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Alteration of macrophage surface in the course of immunological activation: Decay of metabolic response to Concanavalin A
CELLULAR
IMMUNOLOGY
13, 313-321 (1974)
Alteration of Macrophage Surface in the Course of Immunological Activation: Decay of Metabolic Response to Concanavalin A1 D. ROMEO,G.ZABUCCHI,
M. JUG,AND F. ROSSI
Istituto di Chimica Biologica and Istituto di Patologia Generale, Uwiversitd cIi Trieste, 34127 Trieste, Italy Received January 2, 1974 “Activated” macrophages, isolated from lungs of BCG-infected rabbits, exhibited a greater perturbability of oxidative metabolism than their normal counterpart, when exposed iti vitro to unrelated microorganisms or treated with phospholipase C. They were much less metabolically responsive when incubated with Concanavalin A. The metabolic effect of this lectin gradually decayed as time elapsed from the day of infection. These results are interpreted to show a modification of the surface properties of the macrophage in the course of the immunological activation. The possible mechanisms of generation of these surface alterations are discussed.
INTRODUCTION It is well known that macrophages respond to certain aggressions with an increase of their defensive capacities (1, 2). The process of transformation of macrophages into “activated” phagocytes has an immunological basis and involves sensitized lymphocytes through production of chemical mediators (3). In the course of activation the macrophages undergo a variety of morphological and biochemical alterations, such as overdevelopment of Golgi apparatus, increase in number of mitochondria and lysosomes, rise in activity of various lysosomal hydrolases, enhancement of capacity to ingest and kill bacteria and in tendency to spread on glass (l-4). The functional hypertrophy of activated macrophages is accompanied by a greater metabolic perturbability. In fact, they respond to a challenge by phagocytosible matter with a more pronounced increase in glucose oxidation (5-7). The stimulation of oxidative metabolism in macrophages exposed to bacteria is considered to be triggered by biochemical events occurring at the surface of the cell (8, 9). Thus, several modifications of the properties of the macrophage in the process of activation appear to concern functions of the cell periphery. This would suggest that a change in the molecular organization and/or composition has occurred on the surface membrane of the activated macrophages. To test this hypothesis we have analyzed the effect exerted by cell surface perturbing agents on the oxidative metabolism of activated macrophages as compared to their normal counterparts. The agents used were Concanavalin A (Con A) and phospholipase C, which have been shown to mimic the phagocytosis associated 1 This investigation was supported by Research Grant 72.00871.04 from the Italian Consiglio Nazionale delle Ricerche. 313 Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserwd.
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stimulation of glucose oxidation through the hexose monophosphate pathway (HMP) both in macrophages and polymorphonuclear Ieucocytes (10-12). MATERIALS
AND
METHODS
Cells Outbred rabbits weighing about 2.5 kg were infected with Bacihus CaImetteG&in (BCG) kindly supplied by Istituto Vaccinogeno Antitubercolare as a lyophilized powder. Ten milligrams of viable microorganisms, suspended in 1 ml of sterile saline containing 2% (v/v) t ween SO, were injected in the marginal ear vein on two subsequent days. Three weeks later the animals were killed by gaseous embolus and macrophages were removed from the lungs by means of tracheobronchial lavages (13). Cells were centrifuged at 400g for 10 min, treated with 0.2% NaCl for 30-60 set to lyse contaminating red cells, and finally suspended in calcium-free Krebs-Ringer phosphate buffer, pH 7.4 (KRP). These operations were performed with chilled solutions. Differential counts were carried out on MayGriinwald-Giemsa stained smears. Marcophages in cell preparations from BCG infected animals (1.2-2.4 X log cells/rabbit) were SS-91%,, whereas in controls (5-8 X 10’ cells/rabbit) were 9.2-98s. Bacteria and Surface Perturbing
Agents
B. Mycoides were grown on nutrient agar, heat-killed and opsonized in fresh serum derived from control rabbits. Concanavalin A (grade III, Sigma, U.S.A.) was dissolved in distilled water; fluorescein-labelled Con A was prepared according to Smith and Hollers (14) and added to macrophages under the same conditions used for the metabolic studies. For treatment with phospholipase C (CZ. Welchii, E.C. 3. 1. 4.3., Sigma, U.S.A.) cells were resuspended in a medium similar to KRP which contained Tris-HCI instead of phosphate buffer and was supplied with I mM CaCIs (KR-Tris). Glucose Oxidation The oxidation of glucose was determined by incubating the cells at 37°C in 2.5 ml KRP or KR-Tris with 0.2 mM glucose and 0.8 pCi l-14C-glucose or 2 &i 6J4C-glucose. In the case of treatment with Con A, the lectin was usually added 10 min before the addition of the labelled glucose. The 14C02 produced in 10 min of incubation was collected and counted as described elsewhere (8). The HMP activity was calculated as difference between the 14COs generated from l-14Cglucose and that from 6-14C-glucose. Enzyme Assays Acid phosphatase and P-glucuronidase were assayed essentially according to Berthet and de Duve (15) and Gianetto and de Duve (16)) respectively. The activity of peroxidase was determined as described by Romeo et al. (17). RESULTS Reversible Metabolic Stimulatiolz
of Macrophages by Con A
Figure 1 shows that the rate of oxidation of l-14C-glucose in normal lung macrophages is enhanced in the presence of a small amount of Con A in the
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FIG. 1. Stimulation of the “COa production from l-T-glucose in alveolar macrophages by Con A and its reversibility by a-D-methyl mannopyranoside (a-MM) ; 1 X 10’ macrophages were incubated at 37°C in 2.5 ml KRP, containing l-14C-glucose (0.8 &i, 0.5 pmoles) in the absence or in the presence of 37.5 pg of Con A. At the points indicated by arrows (U-MM was added at a final concentration of 50 mM.
medium. If, after the onset of stimulation, ol-methyl-o-mannopyranoside (a-MM), which is known to have a high affinity for Con A (IS), is added to the incubation medium, the rate of glucose oxidation rapidly resumes its resting value. At the concentration used, the lectin induces the formation of small aggregates of macrophages, consisting of 2-4 and, only occasionally, more than 5 cells. The percentage of cells which incur agglutination varies with different cell preparations and does not apparently bear any relation to the extent of the metabolic stimulation. By electron microscopy no formation of pinocytic vesicles is seen in macrophages when treated with Con A under our conditions. This observation appears to rule out the possibility that endocytosed lectin molecules are responsible for the enhancement of glucose oxidation. Perturbability and Con A
of O&dative
Metabolism
of Activated
Macrophages
by Bacteria
Table 1 reports the rate of oxidation of l-14C-glucose and 6-14C-glucose by activated and control macrophages. As already described by others (6, 7), the activated macrophages show a greater metabolic perturbability than control cells when exposed in vitro to bacteria. On the contrary, a treatment with Con A produces almost no change in the oxidation of 6J’C-glucose and enhancement of 14C0, production from 1-14C-glucose, which is smaller than that obtained in control cells. As illustrated in Fig. 2, the increment of HMP activity measured at different Con A concentrations is consistently smaller for activated macrophages than for their normal counterparts. By using fluorescein-tagged Con A a similar pattern of surface distribution of fluorescence is observed for the two types of cells. If a challenge by bacteria is performed on macrophage suspensions in the presence of Con A (12 s/ml), the stimulation of HMP activity is greater than that obtained with Con A alone, but lower than that recorded in the absence of lectin. Thus, the presence of Con A produces an inhibition of the phagocytosis-associated increment
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8.
Con AIp/mll
FIG. 2. Effect of different concentrations of Con A on the activity of the hexose monophospbate pathway (HMP) of normal (0-O) and activated ( O-O ) alveolar macrophages. Activated cells were isolated from rabbits infected with BCG 21 days before. The oxidation of glucose was measured essentially as described in Fig. 1, with 0.8 pCi of l-l”C-glucose or 2 pCi of 6-“C-glucose; HMP activity = [YOz from l-YZ-Glc] - [14COz from 6-I’C-Glc] ; HMP increment = HMP of treated cells-HMP of resting cells (cpm X lO”/rCi of l’C-glucose/l X 10’ cells/l0 min) .
of HMP activity, which averages 25% for the normal macrophages and 42% for the activated cells. This inhibition is probably related to the decreased availability of free cell surface to bacteria due to formation of small clusters of cells. The agglutinability of activated macrophages, however, does not significantly differ from that of controls. Under our conditions, Con A exerts no cytopathic effect on both types of macrophages. Modification of Metabolic Response of Macrophages to a Con A Treatment in the Course of Activation For this series of experiments, macrophages were isolated from rabbits at different times after the infection with BCG. Since there is a good correlation between activation and level of lysosomal enzymes (19, 20), the state of activation of macrophages was monitored by assaying the activity of /I-glucuronidase and acid phosphatase, which peaks in the third week after infection (9). Another enzyme assayed was peroxidase, whose presence in macrophages has been recently described by us (17). The activity of this enzyme progressively decreases from the day of infection, being almost nil at Day 33. The reason for this behavior of peroxidase is obscure ; it might be explained either by a gradual enzyme inactivation or by a specific extracellular release of peroxidase at the site of the inflammatory lesion. The average increment of HMP activity obtained with Con A reacting with macrophages at different stages of activation is shown in Fig. 3. A gradual loss of metabolic perturbability of macrophages is observed as time elapses from the day of infection with BCG. This loss continues also when macrophages derive from a lesion entering the healing stage.
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7 Days
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3. Effect of Con A on the activity of the hexose monophosphate pathway (HMP) of FIG. alveolar macrophages isolated from rabbits at different times from infection with BCG. Each point represents the mean of 4 experiments; the average number of alveolar macrophages per rabbit at Days 0 (control), 7, 14, 21 and 33 were 0.6 X lo’, 7.6 X IO*, 14.8 X 108 and 10.1 X IO’, respectively. The definition of HMP increment is given in Fig. 2.
Metabolic Response of Activated Macrophage to a Treatment with Phospholipase C The split of plasma membrane phospholipids with phospholipase C causes a stimulation of oxidative metabolism, similar to that concomitant with phagocytosis, both in polymorphonuclear leucocytes (11) and macrophages (12). As shown in Fig. 4, a greater stimulation of oxidation of l-14C-glucose by phospholipase C is observed with activated macrophages than with normal cells. The oxidation of 6-14C-glucose is unaffected by the treatment of both types of macrophages with the phospholipid splitting enzyme.
d 10 6 %Gluc LA-
J-a .6
na .2 Units
of
.4 Phospholipase
C
FIG. 4. Effect of phospholipase C on glucose oxidation by normal and activated alveolar macrophages. Activated macrophages derived from rabbits infected with BCG 21 days before. Glucose oxidation was measured as described in Figs. 1 and 2.
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DISCUSSION Macrophages respond to a challenge by bacteria with a rapid enhancement in the rate of respiration, of glucose oxidation through the HMF’, and of Hz02 production (9). The oxidizing product(s) of this metabolic burst are thought to be utilized for bacterial killing (17, 21). Activated macrophages, which kill ingested organism with enhanced efficiency (3)) display a more marked stimulation of their oxidative metabolism than control cells, when exposed to particulate matter. This enhanced perturbability might be ascribed either to a greater activity of oxidative enzyme systems, or to a higher phagocytic rate and capacity, or to a modification of properties of the cell surface, where the triggering mechanism of the metabolic stimulation is very likely generated (8-10). The relevance of the first point cannot be established at the present time, since comparative data concerning the enzymes participating for example in the activation of HMP are not available. Preliminary results obtained in our laboratory suggest, however, that activated macrophages do not possess an enhanced capacity to oxidize NADPH. The rate of oxidation of this nucleotide is considered to be the key factor in the metabolic stimulation of mononuclear and polymorphonuclear phagocytes (8, 9). As for the second point, since also a treatment with phospholipase C induce.; in activated macrophages a stimulation of HMP greater than that produced in their normal counterparts (Fig. 4), the more elevated excitability of the former cells seems not to be linked to an increased phagocytosis. Thus, the more likely explanation of the enhanced metabolic perturbability of the activated macrophage would reside in a modification of the triggering mechanism. This mechanism is assumed to operate at the level of the cell surface (8, 9), and might therefore “feel” a change in the composition or molecular organization of cell membrane. A modification of surface properties of activated macrophages is suggested by their more rapid and extensive spreading on glass (4), by the change in their electrophoretic mobility (22) and by the enhancement of turnover of surface membrane-associated macromolecules (23). A gradual change in the surface properties of macrophages in the process of activation is also consistent with the loss of metabohc response to a treatment with Con A. Apparently, the reduced effect of the lectin is neither due to a failure of binding to activated macrophages, as assessed by the use of fluorescein-labelled Con A, nor to a change in the extent of cell agglutination. The decay of the response of the activated macrophages to Con A might be ascribed to one or more of the following events. (A) Decrease in number or availability of Con A receptors to an extent not detectable by simply using fluorescein-tagged Con A. A drop in the number of receptors might derive either from their shedding into cell environment or from their digestion by hydrolytic enzymes, released from the macrophage itself or from neutrophils present in the pulmonary granuloma. A reduced availability of receptors might be due to insertion of new macromolecules such as cytophilic antibodies (3) in the surface membrane. The possibility exists that molecules competing with normal receptors for Con A might appear on the surface of activated macrophages. It is tempting to speculate that one such molecule might be trehalosed$‘dimycolate (“cord factor”), a complex glycolipid of the tubercle bacilli (24)) which induces pulmonary granulomas and increased resistance to challenge with the
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H37Rv strain of Mycobacterium tubercolosis (25). This hypothesis obviously implies an exchange of lipid between BCG and the host cell membrane. (B) Reduced surface mobility of Con A-receptors. We have advanced the hypothesis (10) that the Con A-induced metabolic stimulation is caused by microaggregation of glycoprotein subunits accompanied by changes of surface properties, such as potential or ion permeability. Trasher et al. (26) have demonstrated that the surface components of macrophages are indeed in a dynamic state on the membrane and form configurations similar to those observed on lymphocytes. A modification of membrane fluidity in the activated macrophages, resulting from a change in the intrinsic properties of the plasma membrane, might be invoked as a perturbing factor in the redistribution of receptor molecules by the lectin. In this connection, it is worthwhile mentioning that the different agglutinability of virus-transformed cells has been shown by Nicholson (27) to be due to a change in fluidity of the surface membrane. (C) Impairment of the mechanism which links Con A-receptor interactions to generation and transmission of the stimulatory signal. This assumption appears to be the least credible since the phagocytosis- and Con A-associated metabolic stimulations share very likely the same mechanism. For the time being we are not in the position to decide among the above alternatives. In fact, a detailed knowledge of the microstructure of the membrane of the activated macrophages as compared to the normal cell is needed to gain a clearer picture of the mechanism of immunological activation of macrophages. An experimental approach of this type is presently in progress in our laboratory. ACKNOWLEDGMENTS The skillful assistance of Mr. G. Orpelli is gratefully acknowledged.
REFERENCES 1. Lurie, M. B., “Resistance to Tubercolosis: Experimental Studies in Native and Acquired Defensive Mechanisms,” Harvard University Press, Cambridge, 1964. 2. Suter, E., and Ramseier, H., Advan. Immunol. 4, 117, 1964. 3. Mackaness, G. B., 1% “Mononuclear Phagocytes” (R. van Furth Ed.), pp. 461-477. Blackwell Scientific Publications, Oxford, 1970. 4. Blanden, R. V., RES: J. Reticuloendothel. Sot. 5, 179, 1968. 5. Evans, E. D., and Myrvik, Q. M., Ann. N.Y. Acad. Sci. 154, 167, 1969. 6. Ratzan, K. R., Musher, D. M., Kensch, G. T., and Weinstein, L., Infect. Immulz. 5, 499, 1972. 7. Stubbs, M., Kuhner, V. A., Glass, E. A., David, J. K., and Karnovsky, M. L., J. Ex@. Med. 137, 537, 1973. 8. Romeo, D., Zabucchi, G., Marzi, T., and Rossi, F., Exp. Cell Res. 78, 423, 1973. 9. Rossi, F., Zabucchi, G., and Romeo, D., In “Mononuclear Phagocytes in Immunity, Infection and Pathology” (R. van Furth, Ed.). Blackwell Scientific Publications, Oxford, England, in press. 10. Romeo, D., Zabucchi, G., and Rossi, F., Nature New Biol. 243, 111, 1973. 11. Patriarca, P., Cramer, R., Marussi, M., Moncalvo, S., and Rossi, F., RES: J. Reticuloendothel. Sot. 10, 251, 1971. 12. Sachs, F. L., and Gee, J. B. C., RES: J. Reticzlloetiothel. Sot. 14, 52, 1973. 13. Myrvik, Q. M., Leake, E. S., and Fariss, B., J. Immunol. 86, 128, 1961. 14. Smith, C. W., and Hollers, J. C., RES: J. Reticuloendothel. Sot. 8, 458, 1970. 15. Berthet, J., andi de Duve, C., Biochem. J. 50, 174, 1951. 16. Gianetto, R., and de Duve, C., Biochem. J. 59, 433, 1955.
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17. Romeo, D., Cramer, R., Marzi, T., Soranzo, M. R., Zabucchi, G., and Rossi, F., RES: J. Reticuloendothel. Sot. 13, 199, 1973. 18. So, L. L., and Goldstein, I. J., Biochim. Biojhys. Acta 165, 398, 1968. 19. Cohn, A. Z., and Wiener, E., J. Exp. Med. 118, 991, 1963. 20. Saito, K., and Suter, E., J. Exp. Med. 121, 727, 1965. 21. Paul, B. B., Strauss, R. R., Selvaraj, R. J., and Sbarra, A. J., Science 181, 849, 1973. 22. Diengdoh, J. V., and Turk, J. L., Znt. Avck Allergy 34, 297, 1968. 23. Hammon& M. E., and Dvorak, H. F., J. Exp. Med. 136, 1518, 1972. 24. Noll, H., Bloch, H., Asselinan, J., and Lederer, E., Biochim. Biophys. Acta 20, 299 1956. 25. Bekierkunst, A., Levij, A. S., Yarkoni, E., Vilkas, E., and Lederer, E., J. Bacterial. 100, 95, 1969. 26. Trasher, S. G., Bigazzi, P., and Cohen, S., Fed. Proc. 32, 977 abs., 1973. 27. Nicolson, G. L., Nature New. Biol. 243, 218, 1973.
IMMUNOLOGY
13, 313-321 (1974)
Alteration of Macrophage Surface in the Course of Immunological Activation: Decay of Metabolic Response to Concanavalin A1 D. ROMEO,G.ZABUCCHI,
M. JUG,AND F. ROSSI
Istituto di Chimica Biologica and Istituto di Patologia Generale, Uwiversitd cIi Trieste, 34127 Trieste, Italy Received January 2, 1974 “Activated” macrophages, isolated from lungs of BCG-infected rabbits, exhibited a greater perturbability of oxidative metabolism than their normal counterpart, when exposed iti vitro to unrelated microorganisms or treated with phospholipase C. They were much less metabolically responsive when incubated with Concanavalin A. The metabolic effect of this lectin gradually decayed as time elapsed from the day of infection. These results are interpreted to show a modification of the surface properties of the macrophage in the course of the immunological activation. The possible mechanisms of generation of these surface alterations are discussed.
INTRODUCTION It is well known that macrophages respond to certain aggressions with an increase of their defensive capacities (1, 2). The process of transformation of macrophages into “activated” phagocytes has an immunological basis and involves sensitized lymphocytes through production of chemical mediators (3). In the course of activation the macrophages undergo a variety of morphological and biochemical alterations, such as overdevelopment of Golgi apparatus, increase in number of mitochondria and lysosomes, rise in activity of various lysosomal hydrolases, enhancement of capacity to ingest and kill bacteria and in tendency to spread on glass (l-4). The functional hypertrophy of activated macrophages is accompanied by a greater metabolic perturbability. In fact, they respond to a challenge by phagocytosible matter with a more pronounced increase in glucose oxidation (5-7). The stimulation of oxidative metabolism in macrophages exposed to bacteria is considered to be triggered by biochemical events occurring at the surface of the cell (8, 9). Thus, several modifications of the properties of the macrophage in the process of activation appear to concern functions of the cell periphery. This would suggest that a change in the molecular organization and/or composition has occurred on the surface membrane of the activated macrophages. To test this hypothesis we have analyzed the effect exerted by cell surface perturbing agents on the oxidative metabolism of activated macrophages as compared to their normal counterparts. The agents used were Concanavalin A (Con A) and phospholipase C, which have been shown to mimic the phagocytosis associated 1 This investigation was supported by Research Grant 72.00871.04 from the Italian Consiglio Nazionale delle Ricerche. 313 Copyright 0 1974 by Academic Press. Inc. All rights of reproduction in any form reserwd.
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ROMEO
ET
AL.
stimulation of glucose oxidation through the hexose monophosphate pathway (HMP) both in macrophages and polymorphonuclear Ieucocytes (10-12). MATERIALS
AND
METHODS
Cells Outbred rabbits weighing about 2.5 kg were infected with Bacihus CaImetteG&in (BCG) kindly supplied by Istituto Vaccinogeno Antitubercolare as a lyophilized powder. Ten milligrams of viable microorganisms, suspended in 1 ml of sterile saline containing 2% (v/v) t ween SO, were injected in the marginal ear vein on two subsequent days. Three weeks later the animals were killed by gaseous embolus and macrophages were removed from the lungs by means of tracheobronchial lavages (13). Cells were centrifuged at 400g for 10 min, treated with 0.2% NaCl for 30-60 set to lyse contaminating red cells, and finally suspended in calcium-free Krebs-Ringer phosphate buffer, pH 7.4 (KRP). These operations were performed with chilled solutions. Differential counts were carried out on MayGriinwald-Giemsa stained smears. Marcophages in cell preparations from BCG infected animals (1.2-2.4 X log cells/rabbit) were SS-91%,, whereas in controls (5-8 X 10’ cells/rabbit) were 9.2-98s. Bacteria and Surface Perturbing
Agents
B. Mycoides were grown on nutrient agar, heat-killed and opsonized in fresh serum derived from control rabbits. Concanavalin A (grade III, Sigma, U.S.A.) was dissolved in distilled water; fluorescein-labelled Con A was prepared according to Smith and Hollers (14) and added to macrophages under the same conditions used for the metabolic studies. For treatment with phospholipase C (CZ. Welchii, E.C. 3. 1. 4.3., Sigma, U.S.A.) cells were resuspended in a medium similar to KRP which contained Tris-HCI instead of phosphate buffer and was supplied with I mM CaCIs (KR-Tris). Glucose Oxidation The oxidation of glucose was determined by incubating the cells at 37°C in 2.5 ml KRP or KR-Tris with 0.2 mM glucose and 0.8 pCi l-14C-glucose or 2 &i 6J4C-glucose. In the case of treatment with Con A, the lectin was usually added 10 min before the addition of the labelled glucose. The 14C02 produced in 10 min of incubation was collected and counted as described elsewhere (8). The HMP activity was calculated as difference between the 14COs generated from l-14Cglucose and that from 6-14C-glucose. Enzyme Assays Acid phosphatase and P-glucuronidase were assayed essentially according to Berthet and de Duve (15) and Gianetto and de Duve (16)) respectively. The activity of peroxidase was determined as described by Romeo et al. (17). RESULTS Reversible Metabolic Stimulatiolz
of Macrophages by Con A
Figure 1 shows that the rate of oxidation of l-14C-glucose in normal lung macrophages is enhanced in the presence of a small amount of Con A in the
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FIG. 1. Stimulation of the “COa production from l-T-glucose in alveolar macrophages by Con A and its reversibility by a-D-methyl mannopyranoside (a-MM) ; 1 X 10’ macrophages were incubated at 37°C in 2.5 ml KRP, containing l-14C-glucose (0.8 &i, 0.5 pmoles) in the absence or in the presence of 37.5 pg of Con A. At the points indicated by arrows (U-MM was added at a final concentration of 50 mM.
medium. If, after the onset of stimulation, ol-methyl-o-mannopyranoside (a-MM), which is known to have a high affinity for Con A (IS), is added to the incubation medium, the rate of glucose oxidation rapidly resumes its resting value. At the concentration used, the lectin induces the formation of small aggregates of macrophages, consisting of 2-4 and, only occasionally, more than 5 cells. The percentage of cells which incur agglutination varies with different cell preparations and does not apparently bear any relation to the extent of the metabolic stimulation. By electron microscopy no formation of pinocytic vesicles is seen in macrophages when treated with Con A under our conditions. This observation appears to rule out the possibility that endocytosed lectin molecules are responsible for the enhancement of glucose oxidation. Perturbability and Con A
of O&dative
Metabolism
of Activated
Macrophages
by Bacteria
Table 1 reports the rate of oxidation of l-14C-glucose and 6-14C-glucose by activated and control macrophages. As already described by others (6, 7), the activated macrophages show a greater metabolic perturbability than control cells when exposed in vitro to bacteria. On the contrary, a treatment with Con A produces almost no change in the oxidation of 6J’C-glucose and enhancement of 14C0, production from 1-14C-glucose, which is smaller than that obtained in control cells. As illustrated in Fig. 2, the increment of HMP activity measured at different Con A concentrations is consistently smaller for activated macrophages than for their normal counterparts. By using fluorescein-tagged Con A a similar pattern of surface distribution of fluorescence is observed for the two types of cells. If a challenge by bacteria is performed on macrophage suspensions in the presence of Con A (12 s/ml), the stimulation of HMP activity is greater than that obtained with Con A alone, but lower than that recorded in the absence of lectin. Thus, the presence of Con A produces an inhibition of the phagocytosis-associated increment
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d V
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8.
Con AIp/mll
FIG. 2. Effect of different concentrations of Con A on the activity of the hexose monophospbate pathway (HMP) of normal (0-O) and activated ( O-O ) alveolar macrophages. Activated cells were isolated from rabbits infected with BCG 21 days before. The oxidation of glucose was measured essentially as described in Fig. 1, with 0.8 pCi of l-l”C-glucose or 2 pCi of 6-“C-glucose; HMP activity = [YOz from l-YZ-Glc] - [14COz from 6-I’C-Glc] ; HMP increment = HMP of treated cells-HMP of resting cells (cpm X lO”/rCi of l’C-glucose/l X 10’ cells/l0 min) .
of HMP activity, which averages 25% for the normal macrophages and 42% for the activated cells. This inhibition is probably related to the decreased availability of free cell surface to bacteria due to formation of small clusters of cells. The agglutinability of activated macrophages, however, does not significantly differ from that of controls. Under our conditions, Con A exerts no cytopathic effect on both types of macrophages. Modification of Metabolic Response of Macrophages to a Con A Treatment in the Course of Activation For this series of experiments, macrophages were isolated from rabbits at different times after the infection with BCG. Since there is a good correlation between activation and level of lysosomal enzymes (19, 20), the state of activation of macrophages was monitored by assaying the activity of /I-glucuronidase and acid phosphatase, which peaks in the third week after infection (9). Another enzyme assayed was peroxidase, whose presence in macrophages has been recently described by us (17). The activity of this enzyme progressively decreases from the day of infection, being almost nil at Day 33. The reason for this behavior of peroxidase is obscure ; it might be explained either by a gradual enzyme inactivation or by a specific extracellular release of peroxidase at the site of the inflammatory lesion. The average increment of HMP activity obtained with Con A reacting with macrophages at different stages of activation is shown in Fig. 3. A gradual loss of metabolic perturbability of macrophages is observed as time elapses from the day of infection with BCG. This loss continues also when macrophages derive from a lesion entering the healing stage.
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I 0
14
7 Days
21
from
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3. Effect of Con A on the activity of the hexose monophosphate pathway (HMP) of FIG. alveolar macrophages isolated from rabbits at different times from infection with BCG. Each point represents the mean of 4 experiments; the average number of alveolar macrophages per rabbit at Days 0 (control), 7, 14, 21 and 33 were 0.6 X lo’, 7.6 X IO*, 14.8 X 108 and 10.1 X IO’, respectively. The definition of HMP increment is given in Fig. 2.
Metabolic Response of Activated Macrophage to a Treatment with Phospholipase C The split of plasma membrane phospholipids with phospholipase C causes a stimulation of oxidative metabolism, similar to that concomitant with phagocytosis, both in polymorphonuclear leucocytes (11) and macrophages (12). As shown in Fig. 4, a greater stimulation of oxidation of l-14C-glucose by phospholipase C is observed with activated macrophages than with normal cells. The oxidation of 6-14C-glucose is unaffected by the treatment of both types of macrophages with the phospholipid splitting enzyme.
d 10 6 %Gluc LA-
J-a .6
na .2 Units
of
.4 Phospholipase
C
FIG. 4. Effect of phospholipase C on glucose oxidation by normal and activated alveolar macrophages. Activated macrophages derived from rabbits infected with BCG 21 days before. Glucose oxidation was measured as described in Figs. 1 and 2.
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DISCUSSION Macrophages respond to a challenge by bacteria with a rapid enhancement in the rate of respiration, of glucose oxidation through the HMF’, and of Hz02 production (9). The oxidizing product(s) of this metabolic burst are thought to be utilized for bacterial killing (17, 21). Activated macrophages, which kill ingested organism with enhanced efficiency (3)) display a more marked stimulation of their oxidative metabolism than control cells, when exposed to particulate matter. This enhanced perturbability might be ascribed either to a greater activity of oxidative enzyme systems, or to a higher phagocytic rate and capacity, or to a modification of properties of the cell surface, where the triggering mechanism of the metabolic stimulation is very likely generated (8-10). The relevance of the first point cannot be established at the present time, since comparative data concerning the enzymes participating for example in the activation of HMP are not available. Preliminary results obtained in our laboratory suggest, however, that activated macrophages do not possess an enhanced capacity to oxidize NADPH. The rate of oxidation of this nucleotide is considered to be the key factor in the metabolic stimulation of mononuclear and polymorphonuclear phagocytes (8, 9). As for the second point, since also a treatment with phospholipase C induce.; in activated macrophages a stimulation of HMP greater than that produced in their normal counterparts (Fig. 4), the more elevated excitability of the former cells seems not to be linked to an increased phagocytosis. Thus, the more likely explanation of the enhanced metabolic perturbability of the activated macrophage would reside in a modification of the triggering mechanism. This mechanism is assumed to operate at the level of the cell surface (8, 9), and might therefore “feel” a change in the composition or molecular organization of cell membrane. A modification of surface properties of activated macrophages is suggested by their more rapid and extensive spreading on glass (4), by the change in their electrophoretic mobility (22) and by the enhancement of turnover of surface membrane-associated macromolecules (23). A gradual change in the surface properties of macrophages in the process of activation is also consistent with the loss of metabohc response to a treatment with Con A. Apparently, the reduced effect of the lectin is neither due to a failure of binding to activated macrophages, as assessed by the use of fluorescein-labelled Con A, nor to a change in the extent of cell agglutination. The decay of the response of the activated macrophages to Con A might be ascribed to one or more of the following events. (A) Decrease in number or availability of Con A receptors to an extent not detectable by simply using fluorescein-tagged Con A. A drop in the number of receptors might derive either from their shedding into cell environment or from their digestion by hydrolytic enzymes, released from the macrophage itself or from neutrophils present in the pulmonary granuloma. A reduced availability of receptors might be due to insertion of new macromolecules such as cytophilic antibodies (3) in the surface membrane. The possibility exists that molecules competing with normal receptors for Con A might appear on the surface of activated macrophages. It is tempting to speculate that one such molecule might be trehalosed$‘dimycolate (“cord factor”), a complex glycolipid of the tubercle bacilli (24)) which induces pulmonary granulomas and increased resistance to challenge with the
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H37Rv strain of Mycobacterium tubercolosis (25). This hypothesis obviously implies an exchange of lipid between BCG and the host cell membrane. (B) Reduced surface mobility of Con A-receptors. We have advanced the hypothesis (10) that the Con A-induced metabolic stimulation is caused by microaggregation of glycoprotein subunits accompanied by changes of surface properties, such as potential or ion permeability. Trasher et al. (26) have demonstrated that the surface components of macrophages are indeed in a dynamic state on the membrane and form configurations similar to those observed on lymphocytes. A modification of membrane fluidity in the activated macrophages, resulting from a change in the intrinsic properties of the plasma membrane, might be invoked as a perturbing factor in the redistribution of receptor molecules by the lectin. In this connection, it is worthwhile mentioning that the different agglutinability of virus-transformed cells has been shown by Nicholson (27) to be due to a change in fluidity of the surface membrane. (C) Impairment of the mechanism which links Con A-receptor interactions to generation and transmission of the stimulatory signal. This assumption appears to be the least credible since the phagocytosis- and Con A-associated metabolic stimulations share very likely the same mechanism. For the time being we are not in the position to decide among the above alternatives. In fact, a detailed knowledge of the microstructure of the membrane of the activated macrophages as compared to the normal cell is needed to gain a clearer picture of the mechanism of immunological activation of macrophages. An experimental approach of this type is presently in progress in our laboratory. ACKNOWLEDGMENTS The skillful assistance of Mr. G. Orpelli is gratefully acknowledged.
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