Br. J. exp. Path. (1978) 59, 213
EFFECT OF CARRAGEENAN ON ACTIVITY OF THE MONONUCLEAR PHAGOCYTE SYSTEM IN THE MOUSE E. F. FOWLER AND A. W. THOMSON From the Department of Pathology, Univer8ity Medical Building8, Foresterhill, Aberdeen AB9 2ZD Received for publication November 28, 1977
Summary.-Injection of 5 mg carrageenan caused hepatosplenomegaly and thymic involution and resulted in temporary blockade of the mononuclear phagocyte system (MPS). Impaired MPS activity was shown by decreased hepatic phagocytosis of i.v. injected colloidal carbon and 51Cr-labelled sheep red blood cells (SRBC). Depression of Kupffer cell activity was dependent on carrageenan dose, route of administration and interval between carrageenan and particle injection. Reduction in hepatic uptake of SRBC was accompanied by increased localization of these cells within the spleen. IT is now well established that the algal polysaccharide carrageenan, a high-molecular-weight sulphated polygalactan, is a potent suppressant of immune responses. It depresses both antibody production (Aschheim and Raffel, 1972; Bice et al.,
1972; Thomson et al., 1976) and cell mediated immunity (Bice et al., 1971; Rios and Simmons, 1972; Schwartz and Catanzaro, 1973) and has recently been shown to promote tumour growth in laboratory animals (Keller, 1976; Thomson and Fowler, 1977). In vitro studies have shown that carrageenan is non-toxic to lymphocytes but is phagocytosed by macrophages with consequent release of lysosomal enzymes and eventual cell death (Allison, Harington and Birbeck, 1966; Catanzaro, Schwartz and Graham, 1971). The in vivo immunosuppressive properties of carrageenan have been tentatively attributed to this selective toxicity for macrophages. However, there have been few attempts to analyse the effects of carrageenan on the intact MPS although preliminary observations by Rumjanek, Watson and Sljivic (1977) indicate that carrageenan interferes with the normal function of cells involved in the immune response. Previous workers have shown that pretreatment of animals with MPS "blockad-
ing" agents causes either suppression (Bradfield, Souhami and Addison, 1974; Smith et al., 1976; Souhami, 1972 )or enhancement (Sljivic, Clark and Warr, 1975) of hepatic phagocytosis. High molecular weight dextran sulphate (like carrageenan a sulphated polysaccharide) causes depression of hepatic phagocytosis and increased splenic uptake of SRBC. This shift in localization of antigenic particles has been correlated with the adjuvant activity of dextran sulphate (Diamantstein, Meinhold and Wagner, 1971). The purpose of this study was to determine the effect of carrageenan on MPS activity by measuring blood clearance of colloidal carbon and organ distribution of radiolabelled SRBC. MATERIALS AND METHODS Animals.-12-24-week-old closed-colony-bred female LACA mice, with a mean weight of 25 g, were used throughout. A minimum of 5 animals was used in each experimental group. They received a commercial diet and tapwater ad libitum. Carrageenan.-Potassium carrageenan (Sigma Chemical Company, Lot No. 71C-9011-9) was dissolved in phosphate-buffered saline as previously described (Fowler, Thomson and Horne, 1977). Mice received i.p. or i.v. injections of carrageenan solution (maximum concentration 10 mg/ml) at various times before injection of colloidal carbon or radiolabelled SRBC.
2)14
E. F. FOWLER AND A. W. THOMSON
Car bon clearance.-A series of diluitions of colloidal carbon (518, Giinther Wagner, PelikanWerke, Hanover, Germany) was prepared as described by gljivid (1969). The optical density of these dilutions was measured on a Cecil U.V. spectrophotometer at 800 nm with a 1 cm path length and a standard curve constructed from these readings. Each mouse received a single i.v. injection via a lateral tail vein of 0-3 ml colloidal carbon containing 100 ,lI original carbon suspension (I part carbon suspension to 2 parts 1% gelatin in distilled water). One hour after injection each animal was first anaesthetized with ether, then weighed and bled from the retro-orbital venous plexus. A 10,ul blood sample was taken using a disposable micropipette (Drummond "Microcaps") and lysed in 3 ml 1 % solution ofTriton X100. The optical density reading was used in conjunction with the standard curve to calculate the amount of carbon in the blood. Each animal was killed by cervical dislocation and liver tissue and spleen were removed and fixed in 10% neutral buffered formalin. Paraffin sections were cut at 5 ,um, then stained with haematoxylin and eosin before histological examination. Sections were coded and assessed "blind". For each liver the number of carbon containing cells in 100 highpower fields (x 270) was counted using an eyepiece graticule which allowed a total area (100 fields) of 1-96 mm2 to be scanned. Distribution of 51Cr-labelled SRBC.-Sheep red blood cells (Difco) were labelled with 51Cr (sodium chromate B.P., Radiochemical Centre, Amersham, England) as described by Smith et al. (1976). A concentration of 500 pCi per ml packed erythrocytes was used and the cells incubated at room temperature for 2 h. Each mouse was injected i.v. via a lateral tail vein with a single dose of 0-1 ml 10% erythrocyte suspension and mice were killed either 1 h or 24 h after injection. Immediately before killing a 50 pl blood sample was withdrawn from the retro-orbital venous plexus. The animals were killed by cervical dislocation and lungs, liver, spleen and kidneys were dissected out. Excess blood was removed using filter paper, and whole organs placed in LP4 plastic tubes (Sarstedt). Radioactivity was measured in a well type scintillation counter (Nuclear-Chicago) and results expressed as a percent total injected dose. Statistics.-Student's t test was used for statistical analysis.
100 90
Mean Spleen weight ± S.E.
80 _ 70 _-
60 _ 0r
50 _
40 _ E *8i
Mean Thymus weight + S.E. Aggregate weight of 10 lymph
30 _ 20
nodes
10 _
0' L-
_
0
1
2 3 4 Days after carrageenan injection
5
FIG. 1.-Effect of 5 mg carrageenan i.p. on spleen, thymus and axillary lymph node weights at various times after injection.
weights over the 5-day period following injection. There was a marked increase in spleen weight (P
EFFECT OF CARRAGEENAN ON ACTIVITY OF THE MONONUCLEAR PHAGOCYTE SYSTEM IN THE MOUSE E. F. FOWLER AND A. W. THOMSON From the Department of Pathology, Univer8ity Medical Building8, Foresterhill, Aberdeen AB9 2ZD Received for publication November 28, 1977
Summary.-Injection of 5 mg carrageenan caused hepatosplenomegaly and thymic involution and resulted in temporary blockade of the mononuclear phagocyte system (MPS). Impaired MPS activity was shown by decreased hepatic phagocytosis of i.v. injected colloidal carbon and 51Cr-labelled sheep red blood cells (SRBC). Depression of Kupffer cell activity was dependent on carrageenan dose, route of administration and interval between carrageenan and particle injection. Reduction in hepatic uptake of SRBC was accompanied by increased localization of these cells within the spleen. IT is now well established that the algal polysaccharide carrageenan, a high-molecular-weight sulphated polygalactan, is a potent suppressant of immune responses. It depresses both antibody production (Aschheim and Raffel, 1972; Bice et al.,
1972; Thomson et al., 1976) and cell mediated immunity (Bice et al., 1971; Rios and Simmons, 1972; Schwartz and Catanzaro, 1973) and has recently been shown to promote tumour growth in laboratory animals (Keller, 1976; Thomson and Fowler, 1977). In vitro studies have shown that carrageenan is non-toxic to lymphocytes but is phagocytosed by macrophages with consequent release of lysosomal enzymes and eventual cell death (Allison, Harington and Birbeck, 1966; Catanzaro, Schwartz and Graham, 1971). The in vivo immunosuppressive properties of carrageenan have been tentatively attributed to this selective toxicity for macrophages. However, there have been few attempts to analyse the effects of carrageenan on the intact MPS although preliminary observations by Rumjanek, Watson and Sljivic (1977) indicate that carrageenan interferes with the normal function of cells involved in the immune response. Previous workers have shown that pretreatment of animals with MPS "blockad-
ing" agents causes either suppression (Bradfield, Souhami and Addison, 1974; Smith et al., 1976; Souhami, 1972 )or enhancement (Sljivic, Clark and Warr, 1975) of hepatic phagocytosis. High molecular weight dextran sulphate (like carrageenan a sulphated polysaccharide) causes depression of hepatic phagocytosis and increased splenic uptake of SRBC. This shift in localization of antigenic particles has been correlated with the adjuvant activity of dextran sulphate (Diamantstein, Meinhold and Wagner, 1971). The purpose of this study was to determine the effect of carrageenan on MPS activity by measuring blood clearance of colloidal carbon and organ distribution of radiolabelled SRBC. MATERIALS AND METHODS Animals.-12-24-week-old closed-colony-bred female LACA mice, with a mean weight of 25 g, were used throughout. A minimum of 5 animals was used in each experimental group. They received a commercial diet and tapwater ad libitum. Carrageenan.-Potassium carrageenan (Sigma Chemical Company, Lot No. 71C-9011-9) was dissolved in phosphate-buffered saline as previously described (Fowler, Thomson and Horne, 1977). Mice received i.p. or i.v. injections of carrageenan solution (maximum concentration 10 mg/ml) at various times before injection of colloidal carbon or radiolabelled SRBC.
2)14
E. F. FOWLER AND A. W. THOMSON
Car bon clearance.-A series of diluitions of colloidal carbon (518, Giinther Wagner, PelikanWerke, Hanover, Germany) was prepared as described by gljivid (1969). The optical density of these dilutions was measured on a Cecil U.V. spectrophotometer at 800 nm with a 1 cm path length and a standard curve constructed from these readings. Each mouse received a single i.v. injection via a lateral tail vein of 0-3 ml colloidal carbon containing 100 ,lI original carbon suspension (I part carbon suspension to 2 parts 1% gelatin in distilled water). One hour after injection each animal was first anaesthetized with ether, then weighed and bled from the retro-orbital venous plexus. A 10,ul blood sample was taken using a disposable micropipette (Drummond "Microcaps") and lysed in 3 ml 1 % solution ofTriton X100. The optical density reading was used in conjunction with the standard curve to calculate the amount of carbon in the blood. Each animal was killed by cervical dislocation and liver tissue and spleen were removed and fixed in 10% neutral buffered formalin. Paraffin sections were cut at 5 ,um, then stained with haematoxylin and eosin before histological examination. Sections were coded and assessed "blind". For each liver the number of carbon containing cells in 100 highpower fields (x 270) was counted using an eyepiece graticule which allowed a total area (100 fields) of 1-96 mm2 to be scanned. Distribution of 51Cr-labelled SRBC.-Sheep red blood cells (Difco) were labelled with 51Cr (sodium chromate B.P., Radiochemical Centre, Amersham, England) as described by Smith et al. (1976). A concentration of 500 pCi per ml packed erythrocytes was used and the cells incubated at room temperature for 2 h. Each mouse was injected i.v. via a lateral tail vein with a single dose of 0-1 ml 10% erythrocyte suspension and mice were killed either 1 h or 24 h after injection. Immediately before killing a 50 pl blood sample was withdrawn from the retro-orbital venous plexus. The animals were killed by cervical dislocation and lungs, liver, spleen and kidneys were dissected out. Excess blood was removed using filter paper, and whole organs placed in LP4 plastic tubes (Sarstedt). Radioactivity was measured in a well type scintillation counter (Nuclear-Chicago) and results expressed as a percent total injected dose. Statistics.-Student's t test was used for statistical analysis.
100 90
Mean Spleen weight ± S.E.
80 _ 70 _-
60 _ 0r
50 _
40 _ E *8i
Mean Thymus weight + S.E. Aggregate weight of 10 lymph
30 _ 20
nodes
10 _
0' L-
_
0
1
2 3 4 Days after carrageenan injection
5
FIG. 1.-Effect of 5 mg carrageenan i.p. on spleen, thymus and axillary lymph node weights at various times after injection.
weights over the 5-day period following injection. There was a marked increase in spleen weight (P
