Exp Toxic Pathol 1992; 44: 144-146 Gustav Fischer' Verlag Jena
I) Institute of Pathological Biochemistry, Medical Academy, Erfurt, Germany 2) Institute of laboratory diagnostics, Hospital St. Georg, Leipzig, Germany 3) Department of Animal Production and Veterinary Medicine, University of Leipzig, Leipzig, Germany
Ex vivo investigation of blood monocyte and platelet behaviour in pigs
maintained on an atherogenic diet S. KRAUSE!), A. POHL 1), C. POHL 1), S, FINKI), K. ROHLING 1), R. KATZEL2), G. VON LENGERKEN 3), and U. TILL!)
s. MAAK 3), W. LOSCHEI)
With 2 tables Received: July 9, 1990; Accepted: August 1, 1990
Address for correspondence: Dr. W. LOSCHE, Institut fUr Pathologische Biochemie, Nordhauser StraBe 74, D-O - 501O Erfurt, Deutschland Key words: monocyte ; platelets; hypercholesteremia; phagocytic capacity; adhesion, monocyte; aggregation , platelets; atherogenetic diet; blood , monocytes; blood, platelets
Summary Domestic pigs aged 4 months were fed for 16 weeks an atherogenic diet rich in cholesterol and saturated fatty acid. The increase of plasma cholesterol and triacylglycerol levels was found to be accompanied by a significant increase in the number of blood monocytes and platelets when compared to control animals. Furthermore, the atherogenic diet produced a small but significant reduction in the blood monocyte phagocytic capacity and adhesion to plastic surface. No significant differences between both groups were found when spontaneous platelet aggregation in whole blood was studied. H,owever, platelets from pigs fed the atherogenic diet had a smaller mean cell volume compared to controls. The results indicate than an atherogenic diet may affect blood monocytes and platelets in pigs.
Introduction Hypercholesterolemia is one of the major risk factors for the development of atherosclerosis. In hypercholesterolemia endothelial cell dysfunction and damage as well as accumulation of cholesterol-rich plasma lipoproteins in the arterial wall are found. On the other hand, interactions of blood cells, mainly monocytes and platelets, with the vascular wall are believed to play also an important role in atherogenesis (10, 17). Monocytes are capable to adhere to the endothelium and to migrate into the subendothelial tissue. There , they differentiate to macrophages, take up cholesterol-rich lipoproteins and , in consequence , become "foam cells" (10 , 12). Upon interaction with the vascular wall platelets produce the so-called platelet-derived growth factor (PDGF), a protein that stimulates proliferation of smooth muscle cells (17)._ Hypercholesterolemia is known to affect the behaviour of endothelial cells as well as of red blood cells and platelets (11, 17 , 20, 21). However, little is known whether hypercholes144
Exp. Toxic, Pathol. 44 (1992) 3
terolemia also alters monocyte behaviour and, by that, may support the interaction of monocytes with the vascular wall. Pigs develop a hypercholesterolemia when maintained on a diet rich in cholesterol and saturated fatty acids, and they are highly susceptible to atherosclerosis under this condition (3 , 10), In order to get more insight into the role of hypercholesterolemia in monocyte behaviour we studied ex vivo the effect of an atherogenic diet on monocyte functions in pigs.
Material and Methods Femal domestic pigs aged 4 months and with an average weight of 45 kg were divided into 2 groups of 8 animals each. One group was fed for 16 weeks a diet rich in cholesterol and saturated fatty acids (2 g/lOO g cholesterol and 20 g/lOO g beef tallow ; C/BT group) . The other group was fed normal food. Blood was taken by puncture of the vena cava cranialis . One part was mixed with 1/10 volume of 0.1 mmolll sodium citrate as anticoagulant and used for preparing mononuclear cells (MNC). Another part was allowed to clot (60 min at room temperature) and used for determination of serum lipids. Serum concentrations of total cholesterol (C) and HDLcholesterol (HDL-C) were measured enzymatically (19), HDLC after precipitation of apo B-containing lipoproteins by phosphotungstate/MgCl 2 (22) . Concentrations of VLDL + LDLcholesterol (VLDL + LDL-C) were calculated by the difference between C and HDL-C. Triacylglycerol (TO) was determined enzymatically after alkaline saponification (7) using a test kit from Laborchemie Apolda, Germany. Counts of white and red blood cells as well as of platelets in sampels of blood as well as in MNC suspensions were performed using a Coulter S plus VI counter. By this technique also values for the mean cellular volume of red blood cells and platelets were obtained.
MNC were prepared by dextran sedimentation of red blood cells and centrifugation on Ficoll-Paque according to the method of B0YUM et al. (2) and finally resuspended in phosphate-buffered saline (pH 7.4). The suspensions of MNC contained about 45 % monocytes and less than I % granulocytes. Phagocytic activity of monocytes was estimated as the capacity to ingest opsonized zymosan particles. The procedure applied was similar to that used for determining phagocytic activity of blood granulocytes described elsewhere (14). After MNC (5 x 106 cells) had been incubated with zymosan (Sigma Chemicals; opsonized with a human serum) for 1 h the number of particles engulfed by the monocytes were counted using a light microscope. For measurements of monocyte adhesion MNC (5 x 10 6 cells) were supplemented with 100 mlll fetal calf serum and incubated for 30 min in plastic multi well cell culture dishes (Falcon; well diameter 1.6 cm). After removing non-adherent cells by rinsing the wells with buffered saline the amount of adherent cells was determined by their DNA content as previously described (13). Spontaneous platelet aggregation that occurred when samples of whole blood were stirred (900 rpm) for 10 min was assessed by counting the numbers of single platelets before and after stirring (8). Platelet counts were performed using the Coulter S plus VI. The results are given as arithmetic means ± sd. Means obtained in the control and C/BT group were proved for significant differences by Student's t-test.
Results Compared to controls the animals of the C/BT group had significantly enhanced plasma concentrations of C, HDL-C and LDL + VLDL-C as well as of TG (table 1). Furthermore, the numbers of blood monocytes and platelets were significantly increased in the C/BT group compared to controls. In contrast to that no differences were observed when blood lymphocytes, granulocytes and red cells were counted (table 2). For red cells and platelets we also determined the mean cellular volumes. Whereas no difference was found with respect to red cells the mean cellular volume of platelets was significantly lower in the C/BT group compared to controls (6.99 ± 0.87 fl versus 7.80 ± 0.90 fl, p < 0.05).
Table 1. Concentrations of serum lipids in control and C/BT pigs. C
HDL-C
VLDL + LDL-C
Control
2.78 ±0.40
1.22 ±0.17
1.56 ±0.36
0.38 ±0.05
C/BT
4.54** ±0.39
2.00** ±0.13
2.54** ±0.41
0.50* ±0.16
TG
All values are given in mmol!l. Significant differences between both groups are indicated: * p
I) Institute of Pathological Biochemistry, Medical Academy, Erfurt, Germany 2) Institute of laboratory diagnostics, Hospital St. Georg, Leipzig, Germany 3) Department of Animal Production and Veterinary Medicine, University of Leipzig, Leipzig, Germany
Ex vivo investigation of blood monocyte and platelet behaviour in pigs
maintained on an atherogenic diet S. KRAUSE!), A. POHL 1), C. POHL 1), S, FINKI), K. ROHLING 1), R. KATZEL2), G. VON LENGERKEN 3), and U. TILL!)
s. MAAK 3), W. LOSCHEI)
With 2 tables Received: July 9, 1990; Accepted: August 1, 1990
Address for correspondence: Dr. W. LOSCHE, Institut fUr Pathologische Biochemie, Nordhauser StraBe 74, D-O - 501O Erfurt, Deutschland Key words: monocyte ; platelets; hypercholesteremia; phagocytic capacity; adhesion, monocyte; aggregation , platelets; atherogenetic diet; blood , monocytes; blood, platelets
Summary Domestic pigs aged 4 months were fed for 16 weeks an atherogenic diet rich in cholesterol and saturated fatty acid. The increase of plasma cholesterol and triacylglycerol levels was found to be accompanied by a significant increase in the number of blood monocytes and platelets when compared to control animals. Furthermore, the atherogenic diet produced a small but significant reduction in the blood monocyte phagocytic capacity and adhesion to plastic surface. No significant differences between both groups were found when spontaneous platelet aggregation in whole blood was studied. H,owever, platelets from pigs fed the atherogenic diet had a smaller mean cell volume compared to controls. The results indicate than an atherogenic diet may affect blood monocytes and platelets in pigs.
Introduction Hypercholesterolemia is one of the major risk factors for the development of atherosclerosis. In hypercholesterolemia endothelial cell dysfunction and damage as well as accumulation of cholesterol-rich plasma lipoproteins in the arterial wall are found. On the other hand, interactions of blood cells, mainly monocytes and platelets, with the vascular wall are believed to play also an important role in atherogenesis (10, 17). Monocytes are capable to adhere to the endothelium and to migrate into the subendothelial tissue. There , they differentiate to macrophages, take up cholesterol-rich lipoproteins and , in consequence , become "foam cells" (10 , 12). Upon interaction with the vascular wall platelets produce the so-called platelet-derived growth factor (PDGF), a protein that stimulates proliferation of smooth muscle cells (17)._ Hypercholesterolemia is known to affect the behaviour of endothelial cells as well as of red blood cells and platelets (11, 17 , 20, 21). However, little is known whether hypercholes144
Exp. Toxic, Pathol. 44 (1992) 3
terolemia also alters monocyte behaviour and, by that, may support the interaction of monocytes with the vascular wall. Pigs develop a hypercholesterolemia when maintained on a diet rich in cholesterol and saturated fatty acids, and they are highly susceptible to atherosclerosis under this condition (3 , 10), In order to get more insight into the role of hypercholesterolemia in monocyte behaviour we studied ex vivo the effect of an atherogenic diet on monocyte functions in pigs.
Material and Methods Femal domestic pigs aged 4 months and with an average weight of 45 kg were divided into 2 groups of 8 animals each. One group was fed for 16 weeks a diet rich in cholesterol and saturated fatty acids (2 g/lOO g cholesterol and 20 g/lOO g beef tallow ; C/BT group) . The other group was fed normal food. Blood was taken by puncture of the vena cava cranialis . One part was mixed with 1/10 volume of 0.1 mmolll sodium citrate as anticoagulant and used for preparing mononuclear cells (MNC). Another part was allowed to clot (60 min at room temperature) and used for determination of serum lipids. Serum concentrations of total cholesterol (C) and HDLcholesterol (HDL-C) were measured enzymatically (19), HDLC after precipitation of apo B-containing lipoproteins by phosphotungstate/MgCl 2 (22) . Concentrations of VLDL + LDLcholesterol (VLDL + LDL-C) were calculated by the difference between C and HDL-C. Triacylglycerol (TO) was determined enzymatically after alkaline saponification (7) using a test kit from Laborchemie Apolda, Germany. Counts of white and red blood cells as well as of platelets in sampels of blood as well as in MNC suspensions were performed using a Coulter S plus VI counter. By this technique also values for the mean cellular volume of red blood cells and platelets were obtained.
MNC were prepared by dextran sedimentation of red blood cells and centrifugation on Ficoll-Paque according to the method of B0YUM et al. (2) and finally resuspended in phosphate-buffered saline (pH 7.4). The suspensions of MNC contained about 45 % monocytes and less than I % granulocytes. Phagocytic activity of monocytes was estimated as the capacity to ingest opsonized zymosan particles. The procedure applied was similar to that used for determining phagocytic activity of blood granulocytes described elsewhere (14). After MNC (5 x 106 cells) had been incubated with zymosan (Sigma Chemicals; opsonized with a human serum) for 1 h the number of particles engulfed by the monocytes were counted using a light microscope. For measurements of monocyte adhesion MNC (5 x 10 6 cells) were supplemented with 100 mlll fetal calf serum and incubated for 30 min in plastic multi well cell culture dishes (Falcon; well diameter 1.6 cm). After removing non-adherent cells by rinsing the wells with buffered saline the amount of adherent cells was determined by their DNA content as previously described (13). Spontaneous platelet aggregation that occurred when samples of whole blood were stirred (900 rpm) for 10 min was assessed by counting the numbers of single platelets before and after stirring (8). Platelet counts were performed using the Coulter S plus VI. The results are given as arithmetic means ± sd. Means obtained in the control and C/BT group were proved for significant differences by Student's t-test.
Results Compared to controls the animals of the C/BT group had significantly enhanced plasma concentrations of C, HDL-C and LDL + VLDL-C as well as of TG (table 1). Furthermore, the numbers of blood monocytes and platelets were significantly increased in the C/BT group compared to controls. In contrast to that no differences were observed when blood lymphocytes, granulocytes and red cells were counted (table 2). For red cells and platelets we also determined the mean cellular volumes. Whereas no difference was found with respect to red cells the mean cellular volume of platelets was significantly lower in the C/BT group compared to controls (6.99 ± 0.87 fl versus 7.80 ± 0.90 fl, p < 0.05).
Table 1. Concentrations of serum lipids in control and C/BT pigs. C
HDL-C
VLDL + LDL-C
Control
2.78 ±0.40
1.22 ±0.17
1.56 ±0.36
0.38 ±0.05
C/BT
4.54** ±0.39
2.00** ±0.13
2.54** ±0.41
0.50* ±0.16
TG
All values are given in mmol!l. Significant differences between both groups are indicated: * p
