Alteration of the Structure and Function of Guinea Pig Peritoneal Macrophages by a Soybean Oil Emulsion Robert C. Strunk, MD, Claire M. Payne, PhD, Ray B. Nagle, MD, and Kathleen Kunke, BS
Studies in humans who have received Intralipid (IL) have demonstrated the presence of a fat pigment and fat droplets in reticuloendothelial phagocytic cells. Clinical data and in vitro studies suggest that these cells do not function normally. We have studied the effect of IL on the morphology and function of guinea pig peritoneal macrophages in vitro. Starch-induced macrophages were exposed to IL for up to 48 hours. Ingestion of increasing amounts of IL over the 48-hour period was confirmed by transmission electron microscopy and by oil red 0 stain. The uptake of the IL was associated with marked morphologic changes characterized by a decreased ability of the cells to spread and by a decrease in the number and degree of complexity of the membrane ruffles. The ingestion of IL also resulted in decreased capacity of the cells to associate with latex beads (5.7 ,u in diameter) or Candida albicans and decreased capacity to adhere to and ingest sheep erythrocytes coated with IgG. After ingestion of latex beads 0.46 ,u in diameter, which are similar in size to IL particles, macrophages had normal morphology and function, indicating that neither the morphologic nor functional abnormalities were due to a nonspecific effect of ingestion of small particles. Alterations of human reticuloendothelial macrophage function similar to the effects observed here could compromise host defense against infection. (Am J Pathol 96:753-770, 1979)
PARENTERAL HYPERALIMENTATION has been widely used in the treatment of adults and children with gastrointestinal abnormalities and malnutrition.' Intralipid (IL), a 10% emulsion of soybean oil stabilized with 1.2% egg yolk phospholipid, is now being used as a major constituent of hyperalimentation fluids."2 Since the introduction of IL in the early 1960s, numerous animal studies have demonstrated deposition of a pigmented material containing unsaturated fatty acids and iron in reticuloendothelial cells, especially of the liver and spleen.3 More recently, studies in humans have also demonstrated the presence of this fat pigment and also fat droplets in reticuloendothelial cells in the liver, spleen, and lung.4-" The fat pigment was seen in liver specimens obtained as long as 5 years after the last infusion of IL.5 Van Haelst and Sengers noted large, solitary, or conglomerated fat droplets within lipophagosomes of Kupffer cells on transmission electron microscopy of a liver biopsy specimen from a 5-week-old child who had received IL for 12 days.6 They speculated that From the Department of Pediatrics, National Jewish Hospital and Research Center, and The University of Colorado Medical School, Denver, and the Department of Pathology, University of Arizona Health Sciences Center, Tucson, Arizona. Supported in part by Grant Al 12970 from the National Institutes of Health. Accepted for publication May 1, 1979. Address reprint requests to Robert C. Strunk, MD, Department of Pediatrics, National Jewish Hospital and Research Center, 3800 East Colfax Avenue, Denver, CO 80206. 0002-9440/79/0910-0753$01 .00 753 © American Association of Pathologists
754
STRUNK ET AL
American Journal of Pathology
the accumulation of the fat might inhibit clearance of particulate elements by these cells. In a previous study on the effects of IL on guinea pig peritoneal macrophages in vitro, We noted that synthesis of the second (C2) and fourth (C4) components of complement was decreased. The purpose of this study is to define the morphologic changes seen after exposure to IL and to correlate morphologic and functional changes. Materials and Methods Materials
Medium 199, penicillin, streptomycin, and fetal calf serum were obtained from Grand Island Biological Company (Santa Clara, Calif). The tissue culture dishes were from Falcon Plastics, Oxnard, Calif. Commercially available IL (Cutter Laboratories, Inc., Berkeley, Calif) was used. Uniform styrene divinylbenzene (latex) beads, 5.7 ,u in diameter (Dow Diagnostics, Indianapolis, Ind); hydrolyzed starch and polystyrene (latex) beads, 0.46,u in diameter (Sigma Chemical Company, St. Louis, Mo); freshly frozen guinea pig serum (Suburban Serum, Rockville, Md); and sheep erythrocytes (Bolin Laboratories, Phoenix, Ariz) were obtained commercially. A pure culture of Candida albicans was the gift of Dr. Ken Ryan at the University of Arizona. Cell Culture Guinea pig (GP) peritoneal exudate cells were obtained as described by Strunk et al.7 Briefly, young adult Hartley GP were injected with 7 ml of a 3% sterile aqueous solution of starch. Ninety-six hours later the GP were killed and the peritoneal cavities were washed with 100 ml of Medium 199 containing penicillin (100 units/ml) and streptomycin (100 ,ug/ml) (MI99). The exudate cells were washed three times with M199 and plated on 35mm dishes at 2 X 106 cells/dish in M199 containing 10% heat-inactivated fetal calf serum (MI99-FCS). For some experiments, cells were plated on glass coverslips which were placed in the plastic dishes. After an initial 2-hour period to permit attachment of the cells, the plates were washed vigorously three times to remove nonadherent cells. The remaining cells (macrophages) were then fed with M199-FCS alone or M199-FCS containing IL at a final concentration of 9.3 or 37.5 mg/dl. Electron Microscopy
Macrophage morphology was evaluated by scanning and transmission electron microscopy. Cells were harvested at 4, 24, and 48 hours following placement in tissue culture. For evaluation by scannirng electron microscopy, cells were grown on glass coverslips. At appropriate intervals, coverslips were washed twice with M199 and fixed with 3.3% glutaraldehyde in 0.1 M phosphate buffer for 1 hour. The specimens were then washed and stored in 0.1 M phosphate buffer (pH 7.2) containing 10% sucrose. Specimens for transmission electron microscopy were removed from the surface of plastic Petri dishes with a rubber policeman and centrifuged at 11OOg in conical centrifuge tubes for 10 minutes at room temperature. The pelleted cells were fixed with 3.3% glutaraldehyde in 0.1 M phosphate buffer. Specimens prepared for scanning electron microscopy were postfixed in 1% osmium tetroxide buffered with s-collidine, dehydrated in a graded series of alcohol, and criticalpoint-dried with liquid CO2. The specimens were attached to mounting stubs with double sticky tape and coated with gold-palladium in a Hummer sputtering device. All specimens were examined in a ETEC scanning electron microscope. Random fields were photographed and the numbers of flattened and rounded cells were determined.
Vol. 96, No. 3 September 1979
ALTERATION OF PERITONEAL MACROPHAGES BY IL
755
Specimens for transmission electron microscopy were postfixed with 1 % osmium tetroxide in 0.1 M phosphate buffer, dehydrated in a graded series of alcohol, and embedded in Spurr's epoxy.8 One-micron-thick sections were stained with toluidine blue and selected areas were thin sectioned on a Sorvall MT2-B ultra-microtome. Twenty random cells from each culture were photographed and examined for the presence of IL. Association of Macrophages With Particles
Association of macrophages with latex beads or yeast was studied by a modification of the method of Schmid and Brune.9 After the specified times the cells were washed twice with M199 to remove nonadherent cells. Two milliliters of M199 containing 3 drops of a 10% suspension of latex beads 5.7 in diameter/10 ml or 1 X 106 yeast/ml (approximately 25 to 50 latex beads or 2 to 3 yeast per adherent cell) were added to each dish. Fresh GP serum (final concentration, 5%) was added as a source of opsonins. The dishes were then incubated for 30 minutes (latex) or 60 minutes (yeast) at 37 C without shaking before the nonadherent particles were removed by washing twice with Dulbecco's phosphate-buffered saline. The adherent cells were scraped from the plastic dish with a rubber policeman. The cell suspension was placed in a hemocytometer and the percentage of cells which were associated with one or more latex beads or yeast was determined. With the latex beads and yeast, ingestion and adherence to the cell membrane were not differentiated. Additional experiments were performed with sheep erythrocytes coated with IgG antibody (IgG-SRBC) to determine the percentage of cells which had ingested particles, as opposed to cells which only had particles adherent to the cell membrane. For these experiments, the method of Newman et al 10 was used.
Results Effect of IL on Macrophage Morphology
Scanning electron microscopy of control cells in culture 4 hours revealed that most of the cells were flattened (Table 1). The rounded cells had a ruffled membrane typical of monocytes and macrophages. The elongated flattened cells had delicate thin veils of cytoplasm spreading over the coverglass surface with smaller filamentous cytoplasmic extensions (Figure 1). Following 4 hours of exposure to IL, there was an increase in the percentage of rounded cells (Table 1). The surface morphology, however, appeared unchanged at this time period (Figure 1). By transmission electron microscopy, approximately 45% of the macrophage cross-sections showed intracellular accumulation of a moderately electrondense lipid-like material which had an electron microscopic appearance similar to IL alone. There was no evidence of intracellular digestion of the lipid within lipophagolysosomes (Figure 2). There were no other ultrastructural differences noted between the IL and control cells. At 24 hours, scanning electron microscopy of control cells revealed that fewer cells were flattened than at 4 hours (Table 1), but the morphology of these cells was similar to that observed at 4 hours. However, the cells exposed to IL showed striking changes. Almost all cells were rounded (Table 1). In addition, there was a striking decrease in the number and degree of complexity of the membrane ruffles (Figure 3). Many of the cells
American Journal of Pathology
STRUNK ET AL
756
Table 1-Effect of Intralipid (IL) on the Morphology of GP Peritoneal Macrophages In Vitro*
Presence of ruffled surface by scanning electron microscopy
Time (hr)t
Control
IL
4 24 48
+ + +
+ -
% Transmission electron microscopy % Flattened macrophages on scanning electron microscopy cross-sections with IL present IL Control 79 25 30
55 6 5
45 80 100
* Results in each of the assays shown were obtained at the stated time, after the addition of M199-FCS alone (control) or M199-FCS containing IL (37.5 mg/dl). t Time in culture in control or IL-containing medium
appeared smooth or had blunted, bossilated surfaces. By transmission electron microscopy, approximately 80% of the macrophage cross-sections showed an accumulation of the lipid material. Those macrophage crosssections which contained visible lipid showed a greater accumulation of the material than at 4 hours (Figure 4). Again there was no evidence of lipid digestion within lipophagolysosomes. At 48 hours there was a decrease in the total number of both control and IL cells per scanning field. The control cells at 48 hours were similar to controls at 24 hours, both in percentage of flattened cells and in surface detail (Table 1) (48-hour cells not shown). Cells exposed to IL for 48 hours showed the same striking surface abnormalities seen with IL-treated cells at 24 hours. By transmission electron microscopy, 100% of the IL-treated macrophage cross-sections revealed a large accumulation of lipid material (Table 1). As in the 4-hour and 24-hour cultures, all the lipid material had a moderately electron-dense appearance and did not show any accompanying myelin figures indicative of intracellular digestion. Effect of IL on the Phagocytic Abilities of the Macrophages
The ability of control cells to associate with opsonized latex beads remained relatively constant through the 48-hour observation period (Table 2). Incubation of the cells with IL (37.5 mg/dl) for 4 hours did not affect the capacity of the cells to associate with the latex. However, after 24 hours of incubation with the IL, this capacity was significantly decreased. Incubation of the cells for 24 hours in a lower concentration of IL (9.3 mg/dl) also affected the cells, but the effect was not as marked: the average number of latex particles per cell (calculation described in Table 2) was decreased (2.79 ± 0.62 for IL cells compared with 3.13 ± 0.44 for
ALTERATION OF PERITONEAL MACROPHAGES BY IL
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STRUNK ET AL
American Journal of Pathology
control cells [P < 0.05]) but the percentage of total cells associating with latex was not affected (71.1 ± 20.4 for IL cells compared with 80.4 ± 15.7 for control cells [P > 0.1]). Cells incubated with IL (37.5 mg/dl) for 48 hours had a capacity to associate with latex similar to the cells incubated with IL only 24 hours (Table 1), indicating that maximal effects were reached by 24 hours. Similar experiments were performed with cells on coverslips to examine the possibility that the results of the above experiments may have been influenced by damage to either IL or control cells during the process of scraping from the plate. Unopsonized latex beads were used in these experiments to avoid obscuring the cells by generalized nonspecific adherence of the opsonized beads to the coverslip. The differences between IL (37.5 mg/dl) and control cells in four separate experiments were similar to those in Table 2 or were even more striking. The percentage of control cells associated with latex was 78.0 ± 11.8 (with 2.67 ± 0.57 beads/cell) compared with 22.5 ± 9.6 (with 1.52 ± 0.17 beads/cell) for IL cells (results for IL cells significantly less than control [P < 0.01] for both determinations). The results using the yeast particles were similar to the latex results (Table 3). The capacity of the control cells remained relatively constant from 4 to 24 hours (the difference between control values at 4 and 24 hours was not significant [P > 0.4]). Incubation of the cells for 4 hours with IL, either 9.3 or 37.5 mg/dl, did not affect this capacity, but both concentrations of IL produced significant effects on the macrophage function after 24 hours. The percentage of macrophages associated with IgG-SRBC was determined after washing the cells with medium to remove nonadherent particles or after washing with water to lyse adherent but noningested particles (Table 4). For both control and IL cells, the percentage of cells adherent to IgG-SRBC was significantly increased compared with the percentage of cells which had ingested IgG-SRBC (Table 4). For the control cells the actual differences were small and almost all the cells had ingested erythrocytes. However, for the IL cells, the percentage of cells adherent to IgG-SRBC was much different than the percentage of cells ingesting the IgG-SRBC. As with the latex and yeast particles, the overall capacity of the IL cells to associate with the IgG-SRBC, either adherence or ingestion, was significantly reduced compared with the control cells (P < 0.001). The reduced capacity of the IL cells is also evident when the number of particles per cell is calculated. Most of the control cells had either ingested or adhered to more than three IgG-SRBC (92, or 99%, of
Vol. 96, No. 3 September 1979
ALTERATION OF PERITONEAL MACROPHAGES BY IL
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Studies in humans who have received Intralipid (IL) have demonstrated the presence of a fat pigment and fat droplets in reticuloendothelial phagocytic cells. Clinical data and in vitro studies suggest that these cells do not function normally. We have studied the effect of IL on the morphology and function of guinea pig peritoneal macrophages in vitro. Starch-induced macrophages were exposed to IL for up to 48 hours. Ingestion of increasing amounts of IL over the 48-hour period was confirmed by transmission electron microscopy and by oil red 0 stain. The uptake of the IL was associated with marked morphologic changes characterized by a decreased ability of the cells to spread and by a decrease in the number and degree of complexity of the membrane ruffles. The ingestion of IL also resulted in decreased capacity of the cells to associate with latex beads (5.7 ,u in diameter) or Candida albicans and decreased capacity to adhere to and ingest sheep erythrocytes coated with IgG. After ingestion of latex beads 0.46 ,u in diameter, which are similar in size to IL particles, macrophages had normal morphology and function, indicating that neither the morphologic nor functional abnormalities were due to a nonspecific effect of ingestion of small particles. Alterations of human reticuloendothelial macrophage function similar to the effects observed here could compromise host defense against infection. (Am J Pathol 96:753-770, 1979)
PARENTERAL HYPERALIMENTATION has been widely used in the treatment of adults and children with gastrointestinal abnormalities and malnutrition.' Intralipid (IL), a 10% emulsion of soybean oil stabilized with 1.2% egg yolk phospholipid, is now being used as a major constituent of hyperalimentation fluids."2 Since the introduction of IL in the early 1960s, numerous animal studies have demonstrated deposition of a pigmented material containing unsaturated fatty acids and iron in reticuloendothelial cells, especially of the liver and spleen.3 More recently, studies in humans have also demonstrated the presence of this fat pigment and also fat droplets in reticuloendothelial cells in the liver, spleen, and lung.4-" The fat pigment was seen in liver specimens obtained as long as 5 years after the last infusion of IL.5 Van Haelst and Sengers noted large, solitary, or conglomerated fat droplets within lipophagosomes of Kupffer cells on transmission electron microscopy of a liver biopsy specimen from a 5-week-old child who had received IL for 12 days.6 They speculated that From the Department of Pediatrics, National Jewish Hospital and Research Center, and The University of Colorado Medical School, Denver, and the Department of Pathology, University of Arizona Health Sciences Center, Tucson, Arizona. Supported in part by Grant Al 12970 from the National Institutes of Health. Accepted for publication May 1, 1979. Address reprint requests to Robert C. Strunk, MD, Department of Pediatrics, National Jewish Hospital and Research Center, 3800 East Colfax Avenue, Denver, CO 80206. 0002-9440/79/0910-0753$01 .00 753 © American Association of Pathologists
754
STRUNK ET AL
American Journal of Pathology
the accumulation of the fat might inhibit clearance of particulate elements by these cells. In a previous study on the effects of IL on guinea pig peritoneal macrophages in vitro, We noted that synthesis of the second (C2) and fourth (C4) components of complement was decreased. The purpose of this study is to define the morphologic changes seen after exposure to IL and to correlate morphologic and functional changes. Materials and Methods Materials
Medium 199, penicillin, streptomycin, and fetal calf serum were obtained from Grand Island Biological Company (Santa Clara, Calif). The tissue culture dishes were from Falcon Plastics, Oxnard, Calif. Commercially available IL (Cutter Laboratories, Inc., Berkeley, Calif) was used. Uniform styrene divinylbenzene (latex) beads, 5.7 ,u in diameter (Dow Diagnostics, Indianapolis, Ind); hydrolyzed starch and polystyrene (latex) beads, 0.46,u in diameter (Sigma Chemical Company, St. Louis, Mo); freshly frozen guinea pig serum (Suburban Serum, Rockville, Md); and sheep erythrocytes (Bolin Laboratories, Phoenix, Ariz) were obtained commercially. A pure culture of Candida albicans was the gift of Dr. Ken Ryan at the University of Arizona. Cell Culture Guinea pig (GP) peritoneal exudate cells were obtained as described by Strunk et al.7 Briefly, young adult Hartley GP were injected with 7 ml of a 3% sterile aqueous solution of starch. Ninety-six hours later the GP were killed and the peritoneal cavities were washed with 100 ml of Medium 199 containing penicillin (100 units/ml) and streptomycin (100 ,ug/ml) (MI99). The exudate cells were washed three times with M199 and plated on 35mm dishes at 2 X 106 cells/dish in M199 containing 10% heat-inactivated fetal calf serum (MI99-FCS). For some experiments, cells were plated on glass coverslips which were placed in the plastic dishes. After an initial 2-hour period to permit attachment of the cells, the plates were washed vigorously three times to remove nonadherent cells. The remaining cells (macrophages) were then fed with M199-FCS alone or M199-FCS containing IL at a final concentration of 9.3 or 37.5 mg/dl. Electron Microscopy
Macrophage morphology was evaluated by scanning and transmission electron microscopy. Cells were harvested at 4, 24, and 48 hours following placement in tissue culture. For evaluation by scannirng electron microscopy, cells were grown on glass coverslips. At appropriate intervals, coverslips were washed twice with M199 and fixed with 3.3% glutaraldehyde in 0.1 M phosphate buffer for 1 hour. The specimens were then washed and stored in 0.1 M phosphate buffer (pH 7.2) containing 10% sucrose. Specimens for transmission electron microscopy were removed from the surface of plastic Petri dishes with a rubber policeman and centrifuged at 11OOg in conical centrifuge tubes for 10 minutes at room temperature. The pelleted cells were fixed with 3.3% glutaraldehyde in 0.1 M phosphate buffer. Specimens prepared for scanning electron microscopy were postfixed in 1% osmium tetroxide buffered with s-collidine, dehydrated in a graded series of alcohol, and criticalpoint-dried with liquid CO2. The specimens were attached to mounting stubs with double sticky tape and coated with gold-palladium in a Hummer sputtering device. All specimens were examined in a ETEC scanning electron microscope. Random fields were photographed and the numbers of flattened and rounded cells were determined.
Vol. 96, No. 3 September 1979
ALTERATION OF PERITONEAL MACROPHAGES BY IL
755
Specimens for transmission electron microscopy were postfixed with 1 % osmium tetroxide in 0.1 M phosphate buffer, dehydrated in a graded series of alcohol, and embedded in Spurr's epoxy.8 One-micron-thick sections were stained with toluidine blue and selected areas were thin sectioned on a Sorvall MT2-B ultra-microtome. Twenty random cells from each culture were photographed and examined for the presence of IL. Association of Macrophages With Particles
Association of macrophages with latex beads or yeast was studied by a modification of the method of Schmid and Brune.9 After the specified times the cells were washed twice with M199 to remove nonadherent cells. Two milliliters of M199 containing 3 drops of a 10% suspension of latex beads 5.7 in diameter/10 ml or 1 X 106 yeast/ml (approximately 25 to 50 latex beads or 2 to 3 yeast per adherent cell) were added to each dish. Fresh GP serum (final concentration, 5%) was added as a source of opsonins. The dishes were then incubated for 30 minutes (latex) or 60 minutes (yeast) at 37 C without shaking before the nonadherent particles were removed by washing twice with Dulbecco's phosphate-buffered saline. The adherent cells were scraped from the plastic dish with a rubber policeman. The cell suspension was placed in a hemocytometer and the percentage of cells which were associated with one or more latex beads or yeast was determined. With the latex beads and yeast, ingestion and adherence to the cell membrane were not differentiated. Additional experiments were performed with sheep erythrocytes coated with IgG antibody (IgG-SRBC) to determine the percentage of cells which had ingested particles, as opposed to cells which only had particles adherent to the cell membrane. For these experiments, the method of Newman et al 10 was used.
Results Effect of IL on Macrophage Morphology
Scanning electron microscopy of control cells in culture 4 hours revealed that most of the cells were flattened (Table 1). The rounded cells had a ruffled membrane typical of monocytes and macrophages. The elongated flattened cells had delicate thin veils of cytoplasm spreading over the coverglass surface with smaller filamentous cytoplasmic extensions (Figure 1). Following 4 hours of exposure to IL, there was an increase in the percentage of rounded cells (Table 1). The surface morphology, however, appeared unchanged at this time period (Figure 1). By transmission electron microscopy, approximately 45% of the macrophage cross-sections showed intracellular accumulation of a moderately electrondense lipid-like material which had an electron microscopic appearance similar to IL alone. There was no evidence of intracellular digestion of the lipid within lipophagolysosomes (Figure 2). There were no other ultrastructural differences noted between the IL and control cells. At 24 hours, scanning electron microscopy of control cells revealed that fewer cells were flattened than at 4 hours (Table 1), but the morphology of these cells was similar to that observed at 4 hours. However, the cells exposed to IL showed striking changes. Almost all cells were rounded (Table 1). In addition, there was a striking decrease in the number and degree of complexity of the membrane ruffles (Figure 3). Many of the cells
American Journal of Pathology
STRUNK ET AL
756
Table 1-Effect of Intralipid (IL) on the Morphology of GP Peritoneal Macrophages In Vitro*
Presence of ruffled surface by scanning electron microscopy
Time (hr)t
Control
IL
4 24 48
+ + +
+ -
% Transmission electron microscopy % Flattened macrophages on scanning electron microscopy cross-sections with IL present IL Control 79 25 30
55 6 5
45 80 100
* Results in each of the assays shown were obtained at the stated time, after the addition of M199-FCS alone (control) or M199-FCS containing IL (37.5 mg/dl). t Time in culture in control or IL-containing medium
appeared smooth or had blunted, bossilated surfaces. By transmission electron microscopy, approximately 80% of the macrophage cross-sections showed an accumulation of the lipid material. Those macrophage crosssections which contained visible lipid showed a greater accumulation of the material than at 4 hours (Figure 4). Again there was no evidence of lipid digestion within lipophagolysosomes. At 48 hours there was a decrease in the total number of both control and IL cells per scanning field. The control cells at 48 hours were similar to controls at 24 hours, both in percentage of flattened cells and in surface detail (Table 1) (48-hour cells not shown). Cells exposed to IL for 48 hours showed the same striking surface abnormalities seen with IL-treated cells at 24 hours. By transmission electron microscopy, 100% of the IL-treated macrophage cross-sections revealed a large accumulation of lipid material (Table 1). As in the 4-hour and 24-hour cultures, all the lipid material had a moderately electron-dense appearance and did not show any accompanying myelin figures indicative of intracellular digestion. Effect of IL on the Phagocytic Abilities of the Macrophages
The ability of control cells to associate with opsonized latex beads remained relatively constant through the 48-hour observation period (Table 2). Incubation of the cells with IL (37.5 mg/dl) for 4 hours did not affect the capacity of the cells to associate with the latex. However, after 24 hours of incubation with the IL, this capacity was significantly decreased. Incubation of the cells for 24 hours in a lower concentration of IL (9.3 mg/dl) also affected the cells, but the effect was not as marked: the average number of latex particles per cell (calculation described in Table 2) was decreased (2.79 ± 0.62 for IL cells compared with 3.13 ± 0.44 for
ALTERATION OF PERITONEAL MACROPHAGES BY IL
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STRUNK ET AL
American Journal of Pathology
control cells [P < 0.05]) but the percentage of total cells associating with latex was not affected (71.1 ± 20.4 for IL cells compared with 80.4 ± 15.7 for control cells [P > 0.1]). Cells incubated with IL (37.5 mg/dl) for 48 hours had a capacity to associate with latex similar to the cells incubated with IL only 24 hours (Table 1), indicating that maximal effects were reached by 24 hours. Similar experiments were performed with cells on coverslips to examine the possibility that the results of the above experiments may have been influenced by damage to either IL or control cells during the process of scraping from the plate. Unopsonized latex beads were used in these experiments to avoid obscuring the cells by generalized nonspecific adherence of the opsonized beads to the coverslip. The differences between IL (37.5 mg/dl) and control cells in four separate experiments were similar to those in Table 2 or were even more striking. The percentage of control cells associated with latex was 78.0 ± 11.8 (with 2.67 ± 0.57 beads/cell) compared with 22.5 ± 9.6 (with 1.52 ± 0.17 beads/cell) for IL cells (results for IL cells significantly less than control [P < 0.01] for both determinations). The results using the yeast particles were similar to the latex results (Table 3). The capacity of the control cells remained relatively constant from 4 to 24 hours (the difference between control values at 4 and 24 hours was not significant [P > 0.4]). Incubation of the cells for 4 hours with IL, either 9.3 or 37.5 mg/dl, did not affect this capacity, but both concentrations of IL produced significant effects on the macrophage function after 24 hours. The percentage of macrophages associated with IgG-SRBC was determined after washing the cells with medium to remove nonadherent particles or after washing with water to lyse adherent but noningested particles (Table 4). For both control and IL cells, the percentage of cells adherent to IgG-SRBC was significantly increased compared with the percentage of cells which had ingested IgG-SRBC (Table 4). For the control cells the actual differences were small and almost all the cells had ingested erythrocytes. However, for the IL cells, the percentage of cells adherent to IgG-SRBC was much different than the percentage of cells ingesting the IgG-SRBC. As with the latex and yeast particles, the overall capacity of the IL cells to associate with the IgG-SRBC, either adherence or ingestion, was significantly reduced compared with the control cells (P < 0.001). The reduced capacity of the IL cells is also evident when the number of particles per cell is calculated. Most of the control cells had either ingested or adhered to more than three IgG-SRBC (92, or 99%, of
Vol. 96, No. 3 September 1979
ALTERATION OF PERITONEAL MACROPHAGES BY IL
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