Vol. 17, No. 1 Printed in U.S.A.
INFZCTION AND IMMUNITY, JUlY 1977, p. 161-166 Copyright © 1977 American Society for Microbiology
Effects of Polynucleotides and Levamisole on Alveolar Macrophage Morphology and Receptor Activity C. C. DAUGHADAY,' M. E. SCHMIDT,2
AND
STEVEN D. DOUGLAS*
Department of Medicine and Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received for publication 18 January 1977
Incubation of rabbit pulmonary alveolar macrophages in vitro with polyinosinic acid-polycytidylic acid [poly(I-C)] or levamisole results in enhanced immunoglobulin G receptor activity in comparison to untreated cells. Electron microscopy of cells treated with levamisole or poly(I-C) revealed mitochondrial swelling and cytoplasmic vacuolization. The modulation of receptor activity by these agents suggests that their immunopotentiating effects are due to direct simulation of the mononuclear phagocyte system. Lavaged alveolar macrophages have the capacity to change membrane function in vitro, and these cells provide a convenient system for studying agents with potential effects on macrophages.
Alterations in macrophage structure and function are a major aspect of modified immunological capacity in animal systems and many clinical disease states. The characteristics of the "activated" macrophage have been extensively examined; mouse peritoneal macrophages stimulated by a variety of substances show enhanced adherence, phagocytosis, and killing capacity for both microbes and tumor cells (10, 21). Peripheral blood monocytes from patients with sarcoidosis and other granulomatous disorders exhibit increased immunoprotein receptor activity in comparison to monocytes from normal subjects (4). Pulmonary alveolar macrophages (PAMs) from rabbits given repeated injections of Freund adjuvant display increased numbers and avidity of binding sites for immunoglobulin G (IgG) in comparison to cells from minimally stimulated animals (1). Macrophages recovered from the lungs of rabbits after respiratory infection with either Listeria monocytogenes or Streptococcus pneumoniae were found to be "activated," as manifested by greater adherence and bacterial activity than cells from uninfected animals (9). One question that remains unanswered by these studies, however, is the ability of a population of resting, unstimulated PAMs to respond directly to stimulation, rather than manifesting a change in function as a result of entry into the alveoli of younger cells that have been activated elsewhere. In the present studies, we have examined the I Present address: Department of Hematology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20012. 2 Present address: Department of Medicine, University of Marburg, Marburg, West Germany.
in vitro effects of double-stranded polyinosinic acid-polycytidylic acid [poly(I-C)] and levamisole on rabbit PAMs. We have recently reported that these immunostimulatory agents enhance human monocyte immunoprotein receptor activity in vitro (19, 20). We have now investigated the capacity of these agents to modulate IgG receptor function of rabbit PAMs to assess whether a more mature type of mononuclear phagocyte is able to undergo pharmacologically induced alterations in function. MATERIALS AND METHODS PAMs. Macrophages were obtained by tracheal lavage of whole lungs of outbred New Zealand rabbits as previously described (2). After washing the cells, they were suspended in RPMI 1640 supplemented with glutamine, penicillin-streptomycin, and 30% heat-inactivated fetal calf serum (mean yield: 105.8 x 106 cells, 94% PAM, 4% lymphocytes, and 2% polymorphonuclear leukocytes). Morphological studies. Cells were incubated in the presence of 100 ±g of levamisole (Janssen R & D, Inc., New Brunswick, N.J.), 100 jig of poly(I-C), or 100 j,g of poly(C) (P-L Biochemicals) per ml for 8 to 12 h at 37°C at 5 to 10 rpm on a Rotorack (Fisher Scientific Co.). Control cells were incubated in medium alone. After incubation with the drugs,
antibody- or antibody-complement-coated sheep erythrocytes (SRBC) and fresh medium were added to cell samples and incubated for 1 to 2 h before fixation. Electron microscopy. Cells were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate and postfixed in 1.0% buffered osmium tetroxide. The preparations were stained en bloc with 1.0% aqueous uranyl acetate, rapidly dehydrated, and embedded in Epon. Thin sections, prepared by LKB Ultratome III, were doubly stained with uranyl acetate and lead citrate and examined in a Siemens 102 electron microscope. 161
I
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DAUGHADAY, SCHMIDT, AND DOUGLAS
Immunoglobulin receptor assays. Two million macrophages in 1.5 ml of medium were layered onto cover slips in Leighton tubes. Because of the high purity of the cell population, no preincubation for attachment to the cover slip was necessary, and the cells were incubated with levamisole (100 ug/ml), poly(I-C) (100 ,ug/ml), or poly(C) (100 ,ug/ml) for 4, 8, 12, and 24 h. In preliminary studies, levamisole, poly(I-C), or poly(C) was added to macrophages at concentrations of 10 to 500 ,ug/ml; the optimal effects were observed at 100 ,mg/ml. The controls were incubated with media alone. At each time point the cover slips were washed twice to remove serum, drugs, and nonadherent cells, and 2 x 108 to 4 x 108 SRBC coated with subagglutinating dilutions of 7S rabbit anti-Forssman antibody (Cordis, Miami, Fla.; 1:400-1:3,200) were added together with fresh Hanks balanced salt solution or RPMI. The coated erythrocytes (EA) were prepared at several dilutions of antibody and in some experiments were prelabeled with 51Cr, according to the methods previously described (2). After incubation with SRBC for 1 h at 370C, the cover slips were washed several times with Hanks balanced salt solution, fixed in methanol, counted in a Packard (model 5120) gamma counter, stained, and assessed morphologically. Two hundred cells on each cover slip were scored for the percentage of rosette formation (three or more SRBC attached to the cell surface), phagocytosis (engulfment of SRBC), and "combined" (both binding and phagocytosis within the same cell). The "total" receptor activity is the total percentage ofthe three types of interaction.
RESULTS Morphology. Electron microscopic observations revealed mitochondrial swelling (Fig. 3) and cytoplasmic vacuolization (Fig. 4) in PAMs incubated with levamisole. Similar effects were seen after treatment with poly(I-C) (Fig. 2), but not in control cells incubated with medium alone (Fig. 1). Changes in mitochondrial architecture consisted of swelling, with widening of the intercristal spaces and loss of cristae (Fig. 1-3, insets). Polynucleotides. Incubation of macrophage monolayers for 4 h in the presence of poly(I-C) resulted in an increase in total IgG receptor activity (as determined by percentage of reactive cells) for EA 1:800, from 81.1 to 88.9% (Table 1). A greater increase occurred in the percentage of cells demonstrating the combined type of interaction, from 26.1 to 51.3%. When EA 1:1,600 was used, small increases in both the total activity and the combined type of interaction were observed. At greater antibody dilutions, no consistent differences were observed at 4 h, but at 8 and 24 h small increases in the total reactive cells occurred with EA 1:3,200 (data not shown). An unexpected finding was that the single-stranded homopolymer, poly(C), also enhanced receptor activity. Cells
INFECT. IMMUN.
incubated for 4 h with poly(C) at the same concentration as poly(I-C) showed about a twofold increase in the percentage of cells that demonstrated the combined type of interaction with EA 1:800 compared to untreated (but incubated) cells [control = 31.5%, poly(C) = 59.3%, P < 0.025]. Control cells generally showed greater receptor activity after 24-h culture in comparison to 4-h control cultures, as detected by more combined-type interaction with EA 1:800 (4 h, 26.1%; 24 h, 65.5%). Thus, it was difficult to distinguish between treated and control cells at 24 h. Levamisole. The most consistent evidence of enhanced receptor activity after incubation with levamisole was seen in the group incubated for 4 h and assayed with EA 1:800 (Table 2). There were increases in the mean numbers of total reactive cells from 56.8 to 82.4%. A significant (P < 0.025) increase in combinedtype interaction occurred (control = 12.4%, levamisole = 38.1%) as well as in the percentage of cells showing SRBC attachment (percent combined + percent rosettes: control = 25.6%, levamisole = 55.9%). Isotopic assay. The assay utilizing 5'Cr-labeled SRBC did not detect changes in receptor activity unless the specific activity of the SRBC was very high. In a representative experiment the counts per minute and the corresponding total receptor activity by morphological assessment for cover slips incubated with ilCr-labeled EA 1:800 are as follows: levamisole, 83.7 cpm, 80.5%; control, 46.5 cpm, 28.5%. DISCUSSION Alveolar macrophages obtained by tracheal lavage have long been utilized in morphological and functional studies; however, there is some doubt as to whether the lavaged cells are representative of the total population of PAMs or are capable of "activation" (12). It can be argued that these cells are senescent, and the lung is, in fact, the graveyard for mononuclear phagocytes. This contention is supported by the difficulty with which PAMs are maintained in tissue culture and their low rate of cell division. Nevertheless, a number of studies have characterized the capacity of PAMs to phagocytize and kill bacteria and carry out other mononuclear phagocyte functions. Evidence for enhanced functional capacity has been derived from studies in which animals have been pretreated with Freund adjuvant or infected with BCG or listeria. Lavaged cells from such animals have been shown to have increased content of lysosomal enzymes (22) and increased numbers and affinity of IgG receptor sites (1) in comparison to untreated or minimally stimulated controls.
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FIG. 1. Control rabbit PAM incubated for 8 h in medium alone. Many lysosomes and phagolysosomes are present. The mitochondria are oval and have a normal complement of cristae. xl1,750. Inset, x32,800. FIG. 2. Rabbit PAM incubated with poly(I-C) for 8 h, showing mitochondrial swelling with loss of cristae. xl ,750. Inset, x32,800. 163
164
DAUGHADAY, SCHMIDT, AND DOUGLAS
INFECT. IMMUN.
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INFZCTION AND IMMUNITY, JUlY 1977, p. 161-166 Copyright © 1977 American Society for Microbiology
Effects of Polynucleotides and Levamisole on Alveolar Macrophage Morphology and Receptor Activity C. C. DAUGHADAY,' M. E. SCHMIDT,2
AND
STEVEN D. DOUGLAS*
Department of Medicine and Microbiology, University of Minnesota Medical School, Minneapolis, Minnesota 55455 Received for publication 18 January 1977
Incubation of rabbit pulmonary alveolar macrophages in vitro with polyinosinic acid-polycytidylic acid [poly(I-C)] or levamisole results in enhanced immunoglobulin G receptor activity in comparison to untreated cells. Electron microscopy of cells treated with levamisole or poly(I-C) revealed mitochondrial swelling and cytoplasmic vacuolization. The modulation of receptor activity by these agents suggests that their immunopotentiating effects are due to direct simulation of the mononuclear phagocyte system. Lavaged alveolar macrophages have the capacity to change membrane function in vitro, and these cells provide a convenient system for studying agents with potential effects on macrophages.
Alterations in macrophage structure and function are a major aspect of modified immunological capacity in animal systems and many clinical disease states. The characteristics of the "activated" macrophage have been extensively examined; mouse peritoneal macrophages stimulated by a variety of substances show enhanced adherence, phagocytosis, and killing capacity for both microbes and tumor cells (10, 21). Peripheral blood monocytes from patients with sarcoidosis and other granulomatous disorders exhibit increased immunoprotein receptor activity in comparison to monocytes from normal subjects (4). Pulmonary alveolar macrophages (PAMs) from rabbits given repeated injections of Freund adjuvant display increased numbers and avidity of binding sites for immunoglobulin G (IgG) in comparison to cells from minimally stimulated animals (1). Macrophages recovered from the lungs of rabbits after respiratory infection with either Listeria monocytogenes or Streptococcus pneumoniae were found to be "activated," as manifested by greater adherence and bacterial activity than cells from uninfected animals (9). One question that remains unanswered by these studies, however, is the ability of a population of resting, unstimulated PAMs to respond directly to stimulation, rather than manifesting a change in function as a result of entry into the alveoli of younger cells that have been activated elsewhere. In the present studies, we have examined the I Present address: Department of Hematology, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC 20012. 2 Present address: Department of Medicine, University of Marburg, Marburg, West Germany.
in vitro effects of double-stranded polyinosinic acid-polycytidylic acid [poly(I-C)] and levamisole on rabbit PAMs. We have recently reported that these immunostimulatory agents enhance human monocyte immunoprotein receptor activity in vitro (19, 20). We have now investigated the capacity of these agents to modulate IgG receptor function of rabbit PAMs to assess whether a more mature type of mononuclear phagocyte is able to undergo pharmacologically induced alterations in function. MATERIALS AND METHODS PAMs. Macrophages were obtained by tracheal lavage of whole lungs of outbred New Zealand rabbits as previously described (2). After washing the cells, they were suspended in RPMI 1640 supplemented with glutamine, penicillin-streptomycin, and 30% heat-inactivated fetal calf serum (mean yield: 105.8 x 106 cells, 94% PAM, 4% lymphocytes, and 2% polymorphonuclear leukocytes). Morphological studies. Cells were incubated in the presence of 100 ±g of levamisole (Janssen R & D, Inc., New Brunswick, N.J.), 100 jig of poly(I-C), or 100 j,g of poly(C) (P-L Biochemicals) per ml for 8 to 12 h at 37°C at 5 to 10 rpm on a Rotorack (Fisher Scientific Co.). Control cells were incubated in medium alone. After incubation with the drugs,
antibody- or antibody-complement-coated sheep erythrocytes (SRBC) and fresh medium were added to cell samples and incubated for 1 to 2 h before fixation. Electron microscopy. Cells were fixed in 1.5% glutaraldehyde in 0.1 M sodium cacodylate and postfixed in 1.0% buffered osmium tetroxide. The preparations were stained en bloc with 1.0% aqueous uranyl acetate, rapidly dehydrated, and embedded in Epon. Thin sections, prepared by LKB Ultratome III, were doubly stained with uranyl acetate and lead citrate and examined in a Siemens 102 electron microscope. 161
I
162
DAUGHADAY, SCHMIDT, AND DOUGLAS
Immunoglobulin receptor assays. Two million macrophages in 1.5 ml of medium were layered onto cover slips in Leighton tubes. Because of the high purity of the cell population, no preincubation for attachment to the cover slip was necessary, and the cells were incubated with levamisole (100 ug/ml), poly(I-C) (100 ,ug/ml), or poly(C) (100 ,ug/ml) for 4, 8, 12, and 24 h. In preliminary studies, levamisole, poly(I-C), or poly(C) was added to macrophages at concentrations of 10 to 500 ,ug/ml; the optimal effects were observed at 100 ,mg/ml. The controls were incubated with media alone. At each time point the cover slips were washed twice to remove serum, drugs, and nonadherent cells, and 2 x 108 to 4 x 108 SRBC coated with subagglutinating dilutions of 7S rabbit anti-Forssman antibody (Cordis, Miami, Fla.; 1:400-1:3,200) were added together with fresh Hanks balanced salt solution or RPMI. The coated erythrocytes (EA) were prepared at several dilutions of antibody and in some experiments were prelabeled with 51Cr, according to the methods previously described (2). After incubation with SRBC for 1 h at 370C, the cover slips were washed several times with Hanks balanced salt solution, fixed in methanol, counted in a Packard (model 5120) gamma counter, stained, and assessed morphologically. Two hundred cells on each cover slip were scored for the percentage of rosette formation (three or more SRBC attached to the cell surface), phagocytosis (engulfment of SRBC), and "combined" (both binding and phagocytosis within the same cell). The "total" receptor activity is the total percentage ofthe three types of interaction.
RESULTS Morphology. Electron microscopic observations revealed mitochondrial swelling (Fig. 3) and cytoplasmic vacuolization (Fig. 4) in PAMs incubated with levamisole. Similar effects were seen after treatment with poly(I-C) (Fig. 2), but not in control cells incubated with medium alone (Fig. 1). Changes in mitochondrial architecture consisted of swelling, with widening of the intercristal spaces and loss of cristae (Fig. 1-3, insets). Polynucleotides. Incubation of macrophage monolayers for 4 h in the presence of poly(I-C) resulted in an increase in total IgG receptor activity (as determined by percentage of reactive cells) for EA 1:800, from 81.1 to 88.9% (Table 1). A greater increase occurred in the percentage of cells demonstrating the combined type of interaction, from 26.1 to 51.3%. When EA 1:1,600 was used, small increases in both the total activity and the combined type of interaction were observed. At greater antibody dilutions, no consistent differences were observed at 4 h, but at 8 and 24 h small increases in the total reactive cells occurred with EA 1:3,200 (data not shown). An unexpected finding was that the single-stranded homopolymer, poly(C), also enhanced receptor activity. Cells
INFECT. IMMUN.
incubated for 4 h with poly(C) at the same concentration as poly(I-C) showed about a twofold increase in the percentage of cells that demonstrated the combined type of interaction with EA 1:800 compared to untreated (but incubated) cells [control = 31.5%, poly(C) = 59.3%, P < 0.025]. Control cells generally showed greater receptor activity after 24-h culture in comparison to 4-h control cultures, as detected by more combined-type interaction with EA 1:800 (4 h, 26.1%; 24 h, 65.5%). Thus, it was difficult to distinguish between treated and control cells at 24 h. Levamisole. The most consistent evidence of enhanced receptor activity after incubation with levamisole was seen in the group incubated for 4 h and assayed with EA 1:800 (Table 2). There were increases in the mean numbers of total reactive cells from 56.8 to 82.4%. A significant (P < 0.025) increase in combinedtype interaction occurred (control = 12.4%, levamisole = 38.1%) as well as in the percentage of cells showing SRBC attachment (percent combined + percent rosettes: control = 25.6%, levamisole = 55.9%). Isotopic assay. The assay utilizing 5'Cr-labeled SRBC did not detect changes in receptor activity unless the specific activity of the SRBC was very high. In a representative experiment the counts per minute and the corresponding total receptor activity by morphological assessment for cover slips incubated with ilCr-labeled EA 1:800 are as follows: levamisole, 83.7 cpm, 80.5%; control, 46.5 cpm, 28.5%. DISCUSSION Alveolar macrophages obtained by tracheal lavage have long been utilized in morphological and functional studies; however, there is some doubt as to whether the lavaged cells are representative of the total population of PAMs or are capable of "activation" (12). It can be argued that these cells are senescent, and the lung is, in fact, the graveyard for mononuclear phagocytes. This contention is supported by the difficulty with which PAMs are maintained in tissue culture and their low rate of cell division. Nevertheless, a number of studies have characterized the capacity of PAMs to phagocytize and kill bacteria and carry out other mononuclear phagocyte functions. Evidence for enhanced functional capacity has been derived from studies in which animals have been pretreated with Freund adjuvant or infected with BCG or listeria. Lavaged cells from such animals have been shown to have increased content of lysosomal enzymes (22) and increased numbers and affinity of IgG receptor sites (1) in comparison to untreated or minimally stimulated controls.
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FIG. 1. Control rabbit PAM incubated for 8 h in medium alone. Many lysosomes and phagolysosomes are present. The mitochondria are oval and have a normal complement of cristae. xl1,750. Inset, x32,800. FIG. 2. Rabbit PAM incubated with poly(I-C) for 8 h, showing mitochondrial swelling with loss of cristae. xl ,750. Inset, x32,800. 163
164
DAUGHADAY, SCHMIDT, AND DOUGLAS
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