Immunology 1979 37 689
Stimulation of phosphatidylinositol turnover in the macrophage plasma membrane: a possible mechanism for signal transmission
HELGA M. OGMUNDSDOTTIR & D. M. WEIR, Department of Bacteriology, University of Edinburgh Medical School
Received 18 December 1978; acceptedfor publication 17 January 1979
Summary. The effect of various stimuli on the rate of phosphatidylinositol turnover by mouse peritoneal macrophages was studied by measuring the incorporation of myo-[2-3H]-inositol. It was found that the macrophage-activating agents endotoxin and Corynebacterium parvum caused an increase in phosphatidylinositol turnover whilst inert particles (Staphylococcus albus, latex and carbon) had no effect even though they were phagocytosed. The stimulation by C. parvum was dependent on the presence of T cells. T lymphocytes from normal mice and mice immunized with C. parvum were equally effective and it was found that both normal and immune T cells could be stimulated by C. parvum as indicated by an increased uptake of [6-3H]-thymidine. Supernatants from normal or immune spleen cells cultured with and without C. parvum were ineffective and could not replace intact T cells, indicating that cell to cell contact is required or a labile factor that acts over a short range. The time course of stimulation of phosphatidylinositol turnover by macrophages was studied using endotoxin. The rate of turnover increased slowly over a few hours and was still rising at 6 h but decreasing again at 24 h. The increase in bacteriostatic activity against Listeria monocytogenes by macrophages exposed to endotoxin
took longer to develop and was more marked at 24 h than at 4 h. The differences between pathways leading to phagocytosis or chemotaxis and those resulting in activation are discussed.
INTRODUCTION The biochemical pathways that are set in motion by substances that act on the macrophage plasma membrane are poorly understood although the widely recognized effects of interaction such as 'macrophage activation' and phagocytosis are of importance in antibacterial and tumour immunity (Keller, 1977). The transmission of signals across the plasma membrane frequently involves ion fluxes and can be mediated by second messengers such as cyclic nucleotides. Interaction with a cell surface receptor often leads to an early increase in the turnover of the phosphorylinositol headgroup of membrane lipid phosphatidylinositol and has been found to occur in many cell types including lymphocytes (Maino, Hayman & Crumpton, 1975; Michell, 1975). A model has been proposed by Michell, Jones & Jafferji (1977) where the surface interaction leads to a transduction step of increased phosphatidylinositol turnover which causes release of membrane bound Ca2+ and opening of membrane Ca2+ gates. This is followed by an amplification step of increasing intracellular concentration of Ca2+ which triggers intracellular responses. The rate of phosphatidylinositol turnover is reflected in the rate
Correspondence: Dr D. M. Weir, Immunology Laboratory, Department of Bacteriology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. 00 1 9-2805/79/0700-0689$02.00
C) 1979 Blackwell Scientific Publications 689
Helga M. Ogmundsdottir & D. M. Weir
690
of uptake of 32Pi and labelled inositol (Michell, 1975) and in this work we have measured myo-[2-3H]inositol incorporation in mouse macrophages exposed to the macrophage activating agents bacterial endotoxin (Alexander & Evans, 1971) and C. parvum (Ghaffar, Cullen, Dunbar & Woodruff, 1974) and compared their effects to those of particles that are phagocytosed but do not cause activation (carbon, latex, and S. albus). MATERIALS AND METHODS Animals C3Hf/Bu mice, SPF, age 6-10 weeks were obtained from the departmental breeding colony. Reagents
Myo-[2-3H]-inositol and [6-3H]-thymidine were obtained from the Radiochemical Centre, Amersham. Lipopolysaccharide W (endotoxin) from E. coli 055: B5 and latex particles (0 81 jm) came from Difco Laboratories, Detroit. A stock suspension of colloidal carbon was a kind gift from the Department of Pathology, University of Edinburgh. Corynebacterium parvum (strain 10390), originally obtained from the National Collection of Type Cultures, Colindale, was grown anaerobically in horse digest broth containing 3% glucose. Staphylococcus albus was grown aerobically in nutrient broth. The bacteria were harvested in log phase, killed by 24 h exposure at 4° to 0 5,' formalin in saline and washed four times with saline. Listeria monocytogenes was grown aerobically in nutrient broth and washed three times in Dulbecco's phosphate-buffered saline (PBS) before use. Bacteria were counted in a Neubauer chamber 0-01 mm depth. Foetal calf serum was obtained from Sera-lab, Crawley Down, Sussex. The serum was heat-inactivated for 30 min at 56° before use. Harvesting of peritoneal exudate cells and culture of macrophages The mice were injected i.p. with 2 5 ml Dulbecco's PBS containing 10 i.u./ml of heparin. After gentle massage the fluid was withdrawn by means of a syringe. Peritoneal fluid from eighteen to twenty-four mice was pooled for each of the inositol-uptake experiments. The cells were kept on ice, centrifuged at 40, 250 g for 7 min and resuspended in Eagle's medium (MEM, Wellcome Research Laboratories, Beckenham) to a con-
centration of approximately 25-30 x 106 cells/ml. This suspension was transferred into 13 x 150 mm culture tubes in 3 ml amounts and incubated on a slope for 90 min at 37°. The tubes were then washed seven times with Dulbecco's PBS containing Ca2+ and Mg2+ ions (Dulbecco's PBS + B) to remove non-adherent cells and the adherent cells (macrophages) were reincubated overnight at 370 in Eagle's medium containing 5% foetal calf serum, before use in the assay. An aliquot of the cell suspension was put on to two 'flying' coverslips which were incubated and washed in the same way as the tubes and the adherent cells counted by inverting them on to a haemocytometer (Improved Neubauer). Each tube usually contained approximately 3 x 106 adherent cells; of these approximately 95% were macrophages as shown by phagocytosis of antibody-coated C. parvum on coverslip preparations. The degree of phagocytosis of test particles used in the inositol-uptake experiments was tested in parallel cultures on coverslips which were gramstained and examined microscopically or left unfixed and unstained and counted using phase contrast optics in the case of latex particles. Immunization with C. parvum C. parvum was injected i.p. into the mice at a dose of 3 x 1010 organisms (0 7 mg dry weight) in 0 I ml saline. Spleens from these animals were used 14 days later at which time there was a marked splenomegaly and a high titre of serum antibodies. The spleen cells from these animals will be referred to as immune cells. Preparation of spleen cell cultures and separation of T cells Spleen cell suspensions from normal and immune mice were prepared by pushing one to two spleens gently through a sterile wire mesh. The cells were centrifuged at 40 for 7 min at 250 g and red blood cells were removed by resuspending the cells in a buffer composed of 0 155 M NH4CI, 0 1 mM EDTA and 0-01 M KHCO3 for 4 min. The cells were centrifuged and finally resuspended in tissue culture medium. For the production of culture supernatants the spleen cell suspension was adjusted to 2 x 106 cells/ml and cultured for 4 h at 37° in 10 ml of Eagle's medium without serum in tissue culture bottles in the presence or absence of C. parvum at a final concentration of 2 x Io8 bact./ml. The supernatants were harvested by centrifuging at 500 g for 15 min at 40 and centrifuging the resulting supernatant again at 2000 g for 20 min at 4°.
Stimulation ofphosphatidylinositol turnover The supernatants were stored overnight at 00 before use. T cells were separated from a spleen cell suspension on a nylon wool column as described by Hunt (1978). Preparation oflipid carrier from livers The lipids were extracted from 25 g of homogenized mouse livers with 50 ml of a 2: 1 mixture of chloroform and methanol for 20 min at 37°. After the addition of 25 ml of 0 5 M MgCI2 the mixture was centrifuged for 10 min at 300 g. The upper aqueous phase was discarded and the organic phase withdrawn from underneath the compressed liver tissue. The organic phase was then equilibrated against 0 5 M MgCl2 in the same way as before, the aqueous phase discarded again and residual water removed from the organic phase with anhydrous Na2SO4. The organic phase was finally evaporated to dryness under vacuum and redissolved in chloroform-methanol to give a concentration of extract from 2 g of liver per ml and stored at -20°. For use in the assay the extract was diluted 1: 10 in chloro-
form-methanol. Assay for uptake of myo-[2-3H]-inositol After the macrophages had been incubated overnight with Eagle's medium with 5% foetal calf serum they were washed with Dulbecco's PBS + B and all subsequent steps performed in Eagle's medium without serum. The cells were then incubated with various test particles and substances usually for 4 h at 37°, 2 ,Ci of myo-[2-3H]-inositol were added in 0 I ml of medium to each tube one hour before the end of the culture period. The cell metabolism was then stopped by putting the tubes into ice. They were then washed 7 times with Dulbecco's PBS and 5 ml of chloroform-methanol added to each tube. The lipids were extracted for 20 min at 370 with vigorous shaking on a Rotamixer every 5 min. Two millilitres of liver lipid extract were then added as a carrier and 3 ml of 0 5 M MgCl2 and mixed well. The phases were separated by centrifugation and the organic phase equilibrated once more against 0 5 M MgCl2 as described above. The organic phase was then transferred into liquid scintillation counting vials and evaporated to dryness at 400 under a stream of N2. Toluene-based scintillation fluid containing PPO and POPOP was added and radioactivity counted in a Packard Scintillation spectrometer. Thin layer chromatography separation of the lipid phase confirmed that all the radioactivity was associated with phosphatidylinositol.
691
Assay of DNA-synthesis by T lymphocytes T lymphocytes, separated on a nylon wool column were incubated in flat bottom microtitre tissue culture plates (Sterilin, Teddington, Middlesex) in 0 2 ml of Eagle's medium with 10% foetal calf serum and 0 04 mM 2-mercaptoethanol. The microtitre plates had been prepared by incubating them for 90 min with peritoneal exudate cells. The non-adherent cells were washed away leaving the macrophages in a final number of approximately 5% of the number of T lymphocytes. C.parvum was added in 10 p1 amounts to give the final ratios of 100 and 500 bacteria per cell. These microtitre plates were incubated for 3 days at 370, 4 h before the end of the culture period the medium was gently removed and replaced by medium containing [6-3H]-thymidine, 0 2 pCi per well. The non-adherent cells were then precipitated on to Whatman glass fibre filters with 10% TCA in a sampling manifold (Millipore Corporation, Bedford, Massachusetts). Radioactivity was then counted in a Packard Scintillation spectrometer as described above. Bacteriostatic effect of macrophages against Listeria monocytogenes Peritoneal exudate macrophages were cultured on 'flying' glass coverslips in the manner described above. The coverslips were incubated with E. coli lipopolysaccharide (60 pg/106 cells) in medium without serum for 4 h or 24 h. The cells were then washed and overlayed with a suspension of Listeria monocytogenes in Eagle's medium containing 15% foetal calf serum but no antibiotics to give a ratio of 7-10 bact./cell. The coverslips were then incubated for 30 min at 370 after which they were washed six times with Dulbecco's PBS + B. Half of the coverslips were then air-dried, heat-fixed and gram-stained and the remaining half reincubated with Eagle's medium with 15% foetal calf serum and no antibiotics for 4 h and then processed in the same way. The coverslips were examined microscopically, the number of cells per field, the number with ingested bacteria and the number of intracellular bacteria were counted; twelve to eighteen fields were counted on each slide. For comparison of bacteriostatic effect the number of intracellular bacteria per total number of cells was calculated as this takes account of the proportion of cells containing bacteria. This figure was used to calculate the growth ratio of L. monocytogenes by dividing the number at 4 h by the number at 30 min. The relative bacteriostatic effect of lipopolysaccharide-treated cells v. controls was calculated as growth ratio in controls/growth ratio in treated cells.
692
Helga M. Ogmundsd6ttir & D. M. Weir
RESULTS Turnover of phosphatidylinositol by macrophages exposed to various stimuli Macrophage monolayers were exposed to various stimuli in serum-free medium in vitro and the effect on the uptake of myo-[2-3H]-inositol measured. The results are shown in Table 1, which also gives the concentrations of substances and particles used. It can be seen that both endotoxin and C. parvum cause an increase in the uptake of labelled inositol indicating an increased rate of phosphatidylinositol turnover. Endotoxin has a stimulatory effect both with and without T lymphocytes whilst C. parvum is effective only in the presence of T cells. The ratio of T cells to macrophages was 1: 5. All the test particles were ingested by macrophages, C. parvum was taken up by 33% of the cells, latex particles by 45%, carbon particles by 16% and S. albus by 23%, all during 4 h in serum-free medium. No difference was observed in the number of adherent cells at the end of the incubation period between control and treated cultures. Requirement for T lymphocytes for stimulation of macrophages by C. parvum The nature of the requirement for T cells for the
stimulation of phosphatidylinositol turnover by macrophages caused by C. parvum was investigated further. The requirement seemed absolute and even prolonged exposure (up to 4 days) to C. parvum without T cells had no stimulatory effect. Table 2 shows that T cells from animals immunized 14 days previously with C. parvum were no more effective than T cells from normal animals. In order to test whether the effect of the T cells was mediated by a soluble factor, supernatants were prepared from spleen cells cultured for 4 h under conditions equivalent to those used in the assay. All the supernatants were inactive regardless of whether they were derived from cultures of normal or immune spleen cells and whether or not they had been exposed to C. parvum during the culture period (see Table 3). It can also be seen that the supernatants failed to substitute for intact T cells when the macrophages were treated with C. parvum. The presence of T cells had no effect on the degree of uptake of C. parvum by macrophages. Small cells were seen to cluster around macrophages at the end of the culture period but after the final washing procedure none of these remained and the number of adherent cells was the same as in control cultures. It is therefore unlikely that T cells contributed to the radioactivity measured.
Table 1. The effect of various stimuli on the uptake of myo-[2-3H]-inositol by macrophages. The figures represent means of pooled data from two to three experiments, each done in triplicate. Significance of differences was determined using Student's t test (two-tailed). P values < 0-05 are considered significant, n.s. = not significant
Uptake of myo-[2-3H]-inositol, percentage of unstimulated controls
Macrophages exposed for 4 h to: 129 1 SE 47 Endotoxin P
Stimulation of phosphatidylinositol turnover in the macrophage plasma membrane: a possible mechanism for signal transmission
HELGA M. OGMUNDSDOTTIR & D. M. WEIR, Department of Bacteriology, University of Edinburgh Medical School
Received 18 December 1978; acceptedfor publication 17 January 1979
Summary. The effect of various stimuli on the rate of phosphatidylinositol turnover by mouse peritoneal macrophages was studied by measuring the incorporation of myo-[2-3H]-inositol. It was found that the macrophage-activating agents endotoxin and Corynebacterium parvum caused an increase in phosphatidylinositol turnover whilst inert particles (Staphylococcus albus, latex and carbon) had no effect even though they were phagocytosed. The stimulation by C. parvum was dependent on the presence of T cells. T lymphocytes from normal mice and mice immunized with C. parvum were equally effective and it was found that both normal and immune T cells could be stimulated by C. parvum as indicated by an increased uptake of [6-3H]-thymidine. Supernatants from normal or immune spleen cells cultured with and without C. parvum were ineffective and could not replace intact T cells, indicating that cell to cell contact is required or a labile factor that acts over a short range. The time course of stimulation of phosphatidylinositol turnover by macrophages was studied using endotoxin. The rate of turnover increased slowly over a few hours and was still rising at 6 h but decreasing again at 24 h. The increase in bacteriostatic activity against Listeria monocytogenes by macrophages exposed to endotoxin
took longer to develop and was more marked at 24 h than at 4 h. The differences between pathways leading to phagocytosis or chemotaxis and those resulting in activation are discussed.
INTRODUCTION The biochemical pathways that are set in motion by substances that act on the macrophage plasma membrane are poorly understood although the widely recognized effects of interaction such as 'macrophage activation' and phagocytosis are of importance in antibacterial and tumour immunity (Keller, 1977). The transmission of signals across the plasma membrane frequently involves ion fluxes and can be mediated by second messengers such as cyclic nucleotides. Interaction with a cell surface receptor often leads to an early increase in the turnover of the phosphorylinositol headgroup of membrane lipid phosphatidylinositol and has been found to occur in many cell types including lymphocytes (Maino, Hayman & Crumpton, 1975; Michell, 1975). A model has been proposed by Michell, Jones & Jafferji (1977) where the surface interaction leads to a transduction step of increased phosphatidylinositol turnover which causes release of membrane bound Ca2+ and opening of membrane Ca2+ gates. This is followed by an amplification step of increasing intracellular concentration of Ca2+ which triggers intracellular responses. The rate of phosphatidylinositol turnover is reflected in the rate
Correspondence: Dr D. M. Weir, Immunology Laboratory, Department of Bacteriology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland. 00 1 9-2805/79/0700-0689$02.00
C) 1979 Blackwell Scientific Publications 689
Helga M. Ogmundsdottir & D. M. Weir
690
of uptake of 32Pi and labelled inositol (Michell, 1975) and in this work we have measured myo-[2-3H]inositol incorporation in mouse macrophages exposed to the macrophage activating agents bacterial endotoxin (Alexander & Evans, 1971) and C. parvum (Ghaffar, Cullen, Dunbar & Woodruff, 1974) and compared their effects to those of particles that are phagocytosed but do not cause activation (carbon, latex, and S. albus). MATERIALS AND METHODS Animals C3Hf/Bu mice, SPF, age 6-10 weeks were obtained from the departmental breeding colony. Reagents
Myo-[2-3H]-inositol and [6-3H]-thymidine were obtained from the Radiochemical Centre, Amersham. Lipopolysaccharide W (endotoxin) from E. coli 055: B5 and latex particles (0 81 jm) came from Difco Laboratories, Detroit. A stock suspension of colloidal carbon was a kind gift from the Department of Pathology, University of Edinburgh. Corynebacterium parvum (strain 10390), originally obtained from the National Collection of Type Cultures, Colindale, was grown anaerobically in horse digest broth containing 3% glucose. Staphylococcus albus was grown aerobically in nutrient broth. The bacteria were harvested in log phase, killed by 24 h exposure at 4° to 0 5,' formalin in saline and washed four times with saline. Listeria monocytogenes was grown aerobically in nutrient broth and washed three times in Dulbecco's phosphate-buffered saline (PBS) before use. Bacteria were counted in a Neubauer chamber 0-01 mm depth. Foetal calf serum was obtained from Sera-lab, Crawley Down, Sussex. The serum was heat-inactivated for 30 min at 56° before use. Harvesting of peritoneal exudate cells and culture of macrophages The mice were injected i.p. with 2 5 ml Dulbecco's PBS containing 10 i.u./ml of heparin. After gentle massage the fluid was withdrawn by means of a syringe. Peritoneal fluid from eighteen to twenty-four mice was pooled for each of the inositol-uptake experiments. The cells were kept on ice, centrifuged at 40, 250 g for 7 min and resuspended in Eagle's medium (MEM, Wellcome Research Laboratories, Beckenham) to a con-
centration of approximately 25-30 x 106 cells/ml. This suspension was transferred into 13 x 150 mm culture tubes in 3 ml amounts and incubated on a slope for 90 min at 37°. The tubes were then washed seven times with Dulbecco's PBS containing Ca2+ and Mg2+ ions (Dulbecco's PBS + B) to remove non-adherent cells and the adherent cells (macrophages) were reincubated overnight at 370 in Eagle's medium containing 5% foetal calf serum, before use in the assay. An aliquot of the cell suspension was put on to two 'flying' coverslips which were incubated and washed in the same way as the tubes and the adherent cells counted by inverting them on to a haemocytometer (Improved Neubauer). Each tube usually contained approximately 3 x 106 adherent cells; of these approximately 95% were macrophages as shown by phagocytosis of antibody-coated C. parvum on coverslip preparations. The degree of phagocytosis of test particles used in the inositol-uptake experiments was tested in parallel cultures on coverslips which were gramstained and examined microscopically or left unfixed and unstained and counted using phase contrast optics in the case of latex particles. Immunization with C. parvum C. parvum was injected i.p. into the mice at a dose of 3 x 1010 organisms (0 7 mg dry weight) in 0 I ml saline. Spleens from these animals were used 14 days later at which time there was a marked splenomegaly and a high titre of serum antibodies. The spleen cells from these animals will be referred to as immune cells. Preparation of spleen cell cultures and separation of T cells Spleen cell suspensions from normal and immune mice were prepared by pushing one to two spleens gently through a sterile wire mesh. The cells were centrifuged at 40 for 7 min at 250 g and red blood cells were removed by resuspending the cells in a buffer composed of 0 155 M NH4CI, 0 1 mM EDTA and 0-01 M KHCO3 for 4 min. The cells were centrifuged and finally resuspended in tissue culture medium. For the production of culture supernatants the spleen cell suspension was adjusted to 2 x 106 cells/ml and cultured for 4 h at 37° in 10 ml of Eagle's medium without serum in tissue culture bottles in the presence or absence of C. parvum at a final concentration of 2 x Io8 bact./ml. The supernatants were harvested by centrifuging at 500 g for 15 min at 40 and centrifuging the resulting supernatant again at 2000 g for 20 min at 4°.
Stimulation ofphosphatidylinositol turnover The supernatants were stored overnight at 00 before use. T cells were separated from a spleen cell suspension on a nylon wool column as described by Hunt (1978). Preparation oflipid carrier from livers The lipids were extracted from 25 g of homogenized mouse livers with 50 ml of a 2: 1 mixture of chloroform and methanol for 20 min at 37°. After the addition of 25 ml of 0 5 M MgCI2 the mixture was centrifuged for 10 min at 300 g. The upper aqueous phase was discarded and the organic phase withdrawn from underneath the compressed liver tissue. The organic phase was then equilibrated against 0 5 M MgCl2 in the same way as before, the aqueous phase discarded again and residual water removed from the organic phase with anhydrous Na2SO4. The organic phase was finally evaporated to dryness under vacuum and redissolved in chloroform-methanol to give a concentration of extract from 2 g of liver per ml and stored at -20°. For use in the assay the extract was diluted 1: 10 in chloro-
form-methanol. Assay for uptake of myo-[2-3H]-inositol After the macrophages had been incubated overnight with Eagle's medium with 5% foetal calf serum they were washed with Dulbecco's PBS + B and all subsequent steps performed in Eagle's medium without serum. The cells were then incubated with various test particles and substances usually for 4 h at 37°, 2 ,Ci of myo-[2-3H]-inositol were added in 0 I ml of medium to each tube one hour before the end of the culture period. The cell metabolism was then stopped by putting the tubes into ice. They were then washed 7 times with Dulbecco's PBS and 5 ml of chloroform-methanol added to each tube. The lipids were extracted for 20 min at 370 with vigorous shaking on a Rotamixer every 5 min. Two millilitres of liver lipid extract were then added as a carrier and 3 ml of 0 5 M MgCl2 and mixed well. The phases were separated by centrifugation and the organic phase equilibrated once more against 0 5 M MgCl2 as described above. The organic phase was then transferred into liquid scintillation counting vials and evaporated to dryness at 400 under a stream of N2. Toluene-based scintillation fluid containing PPO and POPOP was added and radioactivity counted in a Packard Scintillation spectrometer. Thin layer chromatography separation of the lipid phase confirmed that all the radioactivity was associated with phosphatidylinositol.
691
Assay of DNA-synthesis by T lymphocytes T lymphocytes, separated on a nylon wool column were incubated in flat bottom microtitre tissue culture plates (Sterilin, Teddington, Middlesex) in 0 2 ml of Eagle's medium with 10% foetal calf serum and 0 04 mM 2-mercaptoethanol. The microtitre plates had been prepared by incubating them for 90 min with peritoneal exudate cells. The non-adherent cells were washed away leaving the macrophages in a final number of approximately 5% of the number of T lymphocytes. C.parvum was added in 10 p1 amounts to give the final ratios of 100 and 500 bacteria per cell. These microtitre plates were incubated for 3 days at 370, 4 h before the end of the culture period the medium was gently removed and replaced by medium containing [6-3H]-thymidine, 0 2 pCi per well. The non-adherent cells were then precipitated on to Whatman glass fibre filters with 10% TCA in a sampling manifold (Millipore Corporation, Bedford, Massachusetts). Radioactivity was then counted in a Packard Scintillation spectrometer as described above. Bacteriostatic effect of macrophages against Listeria monocytogenes Peritoneal exudate macrophages were cultured on 'flying' glass coverslips in the manner described above. The coverslips were incubated with E. coli lipopolysaccharide (60 pg/106 cells) in medium without serum for 4 h or 24 h. The cells were then washed and overlayed with a suspension of Listeria monocytogenes in Eagle's medium containing 15% foetal calf serum but no antibiotics to give a ratio of 7-10 bact./cell. The coverslips were then incubated for 30 min at 370 after which they were washed six times with Dulbecco's PBS + B. Half of the coverslips were then air-dried, heat-fixed and gram-stained and the remaining half reincubated with Eagle's medium with 15% foetal calf serum and no antibiotics for 4 h and then processed in the same way. The coverslips were examined microscopically, the number of cells per field, the number with ingested bacteria and the number of intracellular bacteria were counted; twelve to eighteen fields were counted on each slide. For comparison of bacteriostatic effect the number of intracellular bacteria per total number of cells was calculated as this takes account of the proportion of cells containing bacteria. This figure was used to calculate the growth ratio of L. monocytogenes by dividing the number at 4 h by the number at 30 min. The relative bacteriostatic effect of lipopolysaccharide-treated cells v. controls was calculated as growth ratio in controls/growth ratio in treated cells.
692
Helga M. Ogmundsd6ttir & D. M. Weir
RESULTS Turnover of phosphatidylinositol by macrophages exposed to various stimuli Macrophage monolayers were exposed to various stimuli in serum-free medium in vitro and the effect on the uptake of myo-[2-3H]-inositol measured. The results are shown in Table 1, which also gives the concentrations of substances and particles used. It can be seen that both endotoxin and C. parvum cause an increase in the uptake of labelled inositol indicating an increased rate of phosphatidylinositol turnover. Endotoxin has a stimulatory effect both with and without T lymphocytes whilst C. parvum is effective only in the presence of T cells. The ratio of T cells to macrophages was 1: 5. All the test particles were ingested by macrophages, C. parvum was taken up by 33% of the cells, latex particles by 45%, carbon particles by 16% and S. albus by 23%, all during 4 h in serum-free medium. No difference was observed in the number of adherent cells at the end of the incubation period between control and treated cultures. Requirement for T lymphocytes for stimulation of macrophages by C. parvum The nature of the requirement for T cells for the
stimulation of phosphatidylinositol turnover by macrophages caused by C. parvum was investigated further. The requirement seemed absolute and even prolonged exposure (up to 4 days) to C. parvum without T cells had no stimulatory effect. Table 2 shows that T cells from animals immunized 14 days previously with C. parvum were no more effective than T cells from normal animals. In order to test whether the effect of the T cells was mediated by a soluble factor, supernatants were prepared from spleen cells cultured for 4 h under conditions equivalent to those used in the assay. All the supernatants were inactive regardless of whether they were derived from cultures of normal or immune spleen cells and whether or not they had been exposed to C. parvum during the culture period (see Table 3). It can also be seen that the supernatants failed to substitute for intact T cells when the macrophages were treated with C. parvum. The presence of T cells had no effect on the degree of uptake of C. parvum by macrophages. Small cells were seen to cluster around macrophages at the end of the culture period but after the final washing procedure none of these remained and the number of adherent cells was the same as in control cultures. It is therefore unlikely that T cells contributed to the radioactivity measured.
Table 1. The effect of various stimuli on the uptake of myo-[2-3H]-inositol by macrophages. The figures represent means of pooled data from two to three experiments, each done in triplicate. Significance of differences was determined using Student's t test (two-tailed). P values < 0-05 are considered significant, n.s. = not significant
Uptake of myo-[2-3H]-inositol, percentage of unstimulated controls
Macrophages exposed for 4 h to: 129 1 SE 47 Endotoxin P
