Phosphatidylcholine participates in the interaction between macrophages and lymphocytes

Anita Nishiyama-Naruke and Rui Curi

Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, CEP 05508-900 Brazil


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The role of phosphatidylcholine molecules as mediator for the control of lymphocyte proliferation by macrophages was investigated. Phosphatidylcholine added to the culture medium inhibited the concanavalin A-stimulated lymphocyte proliferation in a concentration-dependent manner. The potency of this effect was dependent on the presence of arachidonic acid in the phosphatidylcholine molecules. The phosphatidylcholine transfer from macrophages to lymphocytes was then investigated. Macrophages incorporated phosphatidylcholine at a much higher rate than lymphocytes and exported phosphatidylcholine to the culture medium. When cocultured, a significant amount of phosphatidylcholine incorporated by macrophages was transferred to lymphocytes. To examine the possible physiological importance of the transfer process, the lymphocyte proliferation was measured in coculture conditions. Macrophages were treated with phosphatidylcholine and washed, and then these cells were cocultured with concanavalin A-stimulated lymphocytes. The effect observed in coculture was an inhibition of lymphocyte proliferation, which was also dependent on the molecular species of the phosphatidylcholine. Therefore, phosphatidylcholine may act as a mediator of the macrophage effect on lymphocyte proliferation.

transfer; macrophages; lymphocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PREVIOUS STUDIES HAVE SHOWN functional effects of phosphatidylcholine and its products either in cultured macrophages and lymphocytes or in living animals. Phospholipid-containing liposomes downmodulate macrophage antileishmanial activities, suppress tumor necrosis factor production, and increase nitric oxide synthesis (16, 22, 26). Macrophages constitute part of the mononuclear phagocyte system and they are the main site of clearance of liposomes. These cells are most likely to sustain damage after liposome delivery and then the administration of phospholipid-containing liposomes is immunosuppressive, possibly by inhibiting the mononuclear phagocyte system (28). In the lung, alveolar macrophages and lymphocytes are in direct contact with surfactant, presenting a high phosphatidylcholine content (33). Experiments have demonstrated that lung lymphocytes are less responsive to concanavalin A, phytohemagglutinin, and pokeweed mitogen than peripheral lymphocytes (2, 4). The mechanism for the lower proliferative capacity of lung lymphocytes is not clear but part of the reduced activity has been assumed to be accounted for by the presence of phosphatidylcholine (23) in the microenvironment.

Products of phosphatidylcholine hydrolysis, including fatty acids, lysophosphatidylcholine, platelet-activating factor, choline, phosphatidic acid, and diacylglycerol participate in intra- and intercellular signaling that affects macrophage and lymphocyte function and, therefore, immune and inflammatory responses (3, 13, 25, 31, 32).

Considering that both arachidonic acid and phosphatidylcholine can regulate lymphocyte proliferation, it remains to be determined whether the phosphatidylcholine effect depends on its fatty acid composition in this molecule. To examine this point, in the first part of this study the effects on lymphocyte proliferation of an arachidonic acid-rich phosphatidylcholine and an arachidonic acid-poor phosphatidylcholine were compared.

Macrophages and lymphocytes can synthesize lipids, including phospholipids, from glucose and glutamine (12, 18). The lipids produced from glucose and glutamine in lymphocytes and macrophages are incorporated into the cells or exported to the culture medium. It is noteworthy that the amount of lipid exported to the culture medium varies with the activation state of the cell (19). In a recent study, evidence was obtained that cholesterol and fatty acids are exported from macrophages and transferred to lymphocytes (19, 29). Taking into account that lymphocytes and macrophages interact in vivo, the phospholipid transfer between these cells could play an important part in the regulation of immune and inflammatory responses. To address this point we measured the following: incorporation and export capacities of L-3-phosphatidylcholine-1-stearoyl-2-[14C]arachidonoyl and L-3-phosphatidyl[n-methyl-14C]choline-1,2-dipalmitoyl by macrophages and lymphocytes, the transfer of phosphatidylcholine from macrophages to lymphocytes, and lymphocyte proliferation in the presence of arachidonic acid-rich phosphatidylcholine and arachidonic acid-poor phosphatidylcholine-loaded macrophages.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Male Wistar rats weighing 180-220 g (age 3-4 mo) were obtained from the Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo. The animals were housed under a light-dark cycle of 12/12 h at 23 ± 2°C. The rats were fed ad libitum a diet containing 52% carbohydrate, 21% protein, and 4% lipid (Nuvilab CR1, Nuvital Nutrientes, Curitiba, Brazil) and had free access to tap water. Ethical approval was granted for these studies by the Institute of Biomedical Sciences Animal Experimental Committee, University of São Paulo.

Reagents. Organic solvents of analytical grade were obtained from Merck (Darmstadt, Germany). Penicillin (2.5 U/ml) and streptomycin (2.5 µg/ml) were purchased from Sigma (St. Louis, MO). MEM and RPMI 1640 medium, pH 7.4, were obtained from GIBCO-BRL (Gaithersburg, MD). The culture media were supplemented with 10% (vol/vol) heat-inactivated (56°C for 30 min) FCS from Adolfo Lutz Institute, São Paulo, Brazil. The following lipid standards for TLC were obtained from Sigma: cholesterol (CHOL), free fatty acids (FFA), triacylglycerol (TAG), cholesteryl ester (CE), and fatty acid methyl ester (ME). The phospholipids (PL) were as follows: L-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), and phosphatidic acid (PA). Two radiolabeled phosphatidylcholines: L-3-phosphatidylcholine-1-stearoyl-2-[1-14C]arachidonoyl ([14C]AA-PC) and L-3-phosphatidyl[n-methyl-14C]choline-1,2,-dipalmitoyl ([14C]choline-PC) were obtained from Amersham International (Little Chalfont, Bukinghamshire, UK), with a specific activity of 55 and 56 mCi/mmol, respectively. [2-14C]thymidine (54 mCi/mmol) was also obtained from Amersham International. The following PC species: L-alpha -phosphatidylcholine-beta -arachidonoyl-gamma -stearoyl (AA-PC) and L-alpha -phosphatidylcholine from egg yolk (FA-PC) were purchased from Sigma. Analysis of fatty acid composition in HPLC revealed that PC from lecithin contains palmitic acid (30-42%), stearic acid (13-15%), oleic acid (25-32%), and linoleic acid (12-16%).

Culture medium. The culture medium with 10% FCS added contained total phospholipids in a concentration of 45.0 µM, with 55.0% represented by phosphatidylcholine. A HPLC analysis revealed that the FCS was composed of the following fatty acids: 1.3% lauric, 3.6% myristic, 26.1% palmitic, 6.0% palmitoleic, 18.2% stearic, 21.9% oleic, 9.5% linoleic, 1.1% linolenic, 6.1% arachidonic, 1.2% eicosapentaenoic, and 5.0% docosahexaenoic. The concentrations of the cholesterol and triacylglycerol in the medium were 3.1 and 6.6 mg/dl, respectively.

Macrophage and lymphocyte preparation. Lymphocytes were obtained by pressing the mesenteric lymph nodes against a steel screen (10), and resident macrophages were collected from the peritoneal cavity. The cells were diluted in culture medium and macrophages were preincubated for 1 h at 37°C in an artificially humidified atmosphere of 5% CO2 (vol/vol) in air under sterile conditions in a Microprocessor CO2 incubator (Lab-Line Instruments, Melrose Park, IL). This process was carried out to promote the macrophage plate adherence.

Preparation of phospholipids. The phospholipids dissolved in chloroform were placed in a sterile tube. The solvent was removed using a N2 gas flux under sterile conditions. The dried phospholipids were dispersed in a culture medium with a vortex mixer at room temperature for 2 min. The phospholipids were then diluted in various concentrations, as indicated in the experiments, and added to the culture medium. The phospholipid content in macrophages, lymphocytes, and FCS was determined as described by Anderson and Davis (1).

Incorporation of labeled phosphatidylcholine species. Lymphocytes or adhered macrophages were cultured in 24-well plates, 2 × 106 cells/well. Cells were cultured for 24 h in 1.0 ml of MEM containing 10% FCS and 0, 10, 20, 50, 100, and 150 µM [14C]AA-PC. After the culture period, cells were washed three times with PBS and the recovered radioactivity determined.

Phosphatidylcholine export to the culture medium. Macrophages were treated with 7.2 µM [14C]AA-PC and [14C]choline-PC for 24 h. After this period, macrophages were washed several times until radioactivity could not be detected in the washing fluid and a new culture medium was added. Cells were then maintained in culture for an additional period of 24 h and the supernatant was then collected.

Lipid extraction and chromatography. The lipid contents of pretreated macrophages and lymphocytes with 7.2 µM [14C]choline-PC and [14C]AA-PC for 24 h as well as the supernatant from the export experiments were extracted using a modification of the procedure described by Folch et al. (15).

TLC. Recovered radioactivity in total lipid fractions obtained by previous lipid extraction was chromatographed in silica plates. To separate total lipid fractions, a method described by Ohta et al. (27) was used to separate the following standards: CHOL, FFA, TAG, CE, and ME. The polar lipids fraction (POL) refers to the intermediate fraction between the phospholipid and cholesterol in this separation method. The PL band at the origin was scraped from the plate, extracted, and rechromatographed. The phospholipid band was separated in a unidimensional TLC system as described by Fine and Sprecher (14). The following phospholipids were used as reference standards: PC, PS, PI, PE, and PA. All plates were visualized in an atmosphere of iodine vapor. The lipid spots were scraped from the plates and transferred into scintillation vials. Ecolume scintillating cocktail (ICN, Costa Mesa, CA) was used and the radioactivity was determined using a Beckman-LS 5000TD liquid scintillation counter (Beckman Instruments, Fullerton, CA).

Coculture of 14C-labeled macrophages with lymphocytes. Prelabeled macrophages were cocultured with lymphocytes in two-chamber compartments as follows. Macrophages, 2 × 106 cells/well, were initially cultured with 7.2 µM of [14C]AA-PC or [14C]choline-PC for 24 h. The supernatant was discharged, the adhered cells were washed several times with 1 ml of sterile MEM until no radioactivity was found in the fluid, and fresh medium was then added. Polycarbonate membrane inserts (10-mm-diameter; Nunc, Roskilde, Denmark) were placed onto macrophage 24-well plates. Lymphocytes were then added (2 × 106 cells/well) on the upper side of the chamber. Afterward, the cells were cocultured for an additional 24 h. Cells were then collected separately and the pellets containing lymphocytes were washed several times with PBS, pelleted, and disrupted by adding 0.5 ml of methanol.

[2-14C]thymidine incorporation by lymphocytes treated with phosphatidylcholine. The effect of 4, 8, 15, 31, 62, 123, and 247 µM AA-PC and FA-PC on lymphocyte proliferation was tested. The proliferative capacity of lymphocytes, at a density of 2 × 105 lymphocytes/well, was evaluated by incorporation of [2-14C]thymidine into DNA in lymphocytes stimulated by 5 µg/ml concanavalin A as described by Curi et al. (11). After 48 h in coculture, [2-14C]thymidine (0.1 µCi/ml) was added to the medium. The cells were then cultured for an additional 18 h and harvested by using an automatic multiple cell harvester (Skatron Combi, Suffolk, UK). The radioactivity contained in the paper discs (Skatron Combi filter papers) with radiolabeled cells was counted in 2 ml of Ecolume scintillation cocktail.

[2-14C]thymidine incorporation by lymphocytes cocultured with untreated macrophages. Lymphocytes at 2 × 105/well were cocultured with increasing amounts of untreated macrophages, without inserts. The proportions of macrophages added were as follows: 0.3, 0.6, 1.2, 2.5, 5, 10, and 20% of the number of lymphocytes.

[2-14C]thymidine incorporation by lymphocytes cocultured with PC-loaded macrophages. Macrophages were initially cultured in the absence or in the presence of AA-PC or FA-PC (at concentrations of 8, 15, 62, and 123 µM) in 96-well plates containing 2 × 103 cells/well (corresponding to 1% of the lymphocyte number). Macrophages were maintained in culture for 6 h, at 37°C, under sterile conditions. After this period, adhered macrophages were washed several times with RPMI 1640, and then 2 × 105 lymphocytes and concanavalin A were added directly to the culture medium.

Statistical analysis. Values are expressed as means ± SE. Comparisons between groups when applied were made using unpaired Student's t-test; the significance level was set for *P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of phospholipids on lymphocyte proliferation. Both AA-PC and FA-PC inhibited [14C]thymidine incorporation into lymphocyte DNA (Fig. 1). Full inhibition (100%) was found for AA-PC and FA-PC at concentrations of 62 and 247 µM, respectively, whereas the half-maximal inhibitory effect occurred at 18 and 40 µM for AA-PC and FA-PC, respectively. Therefore, the inhibitory effect of AA-PC was clearly more pronounced than that of FA-PC.


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Fig. 1.   Effect of nonradiolabeled phosphatidylcholine species on lymphocyte proliferation. Arachidonic acid-rich phosphatidylcholine (AA-PC) and arachidonic acid-poor phosphatidylcholine (FA-PC) were added to the cell culture at the beginning of the proliferation experiments. [2-14C]thymidine incorporation into DNA in lymphocytes stimulated by concanavalin A was measured after 66 h. Values are presented as % inhibition ± SE of 10 determinations of at least 3 cell preparations. Groups were compared using Student's t-test (* P < 0.05).

Incorporation of radiolabeled phosphatidylcholine into macrophages and lymphocytes. In this set of experiments, the incorporation of [14C]AA-PC into macrophages and lymphocytes in culture was compared. Macrophages incorporated ~10-fold more [14C]AA-PC than lymphocytes when cultured in the presence of 10 µM [14C]AA-PC; this difference gradually declined with the increase in the concentration of phosphatidylcholine, reaching eightfold for 50 µM and sixfold for 150 µM (Fig. 2).


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Fig. 2.   Concentration-dependent phosphatidylcholine incorporation by cells. Macrophages and lymphocytes were treated with increasing concentrations of L-3-phosphatidylcholine-1-stearoyl-2-[14C]arachidonoyl. Incorporation rate was measured after 24 h of culture and results are expressed as picomoles of radiolabeled phosphatidylcholine/107 cells ± SE of 3 cell preparations. Groups were compared using Student's t-test (* P < 0.05).

Distribution of radiolabeled phosphatidylcholine among lipid fractions and phospholipid classes in macrophages and lymphocytes. The distribution of 14C from [14C]AA-PC into lipid fractions of macrophages and lymphocytes by using TLC was determined. The radioactivity was present primarily in PL fractions in both cell types: 76.7% in macrophages and 95.2% in lymphocytes (Table 1). Radioactivity was also found in triacylglycerols (10.6%) and cholesteryl esters (6.2%) in macrophages, whereas in lymphocytes only traces were detected in the same fractions. The radioactivity distribution among the phospholipid classes was then analyzed. The radioactivity from [14C]AA-PC was found in PI, AP, PS, PC, and PE; the values were 3.5, 6.8, 14.5, 46.5, and 28.5% for lymphocytes and 5.0, 9.0, 20.3, 45.0, and 20.8% for macrophages, respectively (Fig. 3). The same experiment was also carried out using [14C]choline-PC. The radioactivity was almost exclusively found in the phospholipid fraction (>90%) for both cell types (Table 2); the proportion found in the phosphatidylcholine class was 93.0% and 81.4% for lymphocytes and macrophages, respectively (Fig. 4).

                              
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Table 1.   Radioactivity distribution from [14C]AA-PC in total lipid fractions of 24-h cultured macrophages and lymphocytes



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Fig. 3.   Radioactivity distribution from L-3-phosphatidylcholine-1-stearoyl-2-[14C]arachidonoyl into various phospholipid classes. Macrophages and lymphocytes were cultured for 24 h and cells were analyzed for their content of phosphatidylinositol (PI), phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Results are presented as % means ± SE of 4 cell preparations. Groups were compared using Student's t-test (* P < 0.05).


                              
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Table 2.   Radioactivity distribution from [14C]choline-PC in total lipid fractions of 24-h cultured macrophages and lymphocytes



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Fig. 4.   Radioactivity distribution from L-3-phosphatidyl-[n-methyl-14C]choline-1,2-dipalmitoyl into different phospholipid classes Macrophages and lymphocytes were cultured for 24 h and cells were analyzed for their content of PI, PA, PS, PC, and PE. Results are presented as % means ± SE of 4 cell preparations. Groups were compared using Student's t-test (* P < 0.05).

Phosphatidylcholine export to the culture medium. The radioactivity found in culture medium released from [14C]AA-PC-loaded macrophages was mainly distributed in FFA and PL fractions. The values found were: 103.9 pmol/107 cells of [14C]AA-PC in FFA fraction and 41.3 pmol/107 cells of [14C]AA-PC in PL fraction after 24 h in culture. The proportion of radioactive PC, PE, PS, and PA plus PI found in the phospholipid fraction was 86.3, 5.9, 5.0, and 2.8%, respectively, after 24 h in culture (Fig. 5). The same experimental procedure to examine phosphatidylcholine export was performed replacing [14C]AA-PC by [14C]choline-PC. Macrophages incorporated [14C]choline-PC and exported to the culture medium basically as radiolabeled phospholipids (>95%), and this fraction was comprised of 95% PC class (Fig. 6).


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Fig. 5.   Radioactivity distribution from L-3-phosphatidylcholine-1-stearoyl-2-[14C]arachidonoyl into various lipid fractions in supernatant of cultured cells. Supernatant was collected after 24 h and analyzed for its content of free fatty acids (FFA), phospholipids (PL), polar lipids (POL), cholesterol (CHOL), triacylglycerol (TAG), cholesteryl esters (CE), and methyl esters (ME). Results are expressed as % means ± SE of 8 cell preparations. Inset represents radioactivity found in PL classes: PI+PA, phosphatidylinositol plus phosphatidic acid. Results are presented as % means ± SE of 4 cell preparations.



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Fig. 6.   Radioactivity distribution from L-3-phosphatidyl-[n-methyl-14C]choline-1,2-dipalmitoyl into various lipid fractions in supernatant of cultured cells. Supernatant was collected after 24 h and analyzed for its content of FFA, PL, POL, CHOL, TAG, CE, and ME. Results are expressed as % means ± SE of 8 cell preparations. Inset represents radioactivity found in phospholipid classes. Results are presented as % means ± SE of 4 cell preparations.

Phosphatidylcholine transfer from macrophages to lymphocytes in coculture. In coculture experiments, macrophages incorporated 410 pmol/107 cells of [14C]AA-PC when cultured in the presence of 7.2 µM for 24 h, and transferred to lymphocytes 7.3 pmol/107 cells for 24 h. When cultured with 7.2 µM [14C]choline-PC, macrophages incorporated 379 pmol/107 cells and transferred to lymphocytes 6.3 pmol/107 cells (Fig. 7). In both cases, the values presented always refer to the radioactive compound.


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Fig. 7.   Radiolabeled phosphatidylcholine transfer from macrophages to lymphocytes in coculture. Bars represent radioactivity found in lymphocytes from 24 h of cocultured macrophages treated with: 7.2 µM L-3-phosphatidylcholine-1-stearoyl-2-[14C]arachidonoyl (A); and 7.2 µM L-3-phosphatidyl-[n-methyl-14C]choline-1,2-dipalmitoyl (B). Results are expressed as pmol/107 cells ± SE of 6 cell preparations.

Effect of untreated macrophages on lymphocyte proliferation. The incorporation of [14C]thymidine by concanavalin A-stimulated lymphocytes was inhibited when these cells were cocultured with untreated macrophages. The control value was 1,490 cpm for [14C]thymidine incorporation by concanavalin A-stimulated lymphocytes in the absence of macrophages. Values were not different from the control for the range of 0.6 to 2.5% of added macrophages. Full inhibition was observed with 20% of added macrophages (Fig. 8).


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Fig. 8.   Effect of cocultivation of untreated macrophages on lymphocyte proliferation. Increasing proportions of macrophages (up to 20% of lymphocytes) were added to concanavalin A-stimulated lymphocytes, and proliferation capacity was evaluated. Values are presented as % inhibition ± SE of 15 determinations of at least 3 cell preparations. Groups were compared using Student's t-test (* P < 0.05).

Effect of PC-treated macrophages on lymphocyte proliferation. The incorporation of [14C]thymidine by concanavalin A-stimulated lymphocytes was markedly inhibited when these cells were cocultured with PC-loaded macrophages (Fig. 9). Macrophages treated with increasing amounts of AA-PC and FA-PC evoked a gradual inhibition of concanavalin A-stimulated lymphocyte proliferation, reaching 41.2 and 65.8% at 123 µM AA-PC and FA-PC treatments, respectively. At this concentration, the difference between AA-PC and FA-PC was found to be statistically significant.


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Fig. 9.   Effect of PC-loaded macrophages on lymphocyte proliferation. Macrophages were loaded with AA-PC and FA-PC in increasing concentrations for 6 h. After being washed several times, lymphocytes and concanavalin A were added to the culture medium and proliferation capacity was evaluated. Values are presented as % inhibition ± SE of 5 determinations. Groups were compared using Student's t-test (* P < 0.05).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

This study was undertaken to extend previous observations that PC could be involved with the impaired proliferative capacity of lymphocytes. Kremlev et al. (23) showed that dipalmitoyl-PC was able to inhibit concanavalin A-activated splenocyte proliferation in a concentration-dependent manner. Otherwise, Caselli et al. (8) found that dioleyl-PC did not produce inhibition in human phytohemagglutinin-activated peripheral blood mononuclear cells. The experimental conditions in these studies were quite different, including the lymphocyte source, the mitogen, and the PC molecular species used. Considering that different experimental protocols were used in these studies and that fatty acids of the PC could have a different effect on lymphocyte proliferative capacity (6, 7), two types of PC were tested in the present study: arachidonic acid-rich phosphatidylcholine (AA-PC) and arachidonic acid-poor phosphatidylcholine (FA-PC) on rat mesenteric lymphocyte proliferative capacity. Both types of PC inhibited lymphocyte proliferation. However, the inhibitory effect of AA-PC on lymphocyte proliferation was approximately twofold more pronounced than that of the FA-PC. This difference in the potency of both PC may be due to the presence of AA in the molecule.

Several mechanisms can be involved in the anti-proliferative effect of PC. It has been shown that PC molecules function as negative regulators of protein kinase C (20, 21), inhibiting the activation of this enzyme. On the other hand, it was demonstrated that PC hydrolysis by PC-specific phospholipase D coupled to TCR/CD3 complex may play a role in driving cells into cycle (30). PC hydrolysis contributes significantly to the total diacylglycerol formed (24) in the cell and it is also implicated in the protein kinase C activation. It has been shown that oxidized phosphatidylcholine presents a platelet-activating factor-like activity that is mitogenic to smooth muscle cells (17). The effect of platelet-activating factor-like activity was not measured but platelet-activating factor is able to inhibit interleukin-2 receptor in T cells.

The radioactivity from [14C]AA-PC was distributed into various phospholipid (PL) fractions in macrophages and lymphocytes. These findings indicate that release of AA from PC and subsequent incorporation into other PL classes occurred. This is probably a consequence of the remodeling of the sn-2 acyl group by an acylation-deacylation process (9), indicating that a significant proportion of PC was in fact internalized by the cells. This phenomenon is clearly more pronounced in macrophages than in lymphocytes. The greater PC incorporation capacity of macrophages than lymphocytes led us to investigate the PC transfer from the former cells to the latter. Macrophages can actively uptake liposomes, lipoproteins containing PC and surfactant components. In the lung, phosphatidylcholine-incorporated macrophages are in close contact with many other cell types. Thus the following question was raised: once taken up and metabolized, can phosphatidylcholine be released from macrophages to the extracellular space and then be taken up by surrounding cells? The next experiments were performed to address this point.

The radioactivity from [14C]AA-PC-loaded macrophages was mainly found in fatty acids and phospholipid fractions in the culture medium after 24 h. On the other hand, the radioactivity from [14C]choline-PC-loaded macrophages, under similar conditions, was almost restricted to the PC fraction. These results support the proposition that PC could potentially be transferred from macrophages to surrounding cells. To investigate this possibility, [14C]AA-PC and [14C]choline-PC-preloaded macrophages were cocultured with lymphocytes. Results revealed that ~2% of the radioactivity from both [14C]AA-PC and [14C]choline-PC incorporated into macrophages was transferred to lymphocytes in 24 h, indicating that PC molecules were, in fact, transferred between these cells. The total content of PL in macrophages and lymphocytes was estimated. Lymphocytes and macrophages present 952 µmol PL/107 cells and 4,124 µmol PL/107 cells, respectively. PC represents 51 and 47% of the total PL in lymphocytes and macrophages, respectively (5). Therefore, the estimated net transfer of PC from macrophages to lymphocytes was 34 µmol/107 cells and 32 µmol/107 cells or [14C]AA-PC and [14C]choline-PC, respectively. These values do not take into account a possible reverse flux of PC from lymphocytes to macrophages. However, the net amount of transferred PC is equivalent to 7.1 and 6.6% of total lymphocyte PC for [14C]AA-PC and [14C]choline-PC, respectively. In addition to the transfer of fatty acids as shown by Peres et al. (29), macrophages can also transfer PC for lymphocytes in coculture.

Considering that the transfer of both types of PC were quite similar under this condition, it is likely that the abundance of AA does not interfere in the process of transfer from macrophages to lymphocytes. The possible mechanism for the transfer of phosphatidylcholine remains to be elucidated, however, some possibilities have to be considered such as: shedding of the macrophage membranes, as shown to occur in tumor cells (22), and transport through calf lipoproteins present in the culture medium.

Assuming that PC inhibits lymphocyte proliferation and it is transferred from macrophages to lymphocytes, we tested whether this transfer process could alter the lymphocyte proliferative capacity. Untreated macrophages added at 1% did not produce significant alterations in the lymphocyte proliferation. A low number of macrophages stimulates lymphocyte proliferation, whereas a high proportion clearly causes inhibition. The mechanism to explain these findings may involve cytokine biosynthesis but the full explanation remains to be found. Therefore, this proportion was chosen to investigate the effect of PC-preloaded macrophages on lymphocyte proliferation in coculture. Results show that PC-loaded macrophages did inhibit lymphocyte proliferation. Moreover, the inhibition potency of the phosphatidylcholine-loaded macrophages is more pronounced for AA-PC. Several mechanisms can be involved in this process, including prostaglandin and leukotriene synthesis. It has to be pointed out that the results of the PC effects presented herein and even the processes of PC incorporation, export, and transfer were influenced by the lipids present in the FCS. Indeed, medium with 10% FCS contains phospholipids, triacylglycerol, and cholesterol. Further studies must be developed to determine the influence of these lipid species on the findings described. Despite these considerations, it was shown in our experiments that phosphatidylcholine is, in fact, transferred and FA-PC-loaded macrophages were also able to inhibit concanavalin A-stimulated lymphocyte proliferation. Therefore, these results support the proposition that molecular species of phosphatidylcholine could act as mediator for the control of lymphocyte proliferation by macrophages. To our knowledge, this is the first indication that PC per se may play a role as an intercellular signal during inflammatory and immunological responses.


    ACKNOWLEDGEMENTS

This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Conselho Nacional de Desenvolvimento Científico e Tecnológico, and Pronex.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: R. Curi, Av. Prof. Lineu Prestes, 1524, CEP 05508-900, São Paulo, SP, Brazil (E-mail: ruicuri{at}fisio.icb.usp.br).

Received 18 June 1999; accepted in final form 13 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Anderson, R., and S. Davis. An organic phosphorus assay which avoids the use of hazardous percloric acid. Clin. Chim. Acta 121: 111-116, 1982[ISI][Medline].

2.   Ansfield, M. J., J. Kaltreider, L. Caldwell, and F. N. Jerskowitz. Hyporesponsiveness of canine bronchoalveolar lymphocytes to mitogens: inhibition of lymphocyte proliferation by alveolar macrophage. J. Immunol. 122: 542-548, 1979[ISI][Medline].

3.   Asaoka, Y., M. Oka, K. Yoshida, Y. Sasaki, and Y. Nishizuka. Role of lysophosphatidylcholine in T-lymphocyte activation: involvement of phospholipase A2 in signal transduction through protein kinase C. Proc. Natl. Acad. Sci. USA 89: 6447-6451, 1992[Abstract].

4.   Becker, S., D. T. Harris, and H. S. Koren. Characterization of normal human lung lymphocytes and interleukin-2-induced lung T cell lines. Am. J. Respir. Cell. Mol. Biol. 3: 441-448, 1990[ISI][Medline].

5.   Brouard, C., and M. Pascaud. Effects of moderate dietary supplementations with n-3 fatty acids on macrophage and lymphocyte phospholipids and macrophage eicosanoid synthesis in the rat. Biochim. Biophys. Acta 1047: 19-28, 1990[ISI][Medline].

6.   Calder, P. C. The effects of fatty acids on lymphocyte functions. Braz. J. Med. Biol. Res. 26: 901-917, 1993[ISI][Medline].

7.   Calder, P. C., L. F. B. P. Costa-Rosa, and R. Curi. Effects of feeding lipids of different fatty acid compositions upon rat lymphocyte proliferation. Life Sci. 56: 455-463, 1995[ISI][Medline].

8.   Caselli, E., O. R. Baricordi, L. Melchiorri, F. Bellini, D. Ponzin, and A. Bruni. Inhibition of DNA synthesis in peripheral blood mononuclear cells treated with phosphatidylserines containing unsaturated acyl chains. Immunopharmacol. 23: 205-213, 1992[ISI][Medline].

9.   Choy, P. C., M. Skrzypczak, D. Lee, and F. T. Jay. Acyl-GPC and alkenyl-/alkyl-GPC:acyl-CoA acyltransferases. Biochim. Biophys. Acta 1348: 124-133, 1997[ISI][Medline].

10.   Curi, R., P. Newsholme, and E. A. Newsholme. Metabolism of pyruvate by isolated rat mesenteric lymphocytes mitochondria and isolated mouse macrophages. Biochem. J. 250: 383-388, 1988[ISI][Medline].

11.   Curi, R., J. A. Bond, P. C. Calder, and E. A. Newsholme. Propionate regulates lymphocyte proliferation and metabolism. Gen. Pharmacol. 24: 591-597, 1993[Medline].

12.   Curi, R., P. Newsholme, T. C. Pithon-Curi, M. Pires de Melo, C. Garcia, P. I. Homem de Bittencourt, Jr., and A. R. P. Guimarães. Metabolic fate of glutamine in lymphocytes, macrophages and neutrophils. Braz. J. Med. Biol. Res. 32: 5-21, 1999.

13.   Exton, J. H. Phospholipase D: enzymology, mechanisms of regulation, and function. Physiol. Rev. 77: 303-320, 1997[Abstract/Free Full Text].

14.   Fine, J. B., and H. Sprecher. Unidimensional thin-layer chromatography of phospholipids on boric acid-impregnated plates. J. Lipid Res. 23: 660-663, 1982[Abstract].

15.   Folch, J., M. Lees, and G. H. S. Stanley. A simple method for the isolation and purification of total lipid from animal tissues. J. Biol. Chem. 226: 497-509, 1957[Free Full Text].

16.   Gilbreath, M. J., C. A. Nancy, D. L. Hoover, C. R. Alving, G. M. Swartz, Jr., and M. S. Meltzer. Macrophage activation for microbicidal activity against Leishmania major: inhibition of lymphokine activation by phosphatidylcholine-phosphatidylserine liposomes. J. Immunol. 134: 3420-3425, 1985[Abstract/Free Full Text].

17.   Heery, J. M., M. Kozak, D. M. Stafforini, D. A. Jones, G. A. Zimmerman, T. M. McIntyre, and S. M. Prescott. Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells. J. Clin. Invest. 96: 2322-2330, 1995[ISI][Medline].

18.   Homem de Bittencourt, P. I., C. M. Peres, M. H. Yano, and R. Curi. Pyruvate is a lipid precursor for rat lymphocytes in culture. Evidence for a lipid exporting capacity. Biochem. Mol. Biol. Int. 30: 631-641, 1993[ISI][Medline].

19.   Homem de Bittencourt, P. I., and R. Curi. Transfer of cholesterol from macrophages to lymphocytes in culture. Biochem. Mol. Biol. Int. 44: 347-361, 1998[ISI][Medline].

20.   Isakov, N. Regulation of T-cell-derived protein kinase C activity by vitamin A derivatives. Cell. Immunol. 115: 288-298, 1988[ISI][Medline].

21.   Kaibuchi, K., Y. Takai, and Y. Nishizuka. Cooperative roles of various membrane phospholipids in the activation of calcium-activated, phospholipid-dependent protein kinase. J. Biol. Chem. 256: 7146-7149, 1981[Abstract/Free Full Text].

22.   Kornbluth, R. S. The immunological potential of apoptotic debris produced by tumor cells and during HIV infection. Immunol. Lett. 43: 125-132, 1994[ISI][Medline].

23.   Kremlev, S. G., T. M. Umstead, and D. S. Phelps. Effects of surfactant protein A and surfactant lipids on lymphocyte proliferation in vitro. Am. J. Physiol. Lung Cell. Mol. Physiol. 267: L357-L364, 1994[Abstract/Free Full Text].

24.   Licastro, F., L. J. Davis, and M. C. Morini. Lectins and superantigens: membrane interactions of these compounds with T lymphocytes affect immune responses. Int. J. Biochem. 25: 845-852, 1993[ISI][Medline].

25.   McIntyre, T., K. D. Patel, P. L. Smiley, D. Stafforini, S. M. Prescott, and G. A. Zimmerman. Oxidized phospholipid with PAF-like bioactivity. J. Lipid Med. 10: 37-40, 1994[ISI].

26.   Miles, P. R., L. Bowman, A. Rengasamy, and L. Huffman. Constitutive nitric oxide production by rat alveolar macrophages. Am. J. Physiol. Lung Cell. Mol. Physiol. 274: L360-L368, 1998[Abstract/Free Full Text].

27.   Ohta, A., M. C. Mayo, N. Kramer, and E. M. Lands. Rapid analysis of fatty acids in plasma lipids. Lipids 25: 742-747, 1990[ISI][Medline].

28.   Palatini, P., G. Viola, E. Bigon, A. M. Menegus, and A. Bruni. Pharmacokinetic characterization of phosphatidylserine liposomes in the rat. Br. J. Pharmacol. 102: 345-350, 1991[Abstract].

29.   Peres, C. M., P. I. Homem de Bittencourt, M. Costa, R. Curi, and J. F. Williams. Evidence for the transfer in culture of [14c]-labelled fatty acids from macrophages to lymphocytes. Biochem. Mol. Biol. Int. 43: 1137-1144, 1997[ISI][Medline].

30.   Reid, P. A., S. D. Gardner, D. M. Williams, and M. M. Harnett. The antigen receptors on mature and immature T lymphocytes are coupled to phosphatidylcholine-specific phospholipase D activation. Immunology 90: 250-256, 1997[ISI][Medline].

31.   Rotondo, D., C. R. Earl, K. J. Laing, and D. O. Kaimakamis. Inhibition of cytokine-stimulated thymic lymphocyte proliferation by fatty acids: the role of eicosanoids. Biochim. Biophys. Acta 1223: 185-194, 1994[ISI][Medline].

32.   Sands, W. A., J. S. Clark, and F. Y. Liew. The role of a phosphatidylcholine-specific phospholipase C in the production of diacylglycerol for nitric oxide synthesis in macrophages activated by IFN-gamma and LPS. Biochem. Biophys. Res. Commun. 199: 461-466, 1994[ISI][Medline].

33.   Wright, J. R. Immunomodulatory functions of surfactant. Physiol. Rev. 77: 931-962, 1997[Abstract/Free Full Text].


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