Vol. 26, No. 3
INFECTION AND IMMUNITY, Dec. 1979, p. 910-915 0019-9567/79/12-0910/06$02.00/0
Phagocytosis-Induced Injury of Normal and Activated Alveolar Macrophages MARIA P. McGEE* AND QUENTIN N. MYRVIK Department ofMicrobiology and Immunology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103 Received for publication 13 July 1979
The present study tested the hypothesis that BCG-activated macrophages become injured when they phagocytose certain particulates. The data indicate that alveolar macrophages obtained from Mycobacterium bovis BCG-sensitized animals were more susceptible to cell death after in vitro incubation with BCG or zymosan than were macrophages from normal animals. Increased susceptibility was dependent on phagocytosis, since incubation with cytochalasin B, a phagocytosis inhibitor, abrogated the effect. Catalase, cytochrome c, and ascorbic acid offered partial protection to the macrophage, suggesting the involvement of free radicals in the generation of cytotoxicity. Not all of the cells from the alveolar populations were equally susceptible to cell death, thus suggesting either heterogeneity in the cell population or a requirement of more than one cell type in the induction of necrosis or both.
Immunological reactions against persisting particulate antigens are characterized by the generation of organized granulomas. Although chronic granulomatous reactions are defensive responses which tend to wall off persistent noxious agents, these reactions can be accompanied by necrosis, with considerable destruction of normal tissue (1, 15). Classic examples of this type of tissue damage are the necroses occurring in tuberculosis and mycotic infections. In addition, similar mechanisms may contribute to the tissue injury present in many other infectious diseases as well as in certain autoimmune conditions (4). The exact mechanisms responsible for the tissue damage are not known, but release of hydrolytic enzymes by the activated infiltrating macrophage has been considered an important effector mechanism of necrosis (1, 4, 5). In this regard, granulomatous reactions containing activated macrophage posessing high levels of hydrolytic enzymes can resolve without apparent tissue damage in some circumstances (12). Therefore, tissue destruction cannot be explained solely by the presence of high levels of hydrolytic enzymes in the lesions. In tuberculosis, the onset of necrosis and delayed hypersensitivity occur essentially simultaneously (1). Moreover, peritoneal cells and spleen explants obtained from animals presensitized or infected with tubercle bacilli are damaged if incubated in vitro in the presence of bacilli or its products (2, 17, 18). These observations, together with the fact that necrosis in
granulomas usually begins at the center of the lesion or site of antigen deposition, suggest that cell sensitization and the presence of antigen are factors which influence the onset of tissue destruction. In the present study, the role that phagocytosis of particulate material by activated alveolar macrophage plays in macrophage injury is evaluated.
MATERIALS AND METHODS Animals and animal sensitization protocols. New Zealand white female rabbits, 2 to 3 kg in weight, were sensitized with 100 ,ug of heat-killed, BCG, suspended in 0.1 ml of light mineral oil by a single intravenous injection. Three weeks after sensitization, the animals were sacrificed by air embolism, and the alveolar cells were collected as described below. Ageand sex-matched normal animals were used as controls. Cell procurement and culture conditions. The lungs were excised, and the alveolar macrophages were recovered by transtracheal lavage with 80 ml of sterile physiological saline as previously described (14). The alveolar macrophage were suspended in minimal essential medium containing 5% fetal calf serum, 100 U of penicillin per ml, and 100 tg of streptomycin per ml. The cell suspensions in control cultures were incubated in petri dishes (5 ml; 60 by 150 mm) or in test tubes (1 ml; 13 by 130 mm) at concentrations of 0.5 x 106 and 1 x 106 per ml, respectively, for 24 h at 37°C in an atmosphere of 5% CO2 in air. Test cultures were prepared in the same manner but contained (i) phagocytic particles, (ii) reducing agents or cytochalasin b, or (iii) both i and ii. After the incubation period, the cells were removed from the cultures by gently scrap910
VOL. 26 1979
PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
911
ing with a sterile rubber policeman; total number of Cultures were prepared in triplicate, and each cells and viability was determined by trypan blue experiment was repeated on at least six different exclusion, using a hemocytometer. occasions, except for the zymosan experiments, Density gradients and cell separation. Contin- which were repeated three times. The absolute uous density gradients were prepared by using previ- number of cells recovered from control cultures ously described techniques, with minor modifications (8). Mixtures of Ficoll, diatrizoate (Renografin), Krebs was 3.31 (± 0.103) x 105 per ml when the starting Ringers salts solutions and distilled water were used cell number was 5 x 105 per ml, and it was 5.40 to prepare the high-density (1.080 g/ml) and low-den- (± 0.32) x 105 when the starting cell number was sity (1.040 g/ml) solutions. The solutions were ad- 1.0 X 106. It can be noted that both zymosan and justed to 290 mosmol with 1Ox Krebs Ringers salts or BCG produced significant reduction in the viadistilled water as needed. Linear gradients were bility of BCG-activated macrophage (Fig. 1). It formed by mixing 14 ml of high-density and 15 ml of is noteworthy that BCG expressed some toxicity low-density solutions at 40C in a gradient maker (Bio- for normal alveolar macrophage but zymosan Rad Laboratories, Richmond, Calif.). The solutions nontoxic. In addition, a dosewere loaded with approximately 5 x 107 cells equally appeared to be was demonstrated for both relationship effect gradients The solutions. the two between distributed However, BCG was approxand zymozan. BCG were centrifuged at 200 x g at 40C for 15 min, and the cells were collected in 2-ml fractions. The number of imately twice as effective as zymozan in inducing cells present in the various regions of the gradient macrophage death on a weight basis. To further determine whether the effect of were estimated by measuring the optical density at 400 nm with a Gilford scanner. BCG on macrophage viability was specific, acPhagocytic particles. Lyophilized BCG (11), zym- tivated macrophage were incubated in the presosan (I.C.N. Pharmaceuticals Inc., Cleveland, Ohio), ence of soluble purified protein derivative, insolListeria monocytogenes (21), and insolubilized pro- ubilized tuberculoprotein, insolubilized albumin, teins (11) were prepared by suspending the particles and L. monocytogenes, at concentrations rangin minimal essential medium, containing 5% fetal calf serum, 100 U of penicillin per ml, and 100 ,tg of ing from 25 to 300 ,Ig/ml. The lowest concentrastreptomycin per ml. The suspensions were main- tions of particles that consistently caused a detained in the frozen state at -210C and were sonicated crease in viability are illustrated in Table 1. On before use to standardize the dispersion. The micro- a per weight basis, BCG and insolubilized tuberculoprotein were the most toxic of the particles organisms were heat-killed before lyophilization. Other reagents. Cytochalasin b was purchased tested. Moreover, the data suggest that the abilfrom Aldrich Chemical Co., Milwaukee, Wis. Stock ity to induce macrophage damage is dependent solutions were prepared in dimethyl sulfoxide. Purified in part on the composition and particulate naprotein derivative (lot 9747741) was obtained from ture of the antigen. It is noteworthy that insolParke, Davis & Co., Detroit, Mich. Ascorbic acid, ubilized tuberculoprotein (50 ,ug/ml) was able to ferricytochrome c (fraction III), catalase from bovine induce an effect similar to that of BCG; insoluliver, and Ficoll were obtained from Sigma Chemical albumin exerted a toxic effect at the bilized were solutions prepared Co., St. Louis, Mo. All stock in minimal essential medium, except Ficoll, which was highest dose (250 ,ug/ml), whereas purified prodissolved in distilled water. Renografin (meglumine tein derivative at 100 ,ug/ml had little effect. diatrizoate and sodium diatrizoate) was obtained from Effect of cytochalasin b on BCG-induced E. R. Squibb & Sons, Inc. Princeton, N.J. macrophage death. To determine the role of phagocytosis on the in vitro-induced necrosis,
RESULTS Effect of BCG and other phagocytic particles on the viability of macrophage. Macrophage obtained from normal and BCG-sensitized animals were incubated in the presence of either BCG or zymosan as described in Materials and Methods. After the incubation period, the cells were gently detached from the cultures with a rubber policeman, washed, and suspended in 0.85% saline containing 0.05% trypan blue. The cells were blended in a Vortex mixer for 10 s to reduce cell clumping and counted in a hemocytometer. Percent viability was calculated by the following formula. Percent viability = [(number of viable cells in test cultures, i.e., with particles)/(number of viable cells in control cultures, i.e., without particles)] x 100.
normal and activated macrophages were incubated in the presence or absence of BCG or cytochalasin b, or both as previously described. Cytochalasin b inhibits phagocytosis and other metabolic activities related to phagocytosis, such as the burst in oxygen consumption and the release of H202 (9, 19). The results are represented in Fig. 2. Cytochalasin b at a concentration of 10 ,g/ml fully protected the macrophages, suggesting that phagocytosis or a phagocytosis-induced mechanism was responsible for the in vitro destruction of macrophages. Effect of antioxidants on BCG-induced necrosis of macrophage. To determine whether or not the by-products of the macrophage-oxidative metabolism played a role in the
912
INFECT. IMMUN.
McGEE AND MYRVIK o
normal cells activated cells
0
normal cells activated cells
control
200;ig/ml K0Opmg 50Opg/ml
IOOpug/ml 50jug/ml 25)jg/ml BCG
Zymozan
FIG. 1. Cell viability after incubation with BCG and zymozan. Normal and BCG-activated macrophages were incubated in petri dishes for 24 h as described in the text. The results represent the mean ± 1 standard deviation of the mean of (a) six separate experiments and (b) three separate experiments.
TABLE 1. Effect ofpurified protein derivative (PPD) and various phagocytic particles on activated macrophages Incubation with: Viabilityb 44.25 ± 2.56 BCG (50 ,ug/ml) Insolubilized tuberculoprotein (50 59.94 ± 5.63 tg/ml) Insolubilized albumin (250 ,ug/ml) 46.87 ± 7.23 79.42 ± 4.20 L. monocytogenes (100 ,ug/ml) 78.33 ± 8.66 Soluble PPD (100 Ag/ml) .... a Incubation conditions were as described in text. bExpressed as percent of control cultures. Each value indicates mean ± 1 standard deviation of the mean. 0 normal cells 3 activated cells
control
100
~90
one of three similar experiments. Catalase which scavenges H202 (20), cytochrome c which oxi-
dizes the superoxide anion (26), and ascorbic acid which scavenges free radicals (13), were found to inhibit significantly the BCG-induced macrophage damage. This suggests that the free radicals and peroxides generated upon phagocytosis (19) are contributing to the macrophage damage. However, since the comparatively large doses of antioxidants used did not effect complete protection, it is possible that other factors are involved in the death of the activated macrophage. Susceptibility of different populations of activated macrophage to BCG-induced death. To establish whether or not all the cells present in the alveolar lavage are equally susceptible to BCG-induced damage, the alveolar cell population was separated according to their TABLE 2. Effect of catalase, cytochrome c and ascorbic acid on macrophage viabilitya Incubation with:
.~80
Control
70
~60
l,ug/ml 50jig/ml Cyto-B
BCG
BCG t
Cyto- B FIG. 2. Effect of cytochalasin b on phagocytosisinduced injury. The results represent the mean ± I standard deviation of the mean of six experiments. Cyto-B, Cytochalasin b, (10 fig/ml). BCG was at 50 ig/ml.
induction of cell damage in vitro, catalase, cytochrome c and ascorbic acid were added to the cultures at concentrations of 250, 500, and 250 ,Lg/ml, respectively. Table 2 shows the results of
Viabilityb 100.00 50.12 ± 4.43 87.62 ± 5.92 96.88 ± 5.34
Protection'
BCG Catalase Cytochrome c Ascorbic acid 94.19 ± 6.63 BCG + catalased 71.61 ± 6.88 41.48 BCG + cytochrome c 69.36 ± 6.05 39.27 BCG + ascorbic acid 69.46 ± 9.51 37.70 a Incubation conditions were as described in the text.
b Expressed as percentage of control cultures; each value indicates mean ± 1 standard deviation of the mean. 'Each value indicates {[(percent viability in cultures containing catalase plus BCG) - (percent viability in cultures with BCG but no catalase)]/[percent viability in cultures containing BCG but no catalase]) x 100. d BCG at 100 ,ug/ml, catalase at 250 tg/ml, ascorbic acid at 250 tg/ml, and cytochrome c at 500 jug/ml.
PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
VOL. 26 1979
density in an isoosmolar linear gradient of Ficoll. The various fractions were pooled in five groups, and the cells from each group were suspended in minimal essential medium-5% fetal calf serum at a concentration of 106 cells per ml. Approximately 80 to 90% of cells in each one of the groups were capable of ingesting latex beads, and 86 to 95% of the viable cells were able to concentrate neutral red. The variations from fraction to fraction were not significantly different. By morphological criteria in Giemsa-stained smears, the more dense fractions (D and E in Fig. 3) contained 10 to 20% of cells with the appearance of lymphocytes, whereas in the less dense fractions only 2 to 10% of the cells morphologically resembled lymphocytes. These results indicated that some of the cells classified as lymphocytes by morphological criteria were probably monocytes or macrophage. Triplicate cultures from each one of the pools were incubated with or without 100 ,sg of BCG per ml during 24 h at 370C as previously described for unseparated populations. Figure 3 represents the typical results of one of six similar experiments. Damage induced by BCG was observed only in fractions containing cells from the high-density region of the gradient, indicating that the susceptibility to undergo necrosis in vitro is present only in a particular subpopulation of macrophage. An alternative possibility is that the presence of lymphocytes or lymphocyte-like cells or both in these fractions plays a role in the induction of macrophage death. DISCUSSION It should be emphasized that large numbers of macrophage, epithelioid cells, and giant cells
913
are destroyed in apparent synchrony when central necrosis of a tubercle occurs. This event is apparently associated with cell-mediated hypersensitivity reactions and can vary from necrosis of dermal reactions to central necrosis of allergic granulomas. For example, in tuberculosis, caseous necrosis is apparently dependent on systemic cell-mediated hypersensitivity elicited by products of tubercle bacilli. This concept is supported by the observations that spleen explants and peritoneal exudate macrophage from sensitized animals are damaged more extensively when incubated with tubercle bacilli than comparable cell preparations from normal animals (2, 17, 18). In addition, the observations of Yamamura et al. (22) clearly indicate that if tuberculin-sensitive rabbits are desensitized with tuberculin, central necrosis of tubercles does not occur after challenge with tubercle bacilli. The mechanisms by which activated macrophage are destroyed in the center of an allergic granuloma are not known. It has been suggested that the liberation of hydrolytic enzymes and the release of toxic factors are the causes of this necrotic reaction (1, 4, 5, 7, 10). It is possible that toxic products generated by oxidative metabolism of activated macrophage could also be responsible. The role that toxic by-products of the oxidative metabolism play on bacteriolysis and self destruction by phagocytic cells has been extensively studied in polymorphonuclear leukocytes (19, 20). It is of interest that the premature death that phagocytosis induces in this cell population can be prevented by catalase and free-radical scavengers (20). R.L 13630 20
Density 1077
9'M
10
z
2
.080
.060
5600 90 80 70 60 50 40
1066
30 20 I0 13500
1057
70 60 50 40 30 13420
1048
90 so
1040
FIG. 3. Viability of BCG-activated macrophages separated in density gradient. BCG-activated alveolar macrophage were fractionated as described in the text. Cells from each fraction were incubated in the test tube-type assay for 24 h. One of six similar experiments is shown. Numbers in parentheses indicate the percent viability of the macrophages after incubation. R.L, Refractive index.
914
McGEE AND MYRVIK
In this regard, the increased killing potential of activated macrophage against bacteria and tumor cells has been attributed to the generation of peroxides and free radicals upon phagocytosis (3). For instance, activated macrophage triggered pharmacologically with phorbol myristate acetate are able to destroy normal and transformed cells. The mechanism responsible for this cytotoxicity was found to be the release of H202 by the activated macrophage (16). In the present study we have found that the susceptibility of an activated macrophage to self destruction is largely dependent on the phagocytic event. Moreover our data suggest that the by-products of oxidative metabolism probably play a role in the induction of damage. However, the importance of this mechanism is relative, since protection with antioxidants was only partial (30 to 40%); other mechanisms are probably involved. It is also possible that the oxidants are the major cause of damage but they are released at sites inaccessible to the antioxidants; the chemical lesion could be occluded by opposing membranes at the site of cell-to-cell contact and not available to the surrounding medium. This seems unlikely, because doses of antioxidants similar to those used in our experiments effectively inhibited phagocytosis-induced destruction of polymorphonuclear leukocytes and the lysis of tumor cells by phorbol myristate acetatetriggered macrophages (16, 20). The in vitro self destruction of activated macrophage was not characterized by the degree of specificity normally associated with immunological reactions. However, insolubilized tuberculoprotein and BCG were more efficient in causing cell lysis than other particles, based on the amount added. It is noteworthy that zymosan at 200 jig/ml induced cell death equivalent to 100 ,jg of BCG per ml in activated cells. In contrast, this dose of zymosan produced no detectable damage to normal cells, whereas 100 ,ug of BCG per ml was significantly toxic (15 to 35% cell death). The primary toxicity of BCG for normal cells could explain why BCG seemed to express some specificity based on the minimum effective dose. Based on the data obtained, it is not possible to rule out lymphocytes as possible accessory cells to this reaction. For example, the 24-h incubation period with the various particulates could have induced lymphocyte activation and subsequent "over-activation" of macrophage that had ingested the particulates. After separation of the alveolar cells in continuous density gradients, the macrophage subpopulations found to be most susceptible corresponded to the fraction in which lymphocyte-like cells were most numerous. Since morphological criteria were un-
INFECT. IMMUN.
reliable for distinguishing between small numbers of immature monocytes and medium-sized lymphocytes, and functional tests demonstrated a similar proportion of phagocytes in all fractions, the possible role of the lymphocyte in this reaction could not be resolved. It is possible that lymphocytes could induce macrophage damage, either by the release of cytotoxic substances, or by the activation of the macrophage to a point at which phagocytosis could trigger death of the macrophage. This second possibility seems more likely, in view of reports which indicate that macrophage are not destroyed by lymphotoxin (6). Furthermore, no lymphokines have been reported, to our knowledge, that are toxic for macrophage. Phagocytosis-induced cytotoxicity of over-activated macrophage could result from localized damage to lysosomal membranes, perhaps mediated by toxic products produced by oxidative metabolism. This damage in turn would cause liberation of lysosomal enzymes into the cytoplasm and ultimately the medium, resulting in an accelerated destruction of neighboring phagocytic cells. Experiments designed to elucidate the possible role of lymphocytes or their products or both on the phagocytosis-induced macrophage destruction as well as the mechanisms involved in this destruction are in progress in our laboratory. LITERATURE CITED 1. Adams, D. 0. 1976. The granulomatous inflammatory response. Am. J. Pathol. 84:164-190. 2. Aronson, J. D. 1931. The specific cytotoxic action of tuberculin in tissue cluture. J. Exp. Med. 54:387-397. 3. Blanden, R. V., A. J. Hapel, P. C. Doherty, and R. M. Zinkernager. 1976. Lymphocyte-macrophage interactions and macrophage activation in the expression of antimicrobial immunity in vivo, p. 367. In D. S. Nelson (ed.), Immunology of the Macrophage. Academic Press Inc., New York. 4. Boros, D. L. 1978. Granulomatous inflammation. Prog. Allergy 24: 183-267. 5. Dannenberg, A. M., and M. Suzimoto. 1976. Liquefaction of caseous foci in tuberculosis. Am. Rev. Respir. Dis. 113:257-259. 6. David, J. R., and R. R. David. 1972. Cellular hypersensitivity and immunity. Prog. Allergy 16:300-449. 7. Davies, P., and A. C. Allison. 1976. Secretion of macrophage enzymes in relation to the pathogenesis of chronic inflammation, p. 427. In D. S. Nelson (ed.), Immunobiology of the macrophage. Academic Press Inc., New York. 8. Hayry, P., and L. Anderson. 1976. Fractionation of immunocompetent cells with different physical properties. Scand. J. Immunol. (Suppl. 5) 5:3144. 9. Malawista, S. E., J. B. L. Gee, and R. C. Bersch. 1971. Cytochalasin B reversibly inhibits phagocytosis: functional metabolic and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages. Yale J. Biol. Med. 44:286-300. 10. Marx, J., and R. Burrel. 1973. Delayed hypersensitivity to beryllium compounds. J. Immunol. 111:590-598. 11. McGee, M. P., Q. N. Myrvik, and E. S. Leake. 1978.
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PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
Organization of allergic granulomas and dependence on insoluble antigen. J. Reticuloendothel. Soc. 24:253-261. 12. McGee, M. P., and Q. N. Myrvik. 1976. Hydrolase levels in necrotizing and non-necrotizing BCG-induced pulmonary granulomas. RES J. Reticuloendothel. Soc. 20: 187-195. 13. Mustafa, M. G., and D. F. Tierney. 1978. Biochemical and metabolic changes in lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118: 1061-1090. 14. Myrvik, Q. N., E. S. Leake, and B. Fariss. 1961. Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to procure them in a high state of purity. J. Immunol. 86:128-132. 15. Myrvik, Q. N., L. A. Kohlweiss, and D. Harpold. 1975. Pathological potential of exaggerated immunological reactions, p. 227-235. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 16. Nathan, C. F., L. H. Brukner, S. C. Silverstein, and Z. A. Cohn. 1978. Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity. J. Exp. Med. 149:100-113.
915
17. Preston, M. P., D. Hart, and I. N. Brown. 1977. Immunologically mediated macrophage aggregation in monolayers of peritoneal cells from BCG sensitized mice. Immunology 32:33-41. 18. Rich, A. R., and M. R. Lewis. 1972. Mechanics of allergy in tuberculosis. Proc. Soc. Exp. Biol. Med. 25:596-598. 19. Root, R. K., and J. A. Metcalf. 1976. Superoxide and hydrogen peroxide formation of human granulocytes: inter-relationships and activation mechanisms. Studies with normal and cytochalasin B treated cells, p. 185. In F. Ross, P. Patriarcz, and D. Romeo (ed.), Movement, metabolism and bactericidal mechanisms of phagocytes. Piccin Medical Books, London. 20. Salin, M. L., and J. M. McCord. 1975. Free radical and inflammation. Protection of phagocytosing leukocytes by superoxide dismutase. J. Clin. Invest. 56:1319-1323. 21. Taplits, M., and Q. N. Myrvik. 1977. Host resistance to the LSV strain of Francisella tularensis in BCG vaccinated mice. J. Reticuloendothel. Soc. 24:297-308. 22. Yamamura, Y., Y. Ogawa, H. Maeda, and Y. Yamamura. 1976. Prevention of tuberculosis cavity formation by desensitization with tuberculin-active peptide. Am. Rev. Respir. Dis. 109:596-601.
INFECTION AND IMMUNITY, Dec. 1979, p. 910-915 0019-9567/79/12-0910/06$02.00/0
Phagocytosis-Induced Injury of Normal and Activated Alveolar Macrophages MARIA P. McGEE* AND QUENTIN N. MYRVIK Department ofMicrobiology and Immunology, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27103 Received for publication 13 July 1979
The present study tested the hypothesis that BCG-activated macrophages become injured when they phagocytose certain particulates. The data indicate that alveolar macrophages obtained from Mycobacterium bovis BCG-sensitized animals were more susceptible to cell death after in vitro incubation with BCG or zymosan than were macrophages from normal animals. Increased susceptibility was dependent on phagocytosis, since incubation with cytochalasin B, a phagocytosis inhibitor, abrogated the effect. Catalase, cytochrome c, and ascorbic acid offered partial protection to the macrophage, suggesting the involvement of free radicals in the generation of cytotoxicity. Not all of the cells from the alveolar populations were equally susceptible to cell death, thus suggesting either heterogeneity in the cell population or a requirement of more than one cell type in the induction of necrosis or both.
Immunological reactions against persisting particulate antigens are characterized by the generation of organized granulomas. Although chronic granulomatous reactions are defensive responses which tend to wall off persistent noxious agents, these reactions can be accompanied by necrosis, with considerable destruction of normal tissue (1, 15). Classic examples of this type of tissue damage are the necroses occurring in tuberculosis and mycotic infections. In addition, similar mechanisms may contribute to the tissue injury present in many other infectious diseases as well as in certain autoimmune conditions (4). The exact mechanisms responsible for the tissue damage are not known, but release of hydrolytic enzymes by the activated infiltrating macrophage has been considered an important effector mechanism of necrosis (1, 4, 5). In this regard, granulomatous reactions containing activated macrophage posessing high levels of hydrolytic enzymes can resolve without apparent tissue damage in some circumstances (12). Therefore, tissue destruction cannot be explained solely by the presence of high levels of hydrolytic enzymes in the lesions. In tuberculosis, the onset of necrosis and delayed hypersensitivity occur essentially simultaneously (1). Moreover, peritoneal cells and spleen explants obtained from animals presensitized or infected with tubercle bacilli are damaged if incubated in vitro in the presence of bacilli or its products (2, 17, 18). These observations, together with the fact that necrosis in
granulomas usually begins at the center of the lesion or site of antigen deposition, suggest that cell sensitization and the presence of antigen are factors which influence the onset of tissue destruction. In the present study, the role that phagocytosis of particulate material by activated alveolar macrophage plays in macrophage injury is evaluated.
MATERIALS AND METHODS Animals and animal sensitization protocols. New Zealand white female rabbits, 2 to 3 kg in weight, were sensitized with 100 ,ug of heat-killed, BCG, suspended in 0.1 ml of light mineral oil by a single intravenous injection. Three weeks after sensitization, the animals were sacrificed by air embolism, and the alveolar cells were collected as described below. Ageand sex-matched normal animals were used as controls. Cell procurement and culture conditions. The lungs were excised, and the alveolar macrophages were recovered by transtracheal lavage with 80 ml of sterile physiological saline as previously described (14). The alveolar macrophage were suspended in minimal essential medium containing 5% fetal calf serum, 100 U of penicillin per ml, and 100 tg of streptomycin per ml. The cell suspensions in control cultures were incubated in petri dishes (5 ml; 60 by 150 mm) or in test tubes (1 ml; 13 by 130 mm) at concentrations of 0.5 x 106 and 1 x 106 per ml, respectively, for 24 h at 37°C in an atmosphere of 5% CO2 in air. Test cultures were prepared in the same manner but contained (i) phagocytic particles, (ii) reducing agents or cytochalasin b, or (iii) both i and ii. After the incubation period, the cells were removed from the cultures by gently scrap910
VOL. 26 1979
PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
911
ing with a sterile rubber policeman; total number of Cultures were prepared in triplicate, and each cells and viability was determined by trypan blue experiment was repeated on at least six different exclusion, using a hemocytometer. occasions, except for the zymosan experiments, Density gradients and cell separation. Contin- which were repeated three times. The absolute uous density gradients were prepared by using previ- number of cells recovered from control cultures ously described techniques, with minor modifications (8). Mixtures of Ficoll, diatrizoate (Renografin), Krebs was 3.31 (± 0.103) x 105 per ml when the starting Ringers salts solutions and distilled water were used cell number was 5 x 105 per ml, and it was 5.40 to prepare the high-density (1.080 g/ml) and low-den- (± 0.32) x 105 when the starting cell number was sity (1.040 g/ml) solutions. The solutions were ad- 1.0 X 106. It can be noted that both zymosan and justed to 290 mosmol with 1Ox Krebs Ringers salts or BCG produced significant reduction in the viadistilled water as needed. Linear gradients were bility of BCG-activated macrophage (Fig. 1). It formed by mixing 14 ml of high-density and 15 ml of is noteworthy that BCG expressed some toxicity low-density solutions at 40C in a gradient maker (Bio- for normal alveolar macrophage but zymosan Rad Laboratories, Richmond, Calif.). The solutions nontoxic. In addition, a dosewere loaded with approximately 5 x 107 cells equally appeared to be was demonstrated for both relationship effect gradients The solutions. the two between distributed However, BCG was approxand zymozan. BCG were centrifuged at 200 x g at 40C for 15 min, and the cells were collected in 2-ml fractions. The number of imately twice as effective as zymozan in inducing cells present in the various regions of the gradient macrophage death on a weight basis. To further determine whether the effect of were estimated by measuring the optical density at 400 nm with a Gilford scanner. BCG on macrophage viability was specific, acPhagocytic particles. Lyophilized BCG (11), zym- tivated macrophage were incubated in the presosan (I.C.N. Pharmaceuticals Inc., Cleveland, Ohio), ence of soluble purified protein derivative, insolListeria monocytogenes (21), and insolubilized pro- ubilized tuberculoprotein, insolubilized albumin, teins (11) were prepared by suspending the particles and L. monocytogenes, at concentrations rangin minimal essential medium, containing 5% fetal calf serum, 100 U of penicillin per ml, and 100 ,tg of ing from 25 to 300 ,Ig/ml. The lowest concentrastreptomycin per ml. The suspensions were main- tions of particles that consistently caused a detained in the frozen state at -210C and were sonicated crease in viability are illustrated in Table 1. On before use to standardize the dispersion. The micro- a per weight basis, BCG and insolubilized tuberculoprotein were the most toxic of the particles organisms were heat-killed before lyophilization. Other reagents. Cytochalasin b was purchased tested. Moreover, the data suggest that the abilfrom Aldrich Chemical Co., Milwaukee, Wis. Stock ity to induce macrophage damage is dependent solutions were prepared in dimethyl sulfoxide. Purified in part on the composition and particulate naprotein derivative (lot 9747741) was obtained from ture of the antigen. It is noteworthy that insolParke, Davis & Co., Detroit, Mich. Ascorbic acid, ubilized tuberculoprotein (50 ,ug/ml) was able to ferricytochrome c (fraction III), catalase from bovine induce an effect similar to that of BCG; insoluliver, and Ficoll were obtained from Sigma Chemical albumin exerted a toxic effect at the bilized were solutions prepared Co., St. Louis, Mo. All stock in minimal essential medium, except Ficoll, which was highest dose (250 ,ug/ml), whereas purified prodissolved in distilled water. Renografin (meglumine tein derivative at 100 ,ug/ml had little effect. diatrizoate and sodium diatrizoate) was obtained from Effect of cytochalasin b on BCG-induced E. R. Squibb & Sons, Inc. Princeton, N.J. macrophage death. To determine the role of phagocytosis on the in vitro-induced necrosis,
RESULTS Effect of BCG and other phagocytic particles on the viability of macrophage. Macrophage obtained from normal and BCG-sensitized animals were incubated in the presence of either BCG or zymosan as described in Materials and Methods. After the incubation period, the cells were gently detached from the cultures with a rubber policeman, washed, and suspended in 0.85% saline containing 0.05% trypan blue. The cells were blended in a Vortex mixer for 10 s to reduce cell clumping and counted in a hemocytometer. Percent viability was calculated by the following formula. Percent viability = [(number of viable cells in test cultures, i.e., with particles)/(number of viable cells in control cultures, i.e., without particles)] x 100.
normal and activated macrophages were incubated in the presence or absence of BCG or cytochalasin b, or both as previously described. Cytochalasin b inhibits phagocytosis and other metabolic activities related to phagocytosis, such as the burst in oxygen consumption and the release of H202 (9, 19). The results are represented in Fig. 2. Cytochalasin b at a concentration of 10 ,g/ml fully protected the macrophages, suggesting that phagocytosis or a phagocytosis-induced mechanism was responsible for the in vitro destruction of macrophages. Effect of antioxidants on BCG-induced necrosis of macrophage. To determine whether or not the by-products of the macrophage-oxidative metabolism played a role in the
912
INFECT. IMMUN.
McGEE AND MYRVIK o
normal cells activated cells
0
normal cells activated cells
control
200;ig/ml K0Opmg 50Opg/ml
IOOpug/ml 50jug/ml 25)jg/ml BCG
Zymozan
FIG. 1. Cell viability after incubation with BCG and zymozan. Normal and BCG-activated macrophages were incubated in petri dishes for 24 h as described in the text. The results represent the mean ± 1 standard deviation of the mean of (a) six separate experiments and (b) three separate experiments.
TABLE 1. Effect ofpurified protein derivative (PPD) and various phagocytic particles on activated macrophages Incubation with: Viabilityb 44.25 ± 2.56 BCG (50 ,ug/ml) Insolubilized tuberculoprotein (50 59.94 ± 5.63 tg/ml) Insolubilized albumin (250 ,ug/ml) 46.87 ± 7.23 79.42 ± 4.20 L. monocytogenes (100 ,ug/ml) 78.33 ± 8.66 Soluble PPD (100 Ag/ml) .... a Incubation conditions were as described in text. bExpressed as percent of control cultures. Each value indicates mean ± 1 standard deviation of the mean. 0 normal cells 3 activated cells
control
100
~90
one of three similar experiments. Catalase which scavenges H202 (20), cytochrome c which oxi-
dizes the superoxide anion (26), and ascorbic acid which scavenges free radicals (13), were found to inhibit significantly the BCG-induced macrophage damage. This suggests that the free radicals and peroxides generated upon phagocytosis (19) are contributing to the macrophage damage. However, since the comparatively large doses of antioxidants used did not effect complete protection, it is possible that other factors are involved in the death of the activated macrophage. Susceptibility of different populations of activated macrophage to BCG-induced death. To establish whether or not all the cells present in the alveolar lavage are equally susceptible to BCG-induced damage, the alveolar cell population was separated according to their TABLE 2. Effect of catalase, cytochrome c and ascorbic acid on macrophage viabilitya Incubation with:
.~80
Control
70
~60
l,ug/ml 50jig/ml Cyto-B
BCG
BCG t
Cyto- B FIG. 2. Effect of cytochalasin b on phagocytosisinduced injury. The results represent the mean ± I standard deviation of the mean of six experiments. Cyto-B, Cytochalasin b, (10 fig/ml). BCG was at 50 ig/ml.
induction of cell damage in vitro, catalase, cytochrome c and ascorbic acid were added to the cultures at concentrations of 250, 500, and 250 ,Lg/ml, respectively. Table 2 shows the results of
Viabilityb 100.00 50.12 ± 4.43 87.62 ± 5.92 96.88 ± 5.34
Protection'
BCG Catalase Cytochrome c Ascorbic acid 94.19 ± 6.63 BCG + catalased 71.61 ± 6.88 41.48 BCG + cytochrome c 69.36 ± 6.05 39.27 BCG + ascorbic acid 69.46 ± 9.51 37.70 a Incubation conditions were as described in the text.
b Expressed as percentage of control cultures; each value indicates mean ± 1 standard deviation of the mean. 'Each value indicates {[(percent viability in cultures containing catalase plus BCG) - (percent viability in cultures with BCG but no catalase)]/[percent viability in cultures containing BCG but no catalase]) x 100. d BCG at 100 ,ug/ml, catalase at 250 tg/ml, ascorbic acid at 250 tg/ml, and cytochrome c at 500 jug/ml.
PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
VOL. 26 1979
density in an isoosmolar linear gradient of Ficoll. The various fractions were pooled in five groups, and the cells from each group were suspended in minimal essential medium-5% fetal calf serum at a concentration of 106 cells per ml. Approximately 80 to 90% of cells in each one of the groups were capable of ingesting latex beads, and 86 to 95% of the viable cells were able to concentrate neutral red. The variations from fraction to fraction were not significantly different. By morphological criteria in Giemsa-stained smears, the more dense fractions (D and E in Fig. 3) contained 10 to 20% of cells with the appearance of lymphocytes, whereas in the less dense fractions only 2 to 10% of the cells morphologically resembled lymphocytes. These results indicated that some of the cells classified as lymphocytes by morphological criteria were probably monocytes or macrophage. Triplicate cultures from each one of the pools were incubated with or without 100 ,sg of BCG per ml during 24 h at 370C as previously described for unseparated populations. Figure 3 represents the typical results of one of six similar experiments. Damage induced by BCG was observed only in fractions containing cells from the high-density region of the gradient, indicating that the susceptibility to undergo necrosis in vitro is present only in a particular subpopulation of macrophage. An alternative possibility is that the presence of lymphocytes or lymphocyte-like cells or both in these fractions plays a role in the induction of macrophage death. DISCUSSION It should be emphasized that large numbers of macrophage, epithelioid cells, and giant cells
913
are destroyed in apparent synchrony when central necrosis of a tubercle occurs. This event is apparently associated with cell-mediated hypersensitivity reactions and can vary from necrosis of dermal reactions to central necrosis of allergic granulomas. For example, in tuberculosis, caseous necrosis is apparently dependent on systemic cell-mediated hypersensitivity elicited by products of tubercle bacilli. This concept is supported by the observations that spleen explants and peritoneal exudate macrophage from sensitized animals are damaged more extensively when incubated with tubercle bacilli than comparable cell preparations from normal animals (2, 17, 18). In addition, the observations of Yamamura et al. (22) clearly indicate that if tuberculin-sensitive rabbits are desensitized with tuberculin, central necrosis of tubercles does not occur after challenge with tubercle bacilli. The mechanisms by which activated macrophage are destroyed in the center of an allergic granuloma are not known. It has been suggested that the liberation of hydrolytic enzymes and the release of toxic factors are the causes of this necrotic reaction (1, 4, 5, 7, 10). It is possible that toxic products generated by oxidative metabolism of activated macrophage could also be responsible. The role that toxic by-products of the oxidative metabolism play on bacteriolysis and self destruction by phagocytic cells has been extensively studied in polymorphonuclear leukocytes (19, 20). It is of interest that the premature death that phagocytosis induces in this cell population can be prevented by catalase and free-radical scavengers (20). R.L 13630 20
Density 1077
9'M
10
z
2
.080
.060
5600 90 80 70 60 50 40
1066
30 20 I0 13500
1057
70 60 50 40 30 13420
1048
90 so
1040
FIG. 3. Viability of BCG-activated macrophages separated in density gradient. BCG-activated alveolar macrophage were fractionated as described in the text. Cells from each fraction were incubated in the test tube-type assay for 24 h. One of six similar experiments is shown. Numbers in parentheses indicate the percent viability of the macrophages after incubation. R.L, Refractive index.
914
McGEE AND MYRVIK
In this regard, the increased killing potential of activated macrophage against bacteria and tumor cells has been attributed to the generation of peroxides and free radicals upon phagocytosis (3). For instance, activated macrophage triggered pharmacologically with phorbol myristate acetate are able to destroy normal and transformed cells. The mechanism responsible for this cytotoxicity was found to be the release of H202 by the activated macrophage (16). In the present study we have found that the susceptibility of an activated macrophage to self destruction is largely dependent on the phagocytic event. Moreover our data suggest that the by-products of oxidative metabolism probably play a role in the induction of damage. However, the importance of this mechanism is relative, since protection with antioxidants was only partial (30 to 40%); other mechanisms are probably involved. It is also possible that the oxidants are the major cause of damage but they are released at sites inaccessible to the antioxidants; the chemical lesion could be occluded by opposing membranes at the site of cell-to-cell contact and not available to the surrounding medium. This seems unlikely, because doses of antioxidants similar to those used in our experiments effectively inhibited phagocytosis-induced destruction of polymorphonuclear leukocytes and the lysis of tumor cells by phorbol myristate acetatetriggered macrophages (16, 20). The in vitro self destruction of activated macrophage was not characterized by the degree of specificity normally associated with immunological reactions. However, insolubilized tuberculoprotein and BCG were more efficient in causing cell lysis than other particles, based on the amount added. It is noteworthy that zymosan at 200 jig/ml induced cell death equivalent to 100 ,jg of BCG per ml in activated cells. In contrast, this dose of zymosan produced no detectable damage to normal cells, whereas 100 ,ug of BCG per ml was significantly toxic (15 to 35% cell death). The primary toxicity of BCG for normal cells could explain why BCG seemed to express some specificity based on the minimum effective dose. Based on the data obtained, it is not possible to rule out lymphocytes as possible accessory cells to this reaction. For example, the 24-h incubation period with the various particulates could have induced lymphocyte activation and subsequent "over-activation" of macrophage that had ingested the particulates. After separation of the alveolar cells in continuous density gradients, the macrophage subpopulations found to be most susceptible corresponded to the fraction in which lymphocyte-like cells were most numerous. Since morphological criteria were un-
INFECT. IMMUN.
reliable for distinguishing between small numbers of immature monocytes and medium-sized lymphocytes, and functional tests demonstrated a similar proportion of phagocytes in all fractions, the possible role of the lymphocyte in this reaction could not be resolved. It is possible that lymphocytes could induce macrophage damage, either by the release of cytotoxic substances, or by the activation of the macrophage to a point at which phagocytosis could trigger death of the macrophage. This second possibility seems more likely, in view of reports which indicate that macrophage are not destroyed by lymphotoxin (6). Furthermore, no lymphokines have been reported, to our knowledge, that are toxic for macrophage. Phagocytosis-induced cytotoxicity of over-activated macrophage could result from localized damage to lysosomal membranes, perhaps mediated by toxic products produced by oxidative metabolism. This damage in turn would cause liberation of lysosomal enzymes into the cytoplasm and ultimately the medium, resulting in an accelerated destruction of neighboring phagocytic cells. Experiments designed to elucidate the possible role of lymphocytes or their products or both on the phagocytosis-induced macrophage destruction as well as the mechanisms involved in this destruction are in progress in our laboratory. LITERATURE CITED 1. Adams, D. 0. 1976. The granulomatous inflammatory response. Am. J. Pathol. 84:164-190. 2. Aronson, J. D. 1931. The specific cytotoxic action of tuberculin in tissue cluture. J. Exp. Med. 54:387-397. 3. Blanden, R. V., A. J. Hapel, P. C. Doherty, and R. M. Zinkernager. 1976. Lymphocyte-macrophage interactions and macrophage activation in the expression of antimicrobial immunity in vivo, p. 367. In D. S. Nelson (ed.), Immunology of the Macrophage. Academic Press Inc., New York. 4. Boros, D. L. 1978. Granulomatous inflammation. Prog. Allergy 24: 183-267. 5. Dannenberg, A. M., and M. Suzimoto. 1976. Liquefaction of caseous foci in tuberculosis. Am. Rev. Respir. Dis. 113:257-259. 6. David, J. R., and R. R. David. 1972. Cellular hypersensitivity and immunity. Prog. Allergy 16:300-449. 7. Davies, P., and A. C. Allison. 1976. Secretion of macrophage enzymes in relation to the pathogenesis of chronic inflammation, p. 427. In D. S. Nelson (ed.), Immunobiology of the macrophage. Academic Press Inc., New York. 8. Hayry, P., and L. Anderson. 1976. Fractionation of immunocompetent cells with different physical properties. Scand. J. Immunol. (Suppl. 5) 5:3144. 9. Malawista, S. E., J. B. L. Gee, and R. C. Bersch. 1971. Cytochalasin B reversibly inhibits phagocytosis: functional metabolic and ultrastructural effects in human blood leukocytes and rabbit alveolar macrophages. Yale J. Biol. Med. 44:286-300. 10. Marx, J., and R. Burrel. 1973. Delayed hypersensitivity to beryllium compounds. J. Immunol. 111:590-598. 11. McGee, M. P., Q. N. Myrvik, and E. S. Leake. 1978.
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PHAGOCYTOSIS-INDUCED INJURY OF MACROPHAGES
Organization of allergic granulomas and dependence on insoluble antigen. J. Reticuloendothel. Soc. 24:253-261. 12. McGee, M. P., and Q. N. Myrvik. 1976. Hydrolase levels in necrotizing and non-necrotizing BCG-induced pulmonary granulomas. RES J. Reticuloendothel. Soc. 20: 187-195. 13. Mustafa, M. G., and D. F. Tierney. 1978. Biochemical and metabolic changes in lung with oxygen, ozone and nitrogen dioxide toxicity. Am. Rev. Respir. Dis. 118: 1061-1090. 14. Myrvik, Q. N., E. S. Leake, and B. Fariss. 1961. Studies on pulmonary alveolar macrophages from the normal rabbit: a technique to procure them in a high state of purity. J. Immunol. 86:128-132. 15. Myrvik, Q. N., L. A. Kohlweiss, and D. Harpold. 1975. Pathological potential of exaggerated immunological reactions, p. 227-235. In D. Schlessinger (ed.), Microbiology-1975. American Society for Microbiology, Washington, D.C. 16. Nathan, C. F., L. H. Brukner, S. C. Silverstein, and Z. A. Cohn. 1978. Extracellular cytolysis by activated macrophages and granulocytes. II. Hydrogen peroxide as a mediator of cytotoxicity. J. Exp. Med. 149:100-113.
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17. Preston, M. P., D. Hart, and I. N. Brown. 1977. Immunologically mediated macrophage aggregation in monolayers of peritoneal cells from BCG sensitized mice. Immunology 32:33-41. 18. Rich, A. R., and M. R. Lewis. 1972. Mechanics of allergy in tuberculosis. Proc. Soc. Exp. Biol. Med. 25:596-598. 19. Root, R. K., and J. A. Metcalf. 1976. Superoxide and hydrogen peroxide formation of human granulocytes: inter-relationships and activation mechanisms. Studies with normal and cytochalasin B treated cells, p. 185. In F. Ross, P. Patriarcz, and D. Romeo (ed.), Movement, metabolism and bactericidal mechanisms of phagocytes. Piccin Medical Books, London. 20. Salin, M. L., and J. M. McCord. 1975. Free radical and inflammation. Protection of phagocytosing leukocytes by superoxide dismutase. J. Clin. Invest. 56:1319-1323. 21. Taplits, M., and Q. N. Myrvik. 1977. Host resistance to the LSV strain of Francisella tularensis in BCG vaccinated mice. J. Reticuloendothel. Soc. 24:297-308. 22. Yamamura, Y., Y. Ogawa, H. Maeda, and Y. Yamamura. 1976. Prevention of tuberculosis cavity formation by desensitization with tuberculin-active peptide. Am. Rev. Respir. Dis. 109:596-601.
