Lipoproteins may provide fatty acids necessary for human lymphocyte proliferation by both low density lipoprotein receptor-dependent and -independent mechanisms.

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptor-mediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (less than 10 micrograms cholesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (greater than or equal to 50 micrograms cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL3 enhanced responses of both normal and familial hypercholesterolemia lymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDL3. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptor-mediated and LDL receptor-independent interactions.

Human lymphocytes respond optimally to mitogenic stimulation when cultured in serum-free medium supplemented with transferrin if fatty acids necessary for maximal proliferation are provided. Either lipoproteins or exogenous fatty acids support optimal lymphocyte responses. The current studies examined the role of cell surface receptors for low density lipoprotein (LDL) in the enhancement of lymphocyte proliferation. Support of lymphocyte growth by limiting concentrations of LDL was found to involve interaction of the lipoprotein with LDL receptors. Thus, modification of LDL by reductive methylation so as to inhibit receptormediated interactions markedly decreased the capacity of LDL to enhance lymphocyte proliferation. Moreover, growth of lymphocytes obtained from patients with LDL receptor-negative homozygous familial hypercholesterolemia was minimal when cultures were supplemented with low concentrations of LDL (<lo pg choIesterol/ml). LDL also enhanced lymphocyte proliferation by a receptor-independent mechanism since high concentrations (250 ~g cholesterol/ml) supported growth of both normal and familial hypercholesterolemia lymphocytes. In contrast, support of lymphocyte proliferation by high density lipoprotein (HDL) subclass 3 was completely independent of LDL receptors. Thus, HDL, enhanced responses of both normal and familial hypercholesterolemia Iymphocytes in an equivalent concentration-dependent manner; this effect was not altered by reductive methylation of HDLB. One function of lipoproteins in this system may be the provision of fatty acids since oleic and linoleic acids enhanced DNA synthesis by both normal and familial hypercholesterolemia lymphocytes in the absence of lipoproteins. These results indicate that lipoproteins may provide fatty acids necessary for optimal proliferation of human lymphocytes by both LDL receptormediated and LDL receptor-independent interactions.
The importance of plasma lipoproteins in cellular lipid metabolism has been well documented. Low density lipoprotein (LDL)' transports cholesterol in the plasma and is the * This work was supported by National Institutes of Health Grant A117653 and by a grant from the American Heart Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. major extracellular source of cholesterol (1-4). Following binding of LDL to specific cell surface receptors, the lipoprotein is internalized by the process of receptor-mediated endocytosis (1)(2)(3)(4), After lysosomal degradation, the cholesterol is released from cholesteryl esters in the core and made available for membrane synthesis (1)(2)(3)(4). Other lipoproteins, including very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and high density lipoprotein (HDL) may also provide cholesterol in a similar manner, if they are able to bind to LDL receptors (4). Receptor-mediated endocytosis of LDL and other lipoproteins also results in the delivery of phospholipid and triglyceride to the cell, as well as cholesterol. Like cholesterol, the phospholipids and triglycerides of the lipoprotein particle are transported to lysosomes in the cell. However, the intracellular fate of these non-sterol lipids i s less well defined.
In previous studies, we found that both LDL and HDL were able to support optimal proliferation of lymphocytes cultured in serum-free medium supplemented with transferrin ( 5 ) . Various proteins, cholesterol, and cholesteryl esters were ineffective a t promoting lymphocyte growth, whereas LDL depleted of cholesteryl esters and other neutral lipids by heptane extraction was able to enhance lymphocyte responses (5). Thus, a component of lipoproteins other than cholesterol or neutral lipid appeared to be responsible for the capacity of these particles to support lymphocyte growth. Phospholipid, triglyceride, and the non-esterified fatty acids, oleic acid and linoleic acid, were each able to replace lipoproteins and enhance DNA synthesis of mitogen-stimulated lymphocytes cultured in transferrin-supplemented serum-free medium, thereby suggesting that these lipid components of lipoproteins accounted for the growth promoting activity ( 5 ) .
The current studies were undertaken to examine the requirement for specific LDL receptors in the enhancement of lymphocyte proliferation by lipoproteins. Previous experiments had established that the provision of cholesterol to proliferating lymphocytes by lipoproteins was absolutely dependent on LDL receptors (6)(7)(8). The results of the present experiments demonstrate that receptor-mediated interaction with LDL may also provide fatty acids necessary for human lymphocyte proliferation. However, unlike the provision of cholesterol, non-receptor-mediated mechanisms appear to be capable of promoting lymphocyte growth. Although LDL receptors enhance the effect of lipoproteins, such receptormediated interactions are not required. In vivo, lipoproteins may play an important role in providing lipids necessary for cellular proliferation by LDL receptor-dependent and independent mechanisms.

Lipoprotein Isolation-The
following lipoprotein fractions were isolated and characterized as detailed previously (5): VLDL + IDL (d < 1.020 g/ml), LDL (d = 1.020-1.050 g/ml), and HDL subclass 3 (HDL3, d = 1.125-1.230 g/ml) Purity of lipoprotein fractions was confirmed by electron microscopy of negatively stained preparations and by analysis of apolipoprotein composition as previously described (8). Total and non-esterified fatty acid contents of lipoprotein preparations were measured by titrimetric assay, and individual fatty acids were quantitated by gas-liquid chromatography (Mayo Medical Laboratories, Rochester, MN). For some experiments, lipoproteins were modified by methylation to alter LDL receptor-mediated recognition of apolipoprotein-B and -E, as described (9).
Reagents-Transferrin, fatty acid-free and fatty acid-containing bovine serum albumin (BSA) and fatty acid-containing human serum albumin were purchased from Sigma and Boehringer Mannheim. Non-esterified fatty acid contents of albumin preparations were measured by titrimetric assay (Smith Kline Bioscience Laboratories, Van Nuys, CA and Mayo Medical Laboratories, Rochester, MN). Fatty acids were obtained from Calbiochem-Behring Corp., LaJolla, CA, dissolved in ethanol and added to cultures as indicated in results.
These cultures were also supplemented with BSA to prevent nonspecific inhibitory effects of fatty acids. BSA concentrations of 7.5 pM were sufficient to prevent inhibitory effects of 5-10 pM nonesterified fatty acids, whereas 75 p~ BSA was required to prevent inhibition by 50 p~ nonesterified fatty acids. The BSA used to prevent inhibition in these experiments contained insufficient free fatty acid to support lymphocyte growth and proliferation alone. Lovastatin, a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme for cholesterol synthesis, was provided by Merck, Sharp and Dohme and dissolved in dimethyl sulfoxide (Aldrich) before addition to cultures as detailed (6,7).
Techniques of Cell Preparation and Culture-Peripheral blood mononuclear cells (PBM) were isolated from heparinized venous blood of healthy adults and from patients with LDL receptor-negative familial hypercholesterolemia by centrifugation over sodium diatrizoate-Ficoll and cultured in medium RPMI 1640 (Whittaker M.A. Bioproducts, Walkersville, MD) as detailed (10,11). The LDL receptor status of familial hypercholesterolemia patients was determined previously by fibroblast studies (1, 3). Cells were incubated in sterile flat-well microtiter plates (Falcon Division, Becton Dickinson and Co., Oxnard, CA), with 50 X lo3 cells/well initially cultured for the assay of lymphocyte 13H]thymidine incorporation and measurement of cell proliferation, as described (10,ll). Phytohemagglutinin (PHA, Wellcome Reagents Ltd., Beckenham, United Kingdom) at the previously determined optimal concentration (0.5 pg/ml) was used as the mitogenic stimulus for all experiments.
For experiments examining provision of non-sterol lipids to proliferating lymphocytes by lipoproteins, cells were cultured in serumfree medium supplemented with transferrin (5-50 pg/ml), the irontransporting serum glycoprotein necessary for DNA synthesis (12, 13). Under these culture conditions, cholesterol may be obtained from endogenous synthesis or exogenously from lipoproteins. However, adequate non-sterol lipids cannot be synthesized endogenously and therefore must be obtained from exogenous sources (5). For experiments examiningprovision of cholesterol to proliferating lymphocytes by lipoproteins, cells were cultured in medium supplemented with 1% lipoprotein-poor plasma (LPP), and endogenous cholesterol synthesis was blocked with lovastatin. Lovastatin (0.5 p~) suppressed de nouo sterol biosynthesis in PHA-stimulated PBM by 78 -C 3% (mean -+ S.E., n = 8). Under these culture conditions, proliferation of lovastatin-blocked lymphocytes is dependent on exogenously supplied cholesterol in lipoproteins (8).
Assay of Lymphocyte Responses-Endogenous sterol synthesis was measured by the incorporation of [l-I4C]acetate into digitonin-precipitahle sterols as detailed (8,10,11). Briefly, cells were incubated with radioisotope for 2 h after which they were saponified, and the sterols were extracted and precipitated with digitonin. Data are expressed as the rate of sterol synthesis (picomoles of acetate incorporated into digitonin-precipitable sterols/hour) per lo6 viable cells. Lymphocyte DNA synthesis was measured by [3H]thymidine incorporation after a 4-day incubation as previously described (10). After a 12-18 h pulse with 1 pCi [3H]thymidine/microtiter well, cells were harvested onto glass fiber filters using a semiautomated device (MASH 11, Whittaker M.A. Bioproducts). Data are expressed as the mean counts/minute of triplicate determinations of PHA-stimulated cultures (unstimulated cultures always incorporated <IO00 cpm).
Lymphocyte proliferation induced by mitogenic stimulation was quantitated after a 7-or 8-day incubation by counting the number of cells in triplicate microtiter wells after disruption of the plasma membrane with a detergent, as previously detailed (11).
Statistics-Statistical analyses were performed using Student's two-tailed t test.

RESULTS
LDL Receptor-mediated Promotion of Lymphocyte Growth by LDL-As previously reported (5), LDL is able to enhance DNA synthesis and proliferation of PHA-stimulated human lymphocytes cultured in serum-free medium with transferrin supplementation (Table I and Fig. 1). Similar results were obtained with transferrin in concentrations of 5-100 pg/ml (data not shown). In the absence of either LDL or transferrin, responses were minimal, whereas responses noted in cultures supplemented with both were comparable to those observed in cultures supplemented with 1%, v/v, human serum that previously have been shown to be maximal (14).
The role of LDL receptors in promotion of lymphocyte proliferation was examined directly by comparing the effect of native LDL with that of LDL modified by reductive methylation (8,9). Methyl-LDL was found to be markedly less effective than native LDL at enhancing mitogen-stimulated lymphocyte DNA synthesis and proliferation in transferrinsupplemented cultures (Table  I and Fig. 1). However, at higher concentrations, methyl-LDL was able to support some lymphocyte responsiveness. These results suggest that blocking the interaction between LDL and cell surface LDL receptors by reductive methylation significantly reduced but did not abolish the capacity of LDL to enhance lymphocyte proliferation.
To confirm that methylation had altered receptor-mediated recognition of LDL particles, experiments were carried out in which mitogen-induced lymphocyte proliferation was made dependent on LDL receptor-mediated delivery of cholesterol. This was accomplished by culturing PBM in cholesteroldepleted medium and blocking endogenous sterol synthesis with lovastatin (6). In the absence of an extracellular cholesterol source, lovastatin suppressed lymphocyte DNA synthesis by 94% (Table 11). The addition of native LDL (5 pg of cholesterol/ml) provided sufficient exogenous cholesterol to restore normal DNA synthesis by lovastatin-blocked lymphocytes. In contrast, methyl-LDL was only minimally able to provide the cholesterol required for lymphocyte responses. Lovastatin also blocked mitogen-induced proliferation of normal lymphocytes cultured in lipoprotein-deficient medium (control = 104,100 -+ 7,000 cells/well; lovastatin 0.5 pM = 19,700 -+ 400 cells/well). LDL but not methyl-LDL restored proliferation (LDL 5 pg of cholesterol/ml = 118,800 f 7,800 cells/well, methyl-LDL 5 pg of cholesterol/ml = 22,500 k 2,800 cells/well, 50 fig cholesterol/ml = 38,100 k 800 cells/ well).
Variable Requirement for LDL Receptors in Promotion of Lymphocyte Proliferation by Lipoproteins-The requirement for LDL receptors in facilitating enhancement of lymphocyte responses by other lipoproteins was also examined. Reductive methylation of lipoproteins was used to differentiate LDL receptor-mediated and receptor-independent enhancement of lymphocyte growth. Preparations of VLDL + IDL were similar to LDL in their support of lymphocyte proliferation. Thus, as shown in Fig. 2, VLDL + IDL enhanced mitogen-induced lymphocyte growth, after methylation there was a significant diminution in this capacity ( p < 0.01). Similar results were obtained with VLDL + IDL fractions isolated from three individual donors (data not shown). All VLDL + IDL preparations were also able to supply cholesterol to lovastatinblocked lymphocytes; this was prevented by methylation (Table 111). The requirement for LDL receptors in the enhancement of lymphocyte proliferation by HDL, was also examined. Methylation did not alter the function of HDL3        Fig. 3). HDL3, unlike other lipoprotein fractions, was unable to provide cholesterol to lovastatin-blocked lymphocytes (8 and data not shown). These observations suggested that lipoproteins could support lymphocyte proliferation by both LDL receptor-dependent and independent mechanisms. Moreover, the data obtained with HDL3 supported the conclusion that an interaction with LDL receptors was not essential for this function.
In order to confirm that HDL, was unable to interact with lymphocyte LDL receptors, the ability of this lipoprotein subfraction to regulate lymphocyte sterol synthesis in serumfree cultures was examined. As shown in Table IV, rates of sterol synthesis in unstimulated PBM cultured in serum-free medium were low and increased markedly with PHA stimulation. When LDL was added to PHA-stimulated cells, rates of sterol synthesis were decreased by 64 k 6% (mean -C S.E., n = 3 ) . VLDL + IDL preparations were also able to regulate cholesterol synthesis, HDLX, in contrast, did not inhibit sterol biosynthetic rates. These results confirm that HDL3 could not interact with LDL receptors and thereby regulate sterol synthesis, although HDL3 was able to support lymphocyte proliferation in serum-free medium.
HDL3 and High Concentrations of LDL Promote Growth of LDL Receptor-negatiue Lymphocytes-The next experiments examined the requirement for functional LDL receptors in lipoprotein-mediated promotion of lymphocyte growth by utilization of cells isolated from patients with LDL receptornegative familial hypercholesterolemia (1,3). In the absence of lipoproteins, mitogen-stimulated proliferation of normal and familial hypercholesterolemia lymphocytes cultured in medium supplemented with transferrin was minimal ( Table  V). The addition of low concentrations of LDL (5-10 pg of cholesterol/ml) markedly enhanced proliferation of normal lymphocytes, whereas growth of familial hypercholesterolemia lymphocytes was not altered. Similar results were obtained with three different LDL preparations (data not shown). In contrast, the addition of low concentrations of HDLs promoted the growth of both normal and familial hypercholesterolemia lymphocytes comparably. When higher concentrations of LDL were added, proliferation of familial hypercholesterolemia lymphocytes was also observed. However, even at high concentrations, enhancement of familial hypercholesterolemia lymphocyte proliferation by LDL was significantly less than that observed with HDL,. These results indicate that LDL receptors markedly facilitate the enhancement of mitogen-induced lymphocyte proliferation by low concentrations of LDL but are not necessary for growth promotion by HDL3 and high concentrations of LDL.  Table VI). Similar results were obtained when lymphocyte proliferation was measured after 7 days (data not shown). In other experiments, palmitic acid (16:O) was found to be equivalent to stearic acid and provided only minimal growth support, whereas arachidonic acid (20:4) inhibited responses (data not shown). Thus, non-esterified fatty acids differed markedly in their ability to enhance mitogen-stimulated lymphocyte proliferation, with oleic and linoleic acids being the most effective. To determine whether oleic and/or linoleic acid could also support the growth of familial hypercholester-      Table VI1 was carried out. As can be seen from these results, LDL was unable to support DNA synthesis of familial hypercholesterolemia lymphocytes, whereas the addition of either oleic or linoleic acid enhanced DNA synthesis by normal and familial hypercholesterolemia lymphocytes in an equivalent manner.

Lipoproteins Provide Fatty Acids for Lymphocyte Growth
More Effectively Than Other Serum Proteins-The next experiments sought to compare the capacity of lipoproteins and alternative serum protein carriers of non-esterified fatty acid to support lymphocyte proliferation. In the first experiment, the capacity of lipoproteins to support growth of lymphocytes in serum-free medium was compared with that of non-esterified fatty acid added directly to the cultures. Oleic acid in concentrations up to 50 PM enhanced lymphocyte DNA syn-  thesis less effectively than LDL (Experiment 2, Table VI); higher concentrations of oleic acid ( X 0 PM) had no additional effect (data not shown). The simultaneous addition of both oleic acid and LDL further increased lymphocyte DNA synthesis above the response obtained with either alone, but these responses did not exceed those supported by higher concentrations of LDL alone (Experiment 2, Table VI).
Whether fatty acids necessary for optimal lymphocyte proliferation are likely to be provided by albumin or by lipoproteins was next examined. For these experiments, commercial preparations of bovine serum albumin containing physiological and supraphysiological molar ratios of fatty acid/albumin (0.81 and 4.81) were employed (15). Both fatty acid-containing albumin and lipoproteins were able to increase lymphocyte DNA synthesis (data not shown) and proliferation (Table   VIII). However, when compared on a molar basis, non-esterified fatty acids bound to albumin were less effective than fatty acids in lipoproteins at enhancing lymphocyte responses. Similar results were obtained when fatty acid-containing human serum albumin was compared with lipoproteins (data not shown). The content of individual fatty acids in BSA was measured to ensure that the lack of effect of the commercial BSA preparation was not the result of a low concentration of oleic and linoleic acid. The BSA lot used in Experiment 3, Table VI11 contained both oleic acid (0.351 oleic acid/albumin molar ratio) and linoleic acid (0.81 molar ratio). These findings support the conclusion that lipoproteins are more effective than fatty acid-containing albumin at promoting growth of lymphocytes, even when the albumin contains more than physiologically attainable concentrations of fatty acid.

DISCUSSION
Optimal proliferation of normal human lymphocytes stimulated by mitogens can be obtained when cells are cultured in serum-free medium supplemented with transferrin and lipoproteins (5). Transferrin, the iron-transporting serum g b oprotein, is necessary for DNA synthesis and thus required for continuing proliferation (5, 12, 13). When added to transferrin-containing medium, lipoproteins completely replace the requirement for serum and support optimal lymphocyte responses (5). Neither exogenous cholesterol nor cholesteryl ester is able to enhance lymphocyte responses, whereas phospholipid, triglyceride, and the fatty acids, oleic and linoleic, promote maximal mitogen-induced lymphocyte DNA synthesis (5). Furthermore, LDL depleted of cholesteryl ester by heptane extraction retains the capacity to support growth ( 5 ) . Thus, the growth-promoting activity of lipoproteins does not result from the provision of protein or cholesterol but rather may result from the ability of lipoproteins to provide fatty acids (5).
Normal peripheral blood lymphocytes have been shown to express functional receptors for LDL (6), but the requirement for these receptors in the support of optimal lymphocyte responses by lipoproteins has not been established. In the current studies, two different methods were used to investigate the requirement for LDL receptors. The first approach was that of chemical modification of the apolipoprotein in order to prevent recognition by and binding to the LDL receptor (9). The studies reported here confirm the previous finding that modification of apolipoproteins by reductive methylation interferes with LDL receptor-mediated provision of cholesterol to lymphocytes by lipoproteins (8). The modest increase in DNA synthesis by lovastatin-blocked normal lymphocytes with the addition of methyl-LDL was equivalent to that observed in cultures of lovastatin-blocked LDL receptornegative familial hypercholesterolemia lymphocytes upon the addition of native LDL (6). Therefore, the minimal increase in [3H]thymidine incorporation observed is secondary to nonreceptor-mediated delivery of cholesterol. Similarly, continuing proliferation of lovastatin-blocked normal lymphocytes, measured after prolonged culture, was not supported by methyl-LDL. The small increase in the number of cultured lymphocytes present after a 7-day incubation with the highest concentration of methyl-LDL could be accounted for by LDL receptor-independent provision of cholesterol, since it was equal to that observed with the addition of native LDL to lovastatin-blocked LDL receptor-negative familial hypercholesterolemia lymphocytes (6). These findings indicate that reductive methylation completely prevented both LDL receptor-dependent lymphocyte DNA synthesis and LDL receptordependent lymphocyte proliferation. Methylation of LDL, thus effectively blocked LDL receptor-dependent uptake of lipoproteins and thereby provided a means to assess the role of LDL receptors in the provision of fatty acids to lymphocytes.
When LDL apolipoproteins were modified by reductive methylation, enhancement of lymphocyte proliferation in serum-free cultures was significantly decreased, indicating that support of lymphocyte growth was similar to provision of cholesterol in that it was dependent on LDL receptormediated processes. The acceptance of this conclusion is dependent on the demonstration that methylation completely prevented receptor-mediated uptake, as noted above, and that the lack of effectiveness of methyl-LDL in this system did not result from a nonspecific alteration of the lipoprotein but rather was related to blocking an interaction with the LDL receptor. Indirect evidence indicates that nonspecific alteration of the LDL did not account for the findings. Thus, methyl-LDL had no significant effect on DNA synthesis and proliferation of mitogen-stimulated lymphocytes cultured in lipoprotein-poor plasma without lovastatin. Furthermore, methyl-LDL did not inhibit responses supported by unmodified LDL (data not shown). Moreover, methylation of other lipoproteins such as HDL, did not alter their capacity to enhance lymphocyte proliferation, additionally indicating that the process of reductive methylation did not result in nonspecific inhibitory effects. The second experimental approach used to examine the role of LDL receptors avoided any potential nonspecific effects of methylation by comparing normal and LDL receptor-negative familial hypercholesterolemia lymphocytes. Low concentrations of both LDL and HDL, substantially increased the proliferation of normal lymphocytes, whereas only HDL3 and high concentrations of LDL were able to enhance the growth of mitogen-stimulated LDL receptor-negative familial hypercholesterolemia lymphocytes. The conclusion arising from this series of experiments is that lipoproteins are able to promote lymphocyte proliferation by both LDL receptor-mediated and receptor-independent mechanisms.
The mechanism of LDL receptor-mediated provision of cholesterol is well established (1-4). The metabolic fate of the other lipid components of the lipoprotein particle, including phospholipids, triglyceride, and fatty acids, is not well defined. The current studies suggest that mitogen-stimulated lymphocytes cultured in serum-free medium internalize lipoproteins and release lipids from the core, since LDL regulated endogenous sterol synthesis. These experiments also suggest that following LDL receptor-mediated internalization, fatty acids in lipoproteins may become available to enhance lymphocyte responses. Alternatively, internalization of lipoproteins via the LDL receptor pathway may increase endogenous synthesis of necessary fatty acids or may allow lymphocyte growth to occur by a mechanism unrelated to fatty acids. Since LDL receptor activity increases with activation and proliferation of lymphocytes (6,16), the potential capacity of lipoproteins to provide both fatty acids and cholesterol is also increased. The need for LDL receptors to provide cholesterol to proliferating lymphocytes may be relatively minor compared with their role in processes dependent on fatty acids, since endogenous synthesis of cholesterol is sufficient to support lymphocyte activation and proliferation when cells are cultured in medium depleted of exogenous cholesterol (6, 10, 11).
Lipoproteins also enhanced lymphocyte growth by LDL receptor-independent mechanisms. Thus, large concentrations of methyl-LDL could support the growth of normal lymphocytes, whereas large concentrations of normal LDL supported proliferation of familial hypercholesterolemia lymphocytes. Moreover, HDL3 supported proliferation of both normal and familial hypercholesterolemia lymphocytes. One possible mechanism by which HDL3 and other lipoproteins may enhance lymphocyte proliferation is by providing fatty acids or fatty acid-containing moieties by an LDL receptorindependent mechanism. Lipoprotein phospholipids may exchange with cell membrane phospholipids or be transferred to the cell membrane, depending on the relative cholesterolphospholipid ratios of the lipoprotein and membrane, and thereby deliver fatty acids (17). Recent studies have suggested that the association of HDL3 with cells is not mediated by a specific receptor or unique apolipoproteins but rather involves interaction of cell membrane lipids with the surface lipids of HDL3 (18). Such an association may then promote transfer of lipids. It is also possible that a lipid transfer protein plays a role in the transfer of cholesteryl esters or triglycerides from lipoproteins to lymphocytes. Lipid transfer protein activity is secreted by human monocyte-derived macrophages (19). This activity may also be secreted by the mononuclear phagocytes of Fatty Acids present in the cultures used in these studies. Supporting this possibility is the finding of mRNA for the major plasma cholesteryl ester transfer protein in spleen (20), suggesting the presence of this protein and its lipid transfer activity in activated lymphocytes or mononuclear phagocytes. Lymphocytes are known to transfer fatty acids from triglycerides to phospholipids by a process that is increased by mitogenic stimulation (21). A similar process may allow transfer of fatty acid-containing lipids from lipoproteins to lymphocytes. Thus, there exists at least two potential mechanisms for the non-receptor-mediated transfer of fatty acids in this system, either dependent on the activity of a specific lipid transfer protein or independent of such an effect. Alternatively, lipoproteins may function to support lymphocyte growth by an LDL receptor-independent mechanism that is not related to the provision of fatty acids, as discussed above for LDL.
Fatty acids circulate in plasma in a non-esterified form bound to albumin or esterified in the phospholipid, triglyceride, and cholesteryl ester of lipoproteins. The current studies demonstrate that both fatty acid-containing albumin and lipoproteins can enhance in vitro lymphocyte responses. A number of pieces of evidence indicate that the action of lipoproteins in supporting lymphocyte growth may be of physiologic relevance and more important than the effect of fatty acid-containing albumin in vivo. Thus, the lipoprotein concentrations that enhanced mitogen-induced lymphocyte proliferation in vitro are in the physiologic range. Interstitial fluid and peripheral lymph concentrations of lipoproteins have been estimated to range between 10 and 30% of the corresponding serum values (22)(23)(24)(25)(26)(27). Thus, for example, the concentration of LDL in peripheral lymph is 10% of the serum concentration (23), whereas the concentrations of LDL used in these experiments are <5% of normal serum concentrations (28). Therefore, physiologically relevant concentrations of lipoproteins support optimal lymphocyte proliferation. The data also support the conclusion that fatty acids in lipoproteins may play a more important role than non-esterified fatty acids bound to albumin in enhancing lymphocyte responses in vivo. Thus, lipoproteins enhanced mitogen-induced lymphocyte proliferation more effectively than did albumin-bound fatty acids. This was particularly noteworthy when the molar concentrations of non-esterified fatty acid in lipoprotein and albumin were compared. Only at fatty acid/ albumin molar ratios exceeding the maximal physiological level of 2:l (29), was albumin-bound fatty acid able to support optimal lymphocyte responses. Increasing the albumin concentration to that observed in interstitial fluid, which is estimated to be 30% of serum levels (24), did not alter the findings (data not shown). The greater effectiveness of lipoproteins was not the result of a higher concentration of oleic acid. Thus, the concentration of oleic acid associated with 50 Hg of lipoprotein cholesterol/ml was 1.1-1.5 WM, whereas that associated with 75 FM BSA was approximately 25 pM. Clearly, lipoprotein-associated oleic acid was more effective than albumin-bound oleic acid at supporting lymphocyte proliferation. Although it is difficult to relate in vitro observations directly to in vivo conditions, the data support the conclusion that lipoproteins are a more effective source of the exogenous fatty acid necessary for lymphocyte growth than albumin.
In summary, the data presented here have demonstrated that LDL receptor-mediated interaction with lipoproteins may result in provision of fatty acids necessary for optimal proliferation of mitogen-stimulated normal lymphocytes. Lipoproteins not interacting with LDL receptors may also provide lipids other than cholesterol to both normal and LDL receptor-negative familial hypercholesterolemia lymphocytes and thereby promote growth in transferrin-containing medium. The data, therefore, indicate that both LDL receptormediated and LDL receptor-independent lipoprotein-cell interactions may provide fatty acids necessary for optimal lymphocyte proliferation.