Motility of Rabbit Alveolar Cells Role of Unsaturated Fatty Acids William S. Lynn, MD, and Chhabirani Mukherjee, PhD
A mechanism for the specific accumulation of macrophages in alveoli or other biologic cavities following injury is presented. The data herein indicate that unsaturated fatty acids, ie, linoleic and linolenic acids, which accumulate in rat pleura following injection of carrageenan or during incubation of rabbit alveolar macrophages (AMs), strongly activate migration in vitro of AMs but not of human polymorphonuclear leukocytes (PMNLs). Other anionic lipids, ie, phosphatidylglycerol, as well as various nonspecific proteins, such as gelatin, or albumin were also shown to be potent activators of migration of AMs and not of PMNLs. These observations suggest that the elaboration of unsaturated fatty acids, as well as of nonspecific proteins, is responsible for the specific accumulation of macrophages in injured body spaces, such as alveoli or pleura. (Am J Pathol 96:663-672, 1979)
THE MASSIVE ACCUMULATION of large numbers of alveolar macrophages (AMs) in mammalian lung, both in the interstitium and in alveoli, is known to be provoked by inhalation or injection of a large number of substances, eg, Freund's adjuvant, silica, aluminum silicate, chlorphentermine, or tobacco smoke. 1-4 Other cells, including polymorphonuclear (PMN) cells, do not accumulate with the above agents. The signal that causes this specific cellular accumulation is unidentified. AMs have been shown to respond chemotactically to many different peptides. These same peptides also activate polymorphonuclear leukocytes (PMNLs).5 Thus, it is unlikely that any of the known nonspecific peptide chemotaxins accounts for the specific accumulation of these cells. Neither the mechanism of motility nor the specific control of migration for various types of cells is understood. Recently, we 6 and Naccache et al 7 have suggested that, in PMNLs, the synthetic chemotactic peptides that activate membrane ruffling, cellular flattening and swelling, and cation fluxes (K+ efflux and Na+ and Ca++ influx) activate migration by activating cation pumps. The above effects of the synthetic peptides (which bind to specific receptors on the plasma membranes of PMNLs 8) are all competitively inhibited by nonspecific proteins, eg, serum albumin, globulin, and ovalbumin.9 However, the effect of these proteins on alveolar cells, as shown in this report, is to activate motility of AMs. In addition, From the Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, North Carolina. Accepted for publication April 18, 1979. Address reprint requests to Dr. William S. Lynn, Department of Medicine, Box 3711, Duke University Medical Center, Durham, NC 27710. 0002-9440/79/0910-0663$01.00 663 i American Association of Pathologists
664
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American Journal of Pathology
polyunsaturated fatty acids, which accumulate in experimental pleural inflammation and in media of incubated macrophages, were shown to be potent activators of migration of AMs. These studies therefore suggest that the accumulation of macrophages in chronic inflammatory processes results from the accumulation of polyunsaturated fatty acids, or other anionic lipids, as well as proteins in body spaces. The source of these fatty acids may be the resident macrophages normally present in body spaces. Materials and Methods Isolation of Cells PMNLs
These cells were isolated from human blood of volunteers or from rabbit blood as before, except that time of hypotonic exposure was 10 seconds or less. Because cells that are exposed to hypertonicity for more than 15 seconds are usually unable to accelerate their rate of random migration upon stimulation, only those serums in which red cells could be removed by a 10-second lysis were used. These cells were 85-91% PMNLs. PMNLs were also isolated from sterile dialysates of uremic patients undergoing peritoneal dialysis. These cells were washed in isotonic NaCl and counted. They were more than 97% granulocytes but contained 20-30% band forms. Similar data were obtained with all preparations of human PMNLs. AMs
These cells were isolated by lung lavage, using 0.14 M NaCl, from rabbits sacrificed by injection of air as before.10 These animals were injected with complete Freund's adjuvant 14 to 25 days before sacrifice; the isolated cells were more than 98% mononuclear cells. AMs were also obtained from uninjected rabbits; however, rates of migration, either stimulated or random, by these cells were so slow under these conditions that meaningful data could not be obtained. Also, after 1 to 2 hours of incubation, migration ceased altogether. This was true whether Nuclepore (10,u) or Millipore (100,u) filters were used. Also, only a small number of the unstimulated cells (5-10%) would adhere to the filters under these conditions. These data on uninjected rabbits are not shown graphically. Migration Assays Migration of both cell types was assayed in Boyden chambers as described previously.9 The incubation media contained 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl, and 1 mM Tris-HCl, pH 7.4. Five-micron Millipore filters were used for both cell types and 2 X 106 cells were used. Incubation time was 120 minutes for AMs and 50 minutes for PMNLs. The filters were stained with hematoxylin; rate of migration was estimated by measuring the leading front of cells at ten different areas. Random migration is motility in the absence of stimuli, activated random migration is observed when equal concentrations of stimuli are present on both sides of the filter, and directed migration is the result of adding the stimulant only to the agent side of the filter. Pleural Exudates Groups of 5 rats, weighing 150-200 g, were injected intrapleurally with 0.5 mg carrageenan in 0.5 ml isotonic NaCI and the pleural exudate obtained by aspiration after sacrificing the rat by exposure to chloroform, as described previously.10 The cells were removed by centrifugation at 250g for 10 minutes.
Vol. 96, No. 3 September 1979
MOTILITY AND FATTY ACIDS
665
Isolation of Free Fatty Acids
Total fatty acids were extracted with ethyl ether 3 times after acidification to pH 3.0 using acetic acid. The ether was dried with sodium sulfate and applied to Silica Gel-G thinlayer chromatography (TLC) plates; the free fatty acids were separated from the other neutral lipids using two solvents, according to the method used by Kaplan, Weiss, and Elsback.'6 The free fatty acids were scraped from the TLC plates, extracted into chloroform-methanol (5:1 by volume), dried under nitrogen, and methylated with diazomethane as before.'1 The methyl esters were applied to a gas-liquid chromatography (GLC) column of OV-275 and the esters separated using a temperature gradient of 160 to 200 C. The esters were quantitated by measuring the areas of the peaks with a CSI-38 Integrator. Methyl dodecanoate was used as internal standard and known amounts of each fatty acid methyl ester were used for quantitation.
Results Effect of Fatty Acids on Macrophages and PMNLs
Unsaturated fatty acids (but not saturated fatty acids), hydrophobic peptides, and proteins are potent activators of motility of rabbit alveolar macrophages (Text-figure 1). In the presence of positive, negative, or no concentration gradient using either type of agent, the migrating response of the cells to these gradients is either positive or negative, depending upon the gradient (Text-figure 1). Since these cells are also activated to migrate in the absence of concentration gradients of all three classes of agents (Text-figure 1), it is clear that all these types of agents primarily activate random migration. The concentrations of the stimuli used in Text-figure 1 were shown to be optimal for these cells. At higher concentrations of these agents, especially on the cell side, migration is markedly inhibited (Text-figure 2). Many fatty acids are active, but arachidonic acid O Control 0 Agent Side O Cell Side Both Sides
~~~~~~~~01
100
TEXT-FIGURE 1 -Effect of fatty acids and peptides on alveolar cell migration. Motility of rabbit alveolar cells obtained 30 days after injection of Freund's adjuvant was assessed using 5-,u Millipore filters and 2 X 106 cells. (See Materials and Methods.) Additions were made to each side and to both sides of the filters as indicated. Four to six experiments were done using different cells. The bars represent 20 standard errors.
80
.C 6 60 40
F-Met-Phe 0.1 ,4g/ml
Gelot n
O.S
Linole,c Aced PolmiticAcid 5,eL/ml 10 ug /MI
American Journal of Pathology
LYNN AND MUKHERJEE
666
80
Agent side 60 -..
-=r
0
o
/
40)
/
>40
20 z
> / \ ,/\
t)
g
0.1
1
10
Agent side
TEXT-FIGURE 2 -Effect of arachidonic acid on AM migration. Motility of rabbit alveolar cells were assessed as in T / V \ rlext-figure 1 with various concentraBoth Sides tions of arachidonic acid on the agent Both Sides side (triangles) or both sides (solid circles) of the filter or with f-met-phe on \ \agent side (open circles) of the filter. ; 20 / / The data are from one cell preparation, but 5 trials produced the same types of curves.
100
Arachidonic Acid or F-Met - Phe Ug/ml
is somewhat more active than other long chain fatty acids. Formylated dipeptides and tripeptides are also very active, but not more so than hydrolysates of collagen or fatty acids (Text-figure 1).12 Anionic phospholipids, eg, phosphatidylglycerol and phosphatidylinositol, also specifically activate migration of AMs (Table 1). Neutral or cationic phospholipids are inefffective (Table 1). Also, the methyl ester of formyl-met-phe is somewhat more active on AM than on blood PMNLs (Table 1). PMNLs are also activated to migrate by arachidonic acid; however, linoleic and linolenic acids are without effect on PMNLs (Table 1). Hydroxy and hydroperoxy derivatives of arachidonic acid are equally effective on both cell types (Table 1). These observations indicate that, although migration of both AMs and PMNLs is readily activated by polyunsaturated fatty acids, only AMs are activated by less polar fatty acids or anionic phospholipids (Table 1). Liberation of Bioactive Fatty Acids from AMs In Vivo and In Vitro
Incubation of AMs in glass tubes caused the release of large amounts of fatty acids. These released fatty acids activate macrophage locomotion but are without effect on PMNLs (Table 2). The composition of the liberated fatty acids included palmitic (31%), stearic (18%), oleic (21%), linoleic (19%), linolenic (3%), and unknown acids (2%). No arachidonic acid was liberated. Incubation of PMNLs under identical conditions failed to result in the liberation of chemotactic fatty acids or peptides (Table 3). However, PMNLs can liberate chemotactically active peptides under certain
MOTILITY AND FATTY ACIDS
Vol. 96, No. 3
September 1979
667
Table 1-Effect of Lipids on Migration of AMs and PMNLs % Stimulation*
Addition
AMs
PMNLs
Palmitic acid, 10 ,g/ml Oleic acid, 10,ug/ml Linoleic acid, 1 0,g/ml Linolenic acid, 1 0Aglml Arachidonic acid, 10 Ag/ml
10 ± 14 70 ± 30 210 ± 21 305 ± 31 420 ± 20
0 0 0 0
Phosphatidylcholine, 10 Aglml
0 100 ± 0 140 ± 0 190 ± 30 ±
Phosphatidylinositol, 10 ug/ml
Phosphatidylethanolamine, 10,ug/ml Phosphatidylglycerol, 10,ug/ml Phosphatidyldimethylethanolamine, 10 Ag/ml Phosphatidic acid, 10 ug/ml 1 -Palmitoyl-lysophosphatidylcholine, 10 Lg/ml PGE2, 10 sg/mI
PGEI, 10lg/mI
PGF2a, 10ug/ml HETE,t 10kg/ml HPETE,t 10 ug/ml F-met-phe, 1 Ag/ml F-met-phe, methyl ester, 1 ug/ml Methyl arachidonate
Cholesterylarachidonate Cholesteryl palmitate
14 12
30 15
150 ± 20 0 0 0 0 0 0 0
50 ± 20 50 ± 16 0 200 ± 19 220 ± 24 370 ± 16 360 ± 22
180 ± 21 150 ± 19 360 ± 40 210 ± 15
110 ± 25 120 ± 35 0
0 0 0
0 0 0
* The above experiments represent average of data obtained from 4 different preparations of rabbit AMs and 6 preparations of human PMNLs. The data are expressed as % change after addition of each stimulant, using each cell preparation as its own control. t HETE = 12-L-hydroxy-/ -5,8,10,14-eicosatetraenoic acid; HPETE is the 12-L-hydroperoxy precursor of HETE.
conditions.'3 PMNLs also liberate proteolytic enzymes, which readily hydrolyze these chemotactic peptides.9,14 The accumulation of macrophages in pleural cavities following intrapleural injection of a foreign substance such as carrageenan is associated with the appearance intrapleurally of agents that specifically attract AMs (Text-figure 3). These specific agents could be quantitatively extracted from the exudates with organic solvents and were shown after isolation by TLC to be a mixture of saturated and unsaturated fatty acids. The major polyunsaturated fatty acid was linoleic acid (Table 3). As previously indicated,2 the cells that accumulate following injection are initially PMNLs (4 to 6 hours), but these cells are largely replaced by macrophages in 16 to 18 hours. Most of the fluid and cells are cleared by 48 to 72 hours. The origin of these pleural chemotactic fatty acids is uncertain. However, since the time course of accumulation of fatty acids in pleura
668
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American Journal of Pathology
Table 2-Migratory Activity of Fatty Acids Liberated From AMs or PMNLs % Stimulation
Total (,ug)
Fatty acids liberated from PMNL Fatty acids liberated from AM
0.41
10.4
AMs
PMNLs
0
0 0
340 ± 22
Alveolar macrophages, 5 x 106 cells, and human PMNLs, 5 x 106f cells, were incubated for 45 min at 37 C in glass test tubes in the migration assay buffer. The cells were then removed by centrifugation and the media used on the agent side of fresh cells to assay for rate of migration, as described in Materials and Methods section.
parallels that of accumulation of macrophages, and since, as shown above, macrophages liberate fatty acids when incubated in vitro, it is possible that these pleural fatty acids are also derived from the resident pleural macrophages. Rate of liberation of these pleural fatty acids is shown in Table 3. The concentration of fatty acids, as well as the percentage of unsaturated fatty acids, in these exudates is highest initially but slowly declines to normal levels over 2 to 4 days (Table 3). Discussion
The observations in this report indicate that the controls of motility in these two cell types, AMs and PMNLs, differ in several crucial ways: a) Macrophages, when incubated, release a factor that stimulates motility of other macrophages but has no effect on PMNLs. This factor was shown to be unsaturated fatty acids, primarily linoleic acid. PMNLs, when incubated under similar conditions, do not release this motility factor. b) Nonspecific proteins, such as gelatin or denatured serum albumin, actiTABLE 3-Release of Fatty Acids Into Pleura After Injection of Carrageenan
Volume (ml) Total fatty acid (,ug) Polyunsaturated fatty acids (%) Pal mitic acid (%) Linoleic acid (%) Hours after injection
7.2 118 20.8 29 16.9 3
14.2 123 28.4 38 22.2 7
14.5 99 18.2 44 14.2 15
14.1 115 11.3 43 7.9 24
Twenty rats were injected with 0.5 mg of carrageenan intrapleurally.'0 At the indicated intervals, 5 rats were anesthetized and total pleural fluid removed by aspiration. The cells were removed by centrifugation and a portion of the fluid used as buffer on the agent side to assay its migratory activity on AMs and PMNLs (see Figure 3). Total fatty acids were removed by extraction with ethyl ether, purified by TLC (see Materials and Methods), and an amount of fatty acid equal to the concentration present in the original exudate assayed for migratory activity on AMs and PMNLs, as in Figure 3. The remaining fatty acids were methylated and separated and quantitated by GLC (see Materials and Methods). Insufficient amounts of fluid (less than 0.5 ml) for analyses were obtained from uninjected rats.
MOTILITY AND FATTY ACIDS .-.,
Vol. 96, No. 3
September 1979
...-
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-,%
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uJ
A mechanism for the specific accumulation of macrophages in alveoli or other biologic cavities following injury is presented. The data herein indicate that unsaturated fatty acids, ie, linoleic and linolenic acids, which accumulate in rat pleura following injection of carrageenan or during incubation of rabbit alveolar macrophages (AMs), strongly activate migration in vitro of AMs but not of human polymorphonuclear leukocytes (PMNLs). Other anionic lipids, ie, phosphatidylglycerol, as well as various nonspecific proteins, such as gelatin, or albumin were also shown to be potent activators of migration of AMs and not of PMNLs. These observations suggest that the elaboration of unsaturated fatty acids, as well as of nonspecific proteins, is responsible for the specific accumulation of macrophages in injured body spaces, such as alveoli or pleura. (Am J Pathol 96:663-672, 1979)
THE MASSIVE ACCUMULATION of large numbers of alveolar macrophages (AMs) in mammalian lung, both in the interstitium and in alveoli, is known to be provoked by inhalation or injection of a large number of substances, eg, Freund's adjuvant, silica, aluminum silicate, chlorphentermine, or tobacco smoke. 1-4 Other cells, including polymorphonuclear (PMN) cells, do not accumulate with the above agents. The signal that causes this specific cellular accumulation is unidentified. AMs have been shown to respond chemotactically to many different peptides. These same peptides also activate polymorphonuclear leukocytes (PMNLs).5 Thus, it is unlikely that any of the known nonspecific peptide chemotaxins accounts for the specific accumulation of these cells. Neither the mechanism of motility nor the specific control of migration for various types of cells is understood. Recently, we 6 and Naccache et al 7 have suggested that, in PMNLs, the synthetic chemotactic peptides that activate membrane ruffling, cellular flattening and swelling, and cation fluxes (K+ efflux and Na+ and Ca++ influx) activate migration by activating cation pumps. The above effects of the synthetic peptides (which bind to specific receptors on the plasma membranes of PMNLs 8) are all competitively inhibited by nonspecific proteins, eg, serum albumin, globulin, and ovalbumin.9 However, the effect of these proteins on alveolar cells, as shown in this report, is to activate motility of AMs. In addition, From the Departments of Biochemistry and Medicine, Duke University Medical Center, Durham, North Carolina. Accepted for publication April 18, 1979. Address reprint requests to Dr. William S. Lynn, Department of Medicine, Box 3711, Duke University Medical Center, Durham, NC 27710. 0002-9440/79/0910-0663$01.00 663 i American Association of Pathologists
664
LYNN AND MUKHERJEE
American Journal of Pathology
polyunsaturated fatty acids, which accumulate in experimental pleural inflammation and in media of incubated macrophages, were shown to be potent activators of migration of AMs. These studies therefore suggest that the accumulation of macrophages in chronic inflammatory processes results from the accumulation of polyunsaturated fatty acids, or other anionic lipids, as well as proteins in body spaces. The source of these fatty acids may be the resident macrophages normally present in body spaces. Materials and Methods Isolation of Cells PMNLs
These cells were isolated from human blood of volunteers or from rabbit blood as before, except that time of hypotonic exposure was 10 seconds or less. Because cells that are exposed to hypertonicity for more than 15 seconds are usually unable to accelerate their rate of random migration upon stimulation, only those serums in which red cells could be removed by a 10-second lysis were used. These cells were 85-91% PMNLs. PMNLs were also isolated from sterile dialysates of uremic patients undergoing peritoneal dialysis. These cells were washed in isotonic NaCl and counted. They were more than 97% granulocytes but contained 20-30% band forms. Similar data were obtained with all preparations of human PMNLs. AMs
These cells were isolated by lung lavage, using 0.14 M NaCl, from rabbits sacrificed by injection of air as before.10 These animals were injected with complete Freund's adjuvant 14 to 25 days before sacrifice; the isolated cells were more than 98% mononuclear cells. AMs were also obtained from uninjected rabbits; however, rates of migration, either stimulated or random, by these cells were so slow under these conditions that meaningful data could not be obtained. Also, after 1 to 2 hours of incubation, migration ceased altogether. This was true whether Nuclepore (10,u) or Millipore (100,u) filters were used. Also, only a small number of the unstimulated cells (5-10%) would adhere to the filters under these conditions. These data on uninjected rabbits are not shown graphically. Migration Assays Migration of both cell types was assayed in Boyden chambers as described previously.9 The incubation media contained 0.14 M NaCl, 1 mM CaCl2, 1 mM MgCl, and 1 mM Tris-HCl, pH 7.4. Five-micron Millipore filters were used for both cell types and 2 X 106 cells were used. Incubation time was 120 minutes for AMs and 50 minutes for PMNLs. The filters were stained with hematoxylin; rate of migration was estimated by measuring the leading front of cells at ten different areas. Random migration is motility in the absence of stimuli, activated random migration is observed when equal concentrations of stimuli are present on both sides of the filter, and directed migration is the result of adding the stimulant only to the agent side of the filter. Pleural Exudates Groups of 5 rats, weighing 150-200 g, were injected intrapleurally with 0.5 mg carrageenan in 0.5 ml isotonic NaCI and the pleural exudate obtained by aspiration after sacrificing the rat by exposure to chloroform, as described previously.10 The cells were removed by centrifugation at 250g for 10 minutes.
Vol. 96, No. 3 September 1979
MOTILITY AND FATTY ACIDS
665
Isolation of Free Fatty Acids
Total fatty acids were extracted with ethyl ether 3 times after acidification to pH 3.0 using acetic acid. The ether was dried with sodium sulfate and applied to Silica Gel-G thinlayer chromatography (TLC) plates; the free fatty acids were separated from the other neutral lipids using two solvents, according to the method used by Kaplan, Weiss, and Elsback.'6 The free fatty acids were scraped from the TLC plates, extracted into chloroform-methanol (5:1 by volume), dried under nitrogen, and methylated with diazomethane as before.'1 The methyl esters were applied to a gas-liquid chromatography (GLC) column of OV-275 and the esters separated using a temperature gradient of 160 to 200 C. The esters were quantitated by measuring the areas of the peaks with a CSI-38 Integrator. Methyl dodecanoate was used as internal standard and known amounts of each fatty acid methyl ester were used for quantitation.
Results Effect of Fatty Acids on Macrophages and PMNLs
Unsaturated fatty acids (but not saturated fatty acids), hydrophobic peptides, and proteins are potent activators of motility of rabbit alveolar macrophages (Text-figure 1). In the presence of positive, negative, or no concentration gradient using either type of agent, the migrating response of the cells to these gradients is either positive or negative, depending upon the gradient (Text-figure 1). Since these cells are also activated to migrate in the absence of concentration gradients of all three classes of agents (Text-figure 1), it is clear that all these types of agents primarily activate random migration. The concentrations of the stimuli used in Text-figure 1 were shown to be optimal for these cells. At higher concentrations of these agents, especially on the cell side, migration is markedly inhibited (Text-figure 2). Many fatty acids are active, but arachidonic acid O Control 0 Agent Side O Cell Side Both Sides
~~~~~~~~01
100
TEXT-FIGURE 1 -Effect of fatty acids and peptides on alveolar cell migration. Motility of rabbit alveolar cells obtained 30 days after injection of Freund's adjuvant was assessed using 5-,u Millipore filters and 2 X 106 cells. (See Materials and Methods.) Additions were made to each side and to both sides of the filters as indicated. Four to six experiments were done using different cells. The bars represent 20 standard errors.
80
.C 6 60 40
F-Met-Phe 0.1 ,4g/ml
Gelot n
O.S
Linole,c Aced PolmiticAcid 5,eL/ml 10 ug /MI
American Journal of Pathology
LYNN AND MUKHERJEE
666
80
Agent side 60 -..
-=r
0
o
/
40)
/
>40
20 z
> / \ ,/\
t)
g
0.1
1
10
Agent side
TEXT-FIGURE 2 -Effect of arachidonic acid on AM migration. Motility of rabbit alveolar cells were assessed as in T / V \ rlext-figure 1 with various concentraBoth Sides tions of arachidonic acid on the agent Both Sides side (triangles) or both sides (solid circles) of the filter or with f-met-phe on \ \agent side (open circles) of the filter. ; 20 / / The data are from one cell preparation, but 5 trials produced the same types of curves.
100
Arachidonic Acid or F-Met - Phe Ug/ml
is somewhat more active than other long chain fatty acids. Formylated dipeptides and tripeptides are also very active, but not more so than hydrolysates of collagen or fatty acids (Text-figure 1).12 Anionic phospholipids, eg, phosphatidylglycerol and phosphatidylinositol, also specifically activate migration of AMs (Table 1). Neutral or cationic phospholipids are inefffective (Table 1). Also, the methyl ester of formyl-met-phe is somewhat more active on AM than on blood PMNLs (Table 1). PMNLs are also activated to migrate by arachidonic acid; however, linoleic and linolenic acids are without effect on PMNLs (Table 1). Hydroxy and hydroperoxy derivatives of arachidonic acid are equally effective on both cell types (Table 1). These observations indicate that, although migration of both AMs and PMNLs is readily activated by polyunsaturated fatty acids, only AMs are activated by less polar fatty acids or anionic phospholipids (Table 1). Liberation of Bioactive Fatty Acids from AMs In Vivo and In Vitro
Incubation of AMs in glass tubes caused the release of large amounts of fatty acids. These released fatty acids activate macrophage locomotion but are without effect on PMNLs (Table 2). The composition of the liberated fatty acids included palmitic (31%), stearic (18%), oleic (21%), linoleic (19%), linolenic (3%), and unknown acids (2%). No arachidonic acid was liberated. Incubation of PMNLs under identical conditions failed to result in the liberation of chemotactic fatty acids or peptides (Table 3). However, PMNLs can liberate chemotactically active peptides under certain
MOTILITY AND FATTY ACIDS
Vol. 96, No. 3
September 1979
667
Table 1-Effect of Lipids on Migration of AMs and PMNLs % Stimulation*
Addition
AMs
PMNLs
Palmitic acid, 10 ,g/ml Oleic acid, 10,ug/ml Linoleic acid, 1 0,g/ml Linolenic acid, 1 0Aglml Arachidonic acid, 10 Ag/ml
10 ± 14 70 ± 30 210 ± 21 305 ± 31 420 ± 20
0 0 0 0
Phosphatidylcholine, 10 Aglml
0 100 ± 0 140 ± 0 190 ± 30 ±
Phosphatidylinositol, 10 ug/ml
Phosphatidylethanolamine, 10,ug/ml Phosphatidylglycerol, 10,ug/ml Phosphatidyldimethylethanolamine, 10 Ag/ml Phosphatidic acid, 10 ug/ml 1 -Palmitoyl-lysophosphatidylcholine, 10 Lg/ml PGE2, 10 sg/mI
PGEI, 10lg/mI
PGF2a, 10ug/ml HETE,t 10kg/ml HPETE,t 10 ug/ml F-met-phe, 1 Ag/ml F-met-phe, methyl ester, 1 ug/ml Methyl arachidonate
Cholesterylarachidonate Cholesteryl palmitate
14 12
30 15
150 ± 20 0 0 0 0 0 0 0
50 ± 20 50 ± 16 0 200 ± 19 220 ± 24 370 ± 16 360 ± 22
180 ± 21 150 ± 19 360 ± 40 210 ± 15
110 ± 25 120 ± 35 0
0 0 0
0 0 0
* The above experiments represent average of data obtained from 4 different preparations of rabbit AMs and 6 preparations of human PMNLs. The data are expressed as % change after addition of each stimulant, using each cell preparation as its own control. t HETE = 12-L-hydroxy-/ -5,8,10,14-eicosatetraenoic acid; HPETE is the 12-L-hydroperoxy precursor of HETE.
conditions.'3 PMNLs also liberate proteolytic enzymes, which readily hydrolyze these chemotactic peptides.9,14 The accumulation of macrophages in pleural cavities following intrapleural injection of a foreign substance such as carrageenan is associated with the appearance intrapleurally of agents that specifically attract AMs (Text-figure 3). These specific agents could be quantitatively extracted from the exudates with organic solvents and were shown after isolation by TLC to be a mixture of saturated and unsaturated fatty acids. The major polyunsaturated fatty acid was linoleic acid (Table 3). As previously indicated,2 the cells that accumulate following injection are initially PMNLs (4 to 6 hours), but these cells are largely replaced by macrophages in 16 to 18 hours. Most of the fluid and cells are cleared by 48 to 72 hours. The origin of these pleural chemotactic fatty acids is uncertain. However, since the time course of accumulation of fatty acids in pleura
668
LYNN AND MUKHERJEE
American Journal of Pathology
Table 2-Migratory Activity of Fatty Acids Liberated From AMs or PMNLs % Stimulation
Total (,ug)
Fatty acids liberated from PMNL Fatty acids liberated from AM
0.41
10.4
AMs
PMNLs
0
0 0
340 ± 22
Alveolar macrophages, 5 x 106 cells, and human PMNLs, 5 x 106f cells, were incubated for 45 min at 37 C in glass test tubes in the migration assay buffer. The cells were then removed by centrifugation and the media used on the agent side of fresh cells to assay for rate of migration, as described in Materials and Methods section.
parallels that of accumulation of macrophages, and since, as shown above, macrophages liberate fatty acids when incubated in vitro, it is possible that these pleural fatty acids are also derived from the resident pleural macrophages. Rate of liberation of these pleural fatty acids is shown in Table 3. The concentration of fatty acids, as well as the percentage of unsaturated fatty acids, in these exudates is highest initially but slowly declines to normal levels over 2 to 4 days (Table 3). Discussion
The observations in this report indicate that the controls of motility in these two cell types, AMs and PMNLs, differ in several crucial ways: a) Macrophages, when incubated, release a factor that stimulates motility of other macrophages but has no effect on PMNLs. This factor was shown to be unsaturated fatty acids, primarily linoleic acid. PMNLs, when incubated under similar conditions, do not release this motility factor. b) Nonspecific proteins, such as gelatin or denatured serum albumin, actiTABLE 3-Release of Fatty Acids Into Pleura After Injection of Carrageenan
Volume (ml) Total fatty acid (,ug) Polyunsaturated fatty acids (%) Pal mitic acid (%) Linoleic acid (%) Hours after injection
7.2 118 20.8 29 16.9 3
14.2 123 28.4 38 22.2 7
14.5 99 18.2 44 14.2 15
14.1 115 11.3 43 7.9 24
Twenty rats were injected with 0.5 mg of carrageenan intrapleurally.'0 At the indicated intervals, 5 rats were anesthetized and total pleural fluid removed by aspiration. The cells were removed by centrifugation and a portion of the fluid used as buffer on the agent side to assay its migratory activity on AMs and PMNLs (see Figure 3). Total fatty acids were removed by extraction with ethyl ether, purified by TLC (see Materials and Methods), and an amount of fatty acid equal to the concentration present in the original exudate assayed for migratory activity on AMs and PMNLs, as in Figure 3. The remaining fatty acids were methylated and separated and quantitated by GLC (see Materials and Methods). Insufficient amounts of fluid (less than 0.5 ml) for analyses were obtained from uninjected rats.
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September 1979
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