for the studies described below. Lower Respiratory Tract (LRT) Infection New Zealand male rabbits were exposed to aerosols of L. monocytogenes (T&R airborne infection apparatus, Tri-R Instruments, Rockville Center, N.Y .) as previously described in detail (6). Bacterial Antigen Overnight cultures of Listeria were washed, resuspended in phosphate-buffered saline (PBS), and disrupted in an ultrasonic apparatus (Lab-Line Instrument, Inc., Melrose Park, Ill.). Supernatants were cleared by centrifugation, dialyzed against PBS, sterilized by membrane filtration, and stored at -20°C (3). Collection of Alveolar Macrophages and Lymph Nodes (LN) Animals were anesthetized with sodium thiopental prior to exsanguination. Cells were collected from the LRT of normal animals by lavage with sterile saline (0.15 M) containing heparin (10 units/ml), penicillin (100 U/ml), and streptomycin (100 pg/ml) (Grand Island Biological Co., Grand Island, N.Y.) (3, 6, 7). Thoracic LN were aseptically removed from infected animals. Incubation of LN Lymphocytes for Lymphokine Production LN cells (lymphocytes) were obtained by teasing nodes from infected animals on stainless steel sieves (100 mesh) (W. S. Tyler, Inc., Mentor, Ohio). Cells were washed twice in tissue culture medium 199 (BBL) containing gentamicin, 25 pg/ml (TC199), but without serum, and then resuspended in the same medium at a concentration of 1 to 2 x lo7 viable cells/ml. Sets of culture tubes containing 2.5 ml of cell suspensions were set up with or without Listeria sonicate antigen (60 pg protein/ml) and placed in a CO2 incubator for 24 hr. After incubation, the supernatants were collected by centrifugation and antigen was added to the control supernatants (4).
238 Fractionation
JOHNSON,
of Migration
HAND,
Inhibitory
AND KING-THOMPSON
Factor
Some lymphocyte culture supernatants were then fractionated on Sephadex G-75 as previously described (3). Volumes (150 ml) of antigen-stimulated and control supernatants were dialyzed against PBS and concentrated to a volume of 10 ml. These concentrated supernatants were then fractionated on a calibrated 2 x 90-cm column of Sephadex G-75 (Pharmacia Fine Chemicals, Inc., Piscataway, N.J.) MIF-containing fractions of antigen-stimulated supernatants (corresponding to a molecular weight of approximately 65,000) and the corresponding control supernatant fractions were collected, concentrated, and used in the macrophage migration assay as described below. Macrophage
Monolayers
Cells from the LRT of normal animals were washed and suspended in TC199 containing 15%normal heat-inactivated rabbit serum (NRS), placed in 35-mm plastic tissue culture dishes (3 to 5 x lo6 cells per dish), and incubated for 4 hr at 37°C in 95% air-5% CO, (4). Assay of Macrophage
Function
Lymphocyte culture supernatants were dialyzed against saline and then against Ca2+-and Mg2+-“free” modified Eagle’s medium (MEM) at 4°C over a 72-hr period. NRS, depleted of divalent cations by exposure to a chelating resin (Chelex 100, Bio-Rad, Richmond, Calif.), was added to these supernatants in a concentration of 15%. Ca2+ (1.8 x 10m3M) and/or Mg2+ (0.8 x lop3 M) were added to these supernatants as indicated below. Divalent cation levels in these preparations were measured with an atomic absorption spectrophotometer (Perkin-Elmer). Ca2+ and Mg2+ concentrations in the divalent cation-“free” salt solutions, media, and supernatants were approximately 1 x 1O-5M. (a) Macropkage adherence and bactericidal activity. After removal of nonadherent cells, monolayers of normal alveolar macrophages were covered with antigenstimulated or control supernatants (with or without Ca2+and Mg2+) and incubated for 72 hr prior to studies of macrophage function. At the conclusion of this incubation period, determinations of adherence to tissue culture dishes and of bactericidal activity were performed as previously described (4). Adherence to culture dishes (cell count) of macrophages incubated in antigen-stimulated supernatants was compared with the corresponding cell count in dishes containing control supernatants and expressed as a percentage: % cell adherence = No. of macrophages on dishes with stimulated supernatants x IO0 No. of macrophages on dishes with control supernatants * Survival of L. monocytogenes phagocytized (during a 30-min period) by macrophage monolayers was determined by comparing the number of viable intracellular bacteria remaining after an additional 2-hr incubation with that
Mg*+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
239
immediately.after the phagocytic period (0 time): % intracellular bacterial survival (2 hr) viable intracellular bacteria at 2 hr x 100. viable intracellular bacteria at 0 time (b) Inhibition of macrophage migration. Whole or fractionated stimulated and control supernatants (in MEM-15% NRS with or without Ca2+ and Mg2+) were added to Sykes-Moore chambers containing capillary tubes filled with normal alveolar macrophages (3). The chambers were incubated at 37°C in 95% air-5% COz. Migration of cells from the capillaries was measured at 24 hr and expressed as percentage inhibition of macrophage migration: % inhibition of migration = 100 -
area of migration in antigen-stimulated supernatant x 100 . area of migration in control supernatant ( 1
In some experiments, the macrophages were washed with 1 mM EDTA, to remove divalent cations bound to cell surface, prior to the migration assay. Macrophage viability, migratory capacity, and response to lymphokine were unaffected by this exposure to EDTA. The role of divalent cations in the binding of MIF to the surface of alveolar macrophages was examined in the following manner. Macrophages were incubated at 4°C for 2 hr in MIF-containing (antigen-stimulated) or control supernatants with or without Ca2+and/or Mg2+, at a concentration of 5 X lo6 cells/ml of supernatant. The cells were then removed by centrifugation, and the supernatants were supplemented with Ca2+ and/or Mg2+, if needed, to achieve physiologic concentrations of each cation. Supernatants were then utilized in studies of alveolar macrophage migration. Statistical Analysis Differences between experimental groups were evaluated with Student’s t test. RESULTS Characterization of LRT Cells This cell population has been previously described in detail (3). Cells (40 to 50 x 106) were obtained by LRT lavage of normal animals. Numbers of cells obtained from animals with L. monocytogenes infection of the LRT were increased two to six times above normal. A mean of 85-90% of LRT cells were macrophages. Viability of these cells, as determined by trypan blue exclusion, was routinely ~95%. Assays of Macrophage Function (a) Macrophage adherence and bactericidal activity. LN lymphocytes, obtained from animals 12 to 14 days after initiation of L. monocytogenes respiratory tract infection, were incubated in the presence or absence of Listeria antigen. Supernatants from antigen-stimulated or control lymphocyte incubations were then
240
JOHNSON, HAND, AND KING-THOMPSON TABLE 1 Function of Alveolar Macrophages after Exposure to Supernatants from Antigen-Stimulated or Control Lymphocyte Incubations Experimental group
Percentage intracellular bacterial survival (2 hr)”
Percentage cell adherence”
Control Antigen stimulated
100
54.0 k 6.1” (13)
191.9 k 31.6’
28.0 ? 3.4” (13)
(20) ” ’ ” ”
Calculated as described under Materials and Methods. Mean + SEM. Number of experiments in parentheses. Significantly different from control (P < 0.01). Significantly different from control (P < 0.005).
placed on monolayers of normal alveolar macrophages. Cells exposed to antigen-stimulated supernatants for 72 hr showed evidence of activation. That is, they exhibited increases in adherence to tissue culture dishes and bactericidal activity against L. monocytogenes as compared with control cells (Table 1). Furthermore, these activated cells manifested enhanced agglutinability or clumping (data not shown). TABLE 2 Bactericidal Activity of Alveolar Macrophages after Exposure to Lymphocyte Incubation Supernatants Divalent cations (M)
Percentage intracellular bacterial survival (2 hr)”
Experimental group
Mg’+
Ca’+
Control Ca’+ and M g2+
0.8 x 10m3
1.8 x IO-”
54.0 t 6.1” (13)
ca2+ 6‘free’ 1
0.8 x lo-”
1 x 10-S
55.8 t 9.5 (5)
Mg’+ “free”
1 x 10-s
1.8 x 10m3
61.0 k 8.4 (7)
0.8 x lO-3
I.8 x lO-3
28.0 k 3.4’ (13)
Ca’+ “free”
0.8 x lO-3
1
20.2 2 5.4” (5)
Mg’+ “free”
1
1.8 x 10m3
Antigen stimulated CaZf and Mg’+
x 10-s
x 10-b
67.5 + 12.8
(8) a Calculated as described under Materials and Methods. b Mean + SEM. Number of experiments in parentheses c Significantly different from control (P < 0.005). d Significantly different from control (P < 0.001).
Mg2+ AND Caz+ IN LYMPHOKINE-MACROPHAGE
INTERACTIONS
241
TABLE 3 Divalent Cations and Lymphokine-Mediated Inhibition of Alveolar Macrophage Migration
Experimental group
Percentage inhibition of migration by lymphokine”
Ca’+ and Mg’+
32.1 + 3.0 (26)* P < 0.00001’
Ca*+ “free”
29.3 + 2.9 (26) P < 0.00001
Mg’+ “free”
24.5 + 2.6 (26) P < 0.00001
Ca2+and Mg2’ “free”
6.7 c 4.8 (19) NS
fl Calculated as described under Materials and Methods. * Mean 2 SEM. Number of experiments in parentheses. c Comparisons of macrophage migration in lymphokine preparations versus control supematants are denoted by P values.
The requirement for specific extracellular divalent cations in this lymphokinemediated activation of alveolar macrophages was then examined. Macrophages were exposed to antigen-stimulated or control supernatants with or without the addition of Caz+and/or Mg2+. Lymphokine (antig en-stimulated supernatants) in the presence of physiological concentrations of Ca*+ and Mg*+, or with physiological Mg*+ alone (Ca*+ “ free”), stimulated macrophage killing of Listeria (Table 2). In contrast, exposure of cells to antigen-stimulated supernatants containing physiological Ca*+, but without Mg*+, produced no augmentation of macrophage bactericidal capability. Macrophages incubated in Ca2+- or Mg*+-“free” control supernatants exhibited bactericidal activity identical to that of control incubations with physiological concentrations of Ca*+ and Mg’+. In addition, the increased adherence of macrophages incubated in antigenstimulated supernatants was also dependent upon the presence of Mg*+. The adherence of macrophages incubated in antigen-stimulated supernatants with supernatants physiological Mg*+ was greater than that in Mgz+-“free” (184.3 & 32.3%, P < 0.05). In contrast, the adherence of macrophages incubated in antigen-stimulated supernatants containing physiological Ca*+ was not significantly different from that of cells in Ca*+-“free” supernatants. (b) Inhibition of macrophage migration. Incubation supernatants of LN lymphocytes from animals with Listeria LRT infections were used in assays of alveolar macrophage migration. Antigen-stimulated supernatants (lymphokine) caused marked inhibition of cell migration at 24 hr as compared to control supernatants. Control assays comparing migration in chambers containing MEM-15% NRS alone versus MEM-15% NRS plus Listeria sonicate antigen revealed no evidence of antigen toxicity. Migration of macrophages was inhibited by lymphokine preparations containing physiological concentrations of Ca*+ and Mg*+, physiological Mg*+ alone (Ca*+ “free”), or physiological Caz+ alone (Mg*+ “free”) (Table 3). In the absence of both Ca*+ and Mg*+ in the extracellular
242
JOHNSON, HAND, AND KING-THOMPSON TABLE 4
Divalent Cations and Fractionated Lymphokine (MIF)-Mediated Inhibition of Alveolar Macrophages Percentage inhibition of migration by MIF”
Experimental group Ca2+and Mg*+
22.6 2 2.3 (5)* P = 0.003’
Ca*+ “free”
5.8 f 3.7 (5) NS
Mg*+ “free”
14.4 2 5.8 (5) NS
Ca2+ and Mg*+ “free”
7.8 + 3.7 (5) NS
a Calculated as described under Materials and Methods. * Mean f SEM. Number of experiments in parentheses. c Comparisons of macrophage migration in fractionated lymphokine preparations (MIF) versus control supernatants are denoted by P values.
medium, inhibition of macrophage migration was erratic and poorly reproducible in antigen-stimulated supernatants. MIF-containing and control Sephadex fractions were also utilized in the macrophage migration assay. Divalent cation requirements for inhibition of migration (EDTA-washed macrophages) were similar to those observed in studies with unfractionated supernatants. Maximal MIF activity occurred only in the presence of both Mg2+ and Ca2+. Once again, no MIF activity was seen in the absence of both cations (Table 4). The possibility that the divalent cation dependence of MIF activity reflects the necessity of Ca2+and Mg2+for binding of the lymphokine to the macrophage surface was then examined. Antigen-stimulated supernatants preincubated with alveolar macrophages in the presence of both Ca2+ and Mg2+ lost their ability to subsequently inhibit macrophage migration, presumably because MIF was removed from solution by adsorption to cells. MIF activity was partially lost during preincubation in the presence of either Ca2+or Mg 2+. MIF activity was unaffected when both Ca2+ and Mg2+ were absent during preincubation (Fig. 1). DISCUSSION It is well known that CM1 produces marked changes in macrophages (1). These alterations in macrophage function are believed to be mediated largely by lymphokines, products of antigen-stimulated sensitized lymphocytes. The effects of lymphokines upon macrophages include inhibition of macrophage migration and initiation of the activation process (2). Despite earlier doubts (9) it is now clear that lymphokines will elicit activation and inhibition of migration in alveolar as well as systemic macrophages (3, 4). The means whereby lymphokines alter macrophage function is uncertain. However, features of the activation process in other cells suggested a potential mechanism for macrophage activation. Specifically, the initiating event in
MgZ+ AND Ca*+ IN LYMPHOKINE-MACROPHAGE &j
Lymphokine
q
Preincubated
Co” Mg”
Co” no Mg*’
INTERACTIONS
243
Lymphokine
no Co*’ Mg”
FIG. 1. Inhibition of alveolar macrophage migration by lymphokine. MIF-containing (antigenstimulated) and control lymphocyte supematants, with or without Ca*+ and/or Mg’+ as indicated, were preincubated in the presence of alveolar macrophages (0). After removal of cells, the supematants were supplemented with divalent cations, as needed to achieve physiological concentrations of Ca2+and Mg*+, and used in assays of alveolar macrophage migration. These results are compared with simultaneous studies utilizing portions of the same supematants, which had not been preincubated with macrophages (a).
of certain cells was shown to be the influx or intracellular redistribution activation of divalent cations (IO- 17). As a result of these findings, we recently examined the effect on alveolar macrophages of A23187, an ionophore which selectively binds divalent cations and passively transports them across biological membranes (18, 19). That study showed that exposure to A23187 initiated activation in alveolar macrophages. This ionophore-mediated activation was dependent upon the presence of extracellular Mgz+ and was apparently associated with Mg2+ influx (5). With this background information, we have examined the role of divalent cations in lymphokine-mediated alterations of alveolar macrophage function. As in the ionophore activation model, the presence of extracellular Mg2+ is required for lymphokine-mediated activation. In contrast extracellular Ca2+is not essential for activation. Thus, Ca2+ influx, which has been described as the initiating event in activation of various cells (12, 14, 16, 17) is not the stimulus for lymphokine-induced macrophage activation. Although the need for Mg 2+ in activation of alveolar macrophages is clear, the means by which this divalent cation augments cellular function is uncertain. There are several possible mechanisms for Mg2+ initiation or support of lymphokineinduced stimulation of cellular processes. First, Mg2+ may be essential for binding ofthe macrophage activating factor(s) to the macrophage plasma membrane. In that case no interaction between lymphokine and macrophage would occur in the absence of Mg2+. Second, increases in cyclic GMP levels have been associated with proliferation or activation in fibroblasts and lymphocytes (20-26). Divalent cations, including Mg2+, are utilized in several steps of cyclic GMP synthesis and control (reviewed in Ref. (27)). Third, Mg2+ stimulation of fibroblast proliferation has been ascribed to the necessity of this cation for transphosphorylation reactions (15,28, 30). The concentration of intracellular free Mg2+ may serve as a limiting factor in cellular protein synthesis, and increasing the availability of Mg2+ may thus activate cellular metabolism. In regard to the potential interactions of Mg2+, cyclic GMP,
244
JOHNSON,
HAND, AND KING-THOMPSON
and transphosphorylation, it is of interest that cyclic GMP-dependent protein kinase is uniquely dependent upon high concentrations of Mg2+ for optimal function (27). We previously postulated (5) that at least A23 187-induced macrophage activation might be initiated by an exchange of two H+ for each Mg*+ transported across the plasma cell membrane (18, 19, 31). This H+ efflux could lead to an increase in intracellular cytoplasmic pH, which has been reported to initiate activation of sea urchin eggs (32). However, we found no change in intracellular pH of alveolar macrophages after exposure to A23187. Furthermore, the intracellular pH of normal alveolar macrophages is similar to that of alveolar macrophages from animals with Listeria LRT infection (R. W. Corwin, J. D. Johnson, and W. L. Hand, unpublished observations). These findings may reflect the ready permeability of the alveolar macrophage plasma cell membrane to H+, CO*, and HCO; (33). Divalent cation requirements for lymphokine (MIF)-mediated inhibition of alveolar macrophage migration are somewhat different from that of lymphokinemediated activation. Thus, the presence of both Ca*+ and Mg*+ is required for MIF activity. Other investigators have noted that divalent cations are needed for MIF activity in assays utilizing guinea pig peritoneal macrophages, but the specificity of the cation requirement (i.e., Ca’+, MgZ+, or both) was not examined (34, 35). Our adsorption experiments strongly suggest that both Ca*+ and Mg*+ are necessary for optimal binding of MIF to alveolar macrophages. Obviously, divalent cations might also play additional roles in the MIF-macrophage interaction. Lastly, the observation that the two lymphokine-mediated macrophage functions evaluated in the present study have different divalent cation requirements might suggest that these functions are controlled by separate lymphocyte mediators. This is of interest since MIF, which causes the inhibition of alveolar macrophage migration, is thought by some investigators to also produce macrophage activation (2,36,37). However, it must be remembered that migration and bactericidal activity are very different cellular functions and are not altered at identical times after lymphokine exposure. Additional study is needed to determine whether the lymphokine activities known as migration inhibitory factor and macrophage activating factor are actually distinct entities or a single factor with multiple effects upon macrophages. REFERENCES 1. Mackaness, G. B., Sem. Hematol. 7, 172, 1970. 2. David, J. R., and Remold, H. G., In “Immunobiology ofthe Macrophage” (D. S. Nelson, Ed.), pp. 401-426. Academic Press, New York, 1976. 3. Cantey, J. R., and Hand, W. L., .I. Clin. Invest. 54, 1125, 1974. 4. Johnson, J. D., Hand, W. L., King, N. L., and Hughes, C. G., J. Immunol. 115, 80, 1975. 5. Hand, W. L., King, N. L., Johnson, J. D., and Lowe, D. A., Nature (London) 265, 543, 1977. 6. Hand, W. I,., and Cantey, J. R., J. Clin. Invest. 53, 354, 1974. 7. Myrvik, Q. N., Leake, E. S., and Fariss, B., J. Immunol. 86, 128, 1961. 8. Brennan, J. K., Mansky, J., Roberts, G., and Lichtman, M. A., In Vitro 11, 354, 1975. 9. Mackaness, G. B., Ann. N.Y. Acad. Sci. 221, 312, 1974. 10. Alford, R. H., J. Zmmunol. 104, 698, 1970. 11. Steinhardt, R. A., Epel, D., and Carroll, E. J., Jr., Nature (London) 252, 41, 1974. 12. Maino, V. C., Green, N. M., and Crumpton, M. J., Nature (London) 251, 324, 1974. 13. Luckasen, J. R., White, J. G., and Kersey, J. H., Proc. Nat. Acad. Sci. USA 71, 5088, 1974. 14. Freedman, M. H., Raff, M. C., and Gomperts, B., Nature (London) 255, 378, 1975.
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