The essential role of L-glutamine in lymphocyte differentiation in vitro

JOURNAL OF CELLULAR PHYSIOLOGY 124:275-282 (1985)

The Essential Role of 1-Glutamine in Lymphocyte Differentiation In Vitro

JEFFREY CRAWFORD* AND HARVEY JAY C O H E N Department of Medicine and Geriatric Research, Education and Clinical Center, Veterans

Administration Medical Center, Duke University Medical Center, and Center for the Study of Aging and Human Development, Durham, North Carolina 27705

The biochemistry of human B lymphocyte differentiation to plasma cells is incompletely understood. L-glutamine appears to be required for both lymphoblastic transformation and plasma cell formation in pokeweed-mitogen- stimulated human peripheral blood mononuclear cell cultures. Cells cultured with pokeweed mitogen in glutamine-deficient RPMI-1640 with 10% heat- inactivated and dialyzed fetal bovine serum were unable to incorporate 3H- thymidine or under o morphologic lymphoblastic transformation assessed at

with as little as 0.08 mM L-glutamine or by using nondialyzed heat-inactivated fetal bovine serum, containing approximately .I mM L-glutamine. In subsequent cultures, using glutamine-deficient RPMI-1640 with 10% nondialyzed heat-inactivated fetal bovine serum, lymphoblastic transformation was equivalent with or without additional L-glutamine supplementation. However, only cultures with 2 mM L-glutamine supplementation underwent plasma cell differentiation as assessed by cytoplasmic staining with fluorescein-conjugated anti-immunoglobulin. When the kinetics of cellular immunoglobulin synthesis and secretion were analyzed by 3H- leucine incorporation into immunoglobulin, synthesis was 2-5 fold greater, and secretion 3-lo-fold greater in cell cultures with 2 mM L-glutamine supplementation. By electron microscopy, only the glutamine-supplemented cells showed development of rough endoplasmic reticulum consistent with active immunoglobulin production. L-glutamine supplementation had no apparent effect on cell recovery, viability, % B cells, % T cells, % monocytes, or % helper and suppressor T cells. Thus, L-glutamine is essential for both lymphoblastic transformation and plasma cell differentiation. Future investigation of the selective nutritional requirements of cultured cells should yield further insights into the biochemical control of immune cell differentiation and function.

72 hours. However, 5 H-thymidine incorporation could be maximally restored

Advances in in vitro culture systems by Eagle in 1955 led to the subsequent investigation of many cell populations, including B lymphocytes. In humans, one of the best-studied systems for the latter has involved stimu- lation of peripheral blood mononuclear cells with the T celldependent polyclonal B cell mitogen pokeweed mitogen (PWM) (Fauci et al., 1980). In these cultures, lymphocyte blastogenesis (LBT) of both B cells and T cells occurs within 3 days, followed by differentiation of a subpopulation of B cells to plasma cells secreting IgM, IgG, or IgA within 5-7 days (Hammarstroem et al., 1979). From studies of such cultures, some of the contri- butions of T cell subsets, monocytes, and their lymphok- ines have been identified (Fauci et al., 1983; Ballieux et al., 1979; Rosenberg and Lipsky, 1981; Muraguchi and Fauci, 1982). Though much has been learned about these cellular and humoral interactions, the biochemical regulation of B cell differentiation, plasma cell formation, and immunoglobulin synthesis and secretion remain less well understood.

Manipulation of individual nutrients in human lymphocyte cultures in vitro provides an alternate means to

study the biochemistry of B cell differentiation. One such nutrient, L-glutamine, is an important tissue culture supplement necessary for cellular proliferation in a variety of cell systems. (Eagle, 1955; McKeehan, 1982; Kovacevic and McGiran, 19831, including human fibroblasts and rat lymphocytes (Ardawi and Newsholme, 1983). We will confirm the importance of L-glutamine for human lymphocyte transformation, as well as extend these observations to show an additional essential role for L-glutamine in plasma cell differentiation and immunoglobulin synthesis and secretion in vitro.

MATERIALS AND METHODS Cell separation and culture conditions

After obtaining informed consent as approved by the institutional human studies committee, heparinized venous blood was removed from healthy adult volun-

Received July 11, 1984; accepted March 1,1985. Presented in part at the American Society of Hematology, San Antonio, Texas, December, 1981 (Blood 58:83a, 1981). *To whom reprint requests/correspondence should be addressed.

0 1985 ALAN R. LISS, INC.

276 CRAWFORD AND COHEN

teers. Mononuclear cells were obtained by density centrifugation as previously described (Cohen, 1975). These cells were suspended at a concentration of 1 x lo6 per ml in RPMI 1640 Medium without L-glutamine (Gibco Laboratories, Grand Island Biologic Co., Grand Island, NY) with 10% dialyzed or nondialyzed heat-inactivated fetal bovine serum (Gibco) with (G+) or without (G-),2 mM L-glutamine (Gibco) supplementation. All references to glutamine Supplementation describe supplements added at the initiation of cell culture. Penicillin (50 units/ ml) and streptomycin (50 pg/ml) were routinely added to all cultures. Pokeweed mitogen (Gibco) was added at 1:lOO dilution. Cell cultures were placed in stationary tilted Falcon 2051 tubes (Falcon Products, Oxnard, CA) in 24-ml volumes and incubated in a humidified 37°C 10% COz incubator. Cell recovery was generally 70-80% at the end of 7 days with greater than 70% viability by trypan blue exclusion (George and Cohen, 1979).

3H-thymidine and 3H-leucine studies Cell cultures were assessed for 3H-thymidine (3HT)

incorporation after 72 hours by the addition of 2 pCi/ml of 3HT (specific activity 6.7 ci/mM, New England Nu- clear, Boston, MA) for 8 hours (Shankey et al., 1981). The cells were washed and then lysed with 0.1 M sodium hydroxide (NaOH) followed by precipitation with 10% trichloroacetic acid (TCA). These TCA precipitates were then washed, solubilized, and transferred to a phosphor fluid for counting in a Beckman LS 9000 Liquid Scintil- lation Counter. Light microscopy of Wright-stained cytocentrifuge-prepared slides (Shannon, Ltd., London, England) of cultured cells was used to confirm lymphoblastic transformation morphologically. Protein and immunoglobulin synthesis and secretion studies were performed on day 0 and day 7 unstimulated and day 7 pokeweed-mitogen-stimulated peripheral blood mononuclear cell cultures (George and Cohen, 1979). Cell cultures were washed and readjusted to 2 x lo6 cells per ml in leucine-free media, and 10 pCi/ml 3H-leucine (New England Nuclear, specific activity 57.4 Ci per mM) was added. Over a 9-hour period, aliquots were removed and supernatants (extracellular fluid [ECF]) were sepa- rated from cells by centrifugation. Intracellular fluid (ICF) was obtained by washing the cell pellet three times in phosphate-buffered saline, then solubilizing with 0.75% deoxycholate (Sigma, St. Louis, MO). ECF and ICF were exhaustively dialyzed and 3H- leucine incorporation into protein (3K-Leu-prot) was determined by liquid scintillation counting of 10% TCA precipitates of aliquots of ECF and ICF samples.

3H- leucine incorporation into immunoglobulin (3H- Leu- Ig) was measured by the immune coprecipitation assay (George and Cohen, 1979). Briefly, aliquots of ECF and ICF wee incubated at 37°C for 1 hour with human polyvalent immunoglobulin (human gamma globulin FR II, Miles Labs, Kanakee, IL) and goat antihuman immunoglobulin (Gibco) at equivalence to enhance precipitation. The resultant immune precipitates were then processed for liquid scintillation counting as described above. A control precipitate was performed with aliquots of ECF and ICF incubated with egg albumin and rabbit antiegg albumin (Miles-Yeda, Ltd.). The specific cpm incorporated into 3H-Leu- Ig were determined by sub- tracting the nonspecific cpm for the control precipitate.

Cell surface and cytoplasmic fluorescence studies B cell percentage was determined by incubation of the

cells with fluorescein-conjugated goat polyvalent antihuman immunoglobulin, Fab fragment (Kallestad, Chaska, MN), and examination with a Leitz Ortholux fluorescent microscope (Cohen, 1978). Percent T cells was determined by the ability of cells to form rosettes with neuraminidase-treated sheep red blood cells. Helper and suppressor T cell percentage and percent monocytes were determined by initial incubation of the cells with T4, T8, or Mo2 monoclonal antibodies (Coulter Clone, Hialeah, FL) followed by fluorescein-conjugated goat an- timouse IgG (Meloy Labs, Springfield, VA), washing, and examination using the fluorescent microscope. Cells were washed in phosphate-buffered saline and 10% fetal bovine serum and 0.5% EDTA. Cytofuge preparations of approximately 1 x lo6 cells were prepared using the cytocentrifuge. The slides were fixed in 5% acetic acid and 95% ethanol. The slides were then washed with phosphate-buffered saline and incubated with fluorescein-conjugated goat polyvalent antihuman immunoglobulin, Fab fragment (Kallestad) at 37°C for 45 minutes. The cells were subsequently extensively washed with phosphate-buffered saline followed by dis- tilled water. The slides were then examined with the Leitz fluorescent microscope.

Electron microscopy Cells from selected samples were fixed at room tem-

perature in 0.1 M sodium cacodylate-buffered 3% glutar- aldehyde overnight and postfixed in 1% osmium tetroxide in the same buffer. The cells were then dehy- drated through a graded series of alcohol and embedded in Epon. Thin sections were stained with lead citrate and uranyl acetate and examined at 80 kV with a JEOL lOOB Electron Microscope by the Diagnostic Electron Microscopy Laboratory (VAMC, Durham, NC).

RESULTS Lymphocyte blastogenesis

Table 1 shows the influence of L-glutamine on peripheral blood mononuclear cells cultured in glutamine-deficient RPMI-1640 in the presence of pokeweed mitogen and assayed €or 3H- thymidine incorporation at 72 hours. In the presence of dialyzed heat- inactivated fetal bovine serum (FBS) and no supplemental L-glutamine, 3H- thymidine inco oration was negligible (Table 1, line 1). By comparison?H- thymidine incorporation for normal peripheral blood mononuclear cells cultured without pokeweed mitogen but in the presence of L-glutamine is normally approximately 3,000 counts per minute (CPM)/ lo6 cells (data not shown). In these pokeweek-mitogen- stimulated experiments as little as 0.08 mM L-glutamine supplementation resulted in near-maximal response in terms of 3H- thymidine incorporation (Table 1, line 3). High concentrations of L-glutamine (10 mM) actually decreased thymidine incorporation (line 6). In cells cultured in the presence of nondialyzed heat- inac- tivated FBS lot #1 without supplemental L-glutamine (G- , line 71, additional L-glutamine su plementation in

corporation (line 8). This lack of difference in thymidine incorporation between the G- and G+ cultures appears

the G+ cell culture did not influence 8 H-thymidine in-

L-GLUTAMINE IN LYMPHOCYTE DIFFERENTIATION 277

TABLE 1. The influence of FBS and L-glutamine concentration on 3H-thymidine incorporation in day 3 pokeweed-mitogen-stimulated mononuclear cell cultures

Supplemental Donor cpm/106 cells' L-glutamine FBS FBS

Fetal bovine serum (mM) lot #1 lot #2

1) Dialyzed and heat-inactivated 0.0 546 615 2) Dialyzed and heat-inactivated 0.016 6,545 7,588 3) Dialyzed and heat-inactivated 0.08 17,638 15,649 4) Dialyzed and heat-inactivated 0.40 16,584 15,419 5) Dialyzed and heat-inactivated 2.0 13,611 12,090 6) Dialyzed and heat-inactivated 10.0 8,920 10,104 7) Non-dialyzed and (G-) 0.0 23,160 17,310

heat-inactivated

heat-inactivated 8) Non-dialyzed and (G+) 2.0 23,927 24,745

'Average of duplicate sample CPM of 3H-thymidine incorporation at day 3 in representative donor pokeweed-mitogen-stimulated mononuclear cell population initially cultured in glutamine-deficient RPMI-1640 plus two different lots of dialyzed, heat-inactivated, or nondialyzed, heat-inactivated fetal bovine serum with varying concentrations of supplemental L-glutamine.

TABLE 2. Selective amino acid deficiency and 3H-thyrnidine incoruoration in Dav 3 uokeweed-mitoaen-stimulated cell cultures

Donor cpd106 cells' L-Glutamine L-Glutamic acid L-Asparagine

(2 mM) C.13 mM) (.37 mM) 1 2

+ 31,965 31,965 + 39,600 37,511 - 37,511 32,234 + 1,052 1,215 + 996 1,044 - 119 177

'Results represent average duplicate sample CPM of 3H-thymidine incorporation at day 3 into two different donor pokeweed-mitogen-stimulated mononuclear cell populations initially cultured in RPMI-1640 media plus 10% dialyzed heat inactivated fetal bovine serum deficient in (-1 or supplemented with (+) L- glutamine, L-glutamic acid, and L-asparagine in concentrations generally used in RPMI-1640.

to be due to residual L-glutamine content in the nondialyzed heat-inactivated FBS. The average L-glutamine content of FBS is approximately 0.7 mM or 0.07 mM at a 10% concentration in media (personal communication from Hyclone Sera, Logan, UT). Spontaneous decomposition of L-glutamine in serum or media is time and temperature dependent (Tritsch and Moore, 1962). This may explain the inability of nondialyzed FBS lot #2 to provide maximal thymidine incorporation in the G- cell culture (line 7) without further L-glutamine supplementation (line 8). The failure to reach the same level of 3H- thymidine incorporation in the cell cultures containing dialyzed serum versus those containing nondialyzed serum may represent loss of other important nutrients through dialysis or the introduction of inhibitory factors.

Heat- inactivated nondialyzed FBS lot #1 was used in all subsequent experiments. In seven different donor pokeweed-mitogen-stimulated cell cultures, initially placed in glutamine- deficient RPMI 1640 plus lo'% heat- inactivated fetal bovine serum (lot #1) with or without 2 mM L-glutamine, 3H- thymidine was assessed after 72 hours. The degree of thymidine incorporation varied from donor to donor (range 23, 918-91,500 c p d lo6 cells) but was equivalent for individual donor cell cultures in the absence or presence of additional L-glutamine supplementation, confirming the results in Ta- ble 1 (lines 7 and 8).

Specificity of L-glutamine for lymphoblastic transformation

To examine whether or not L-glutamine or an amide donor, L-asparagine, could reproduce the effects of L- glutamine on thymidine incorporation. Selectamine Kits (Gibco) were used to create RPMI- 1640 media deficient in L-glutamine, L-glutamic acid, and/or L-asparagine. Donor mononuclear cells were cultured in these media supplemented with 10% dial zed, heat-inactivated FBS

after 72 hours (Table 2). While the absence of L-glutamine abolished 3H-thymidine incorporation (lines 4-6), the absence of L-glutamic acid (line 2) or L-asparagine (line 3) in the presence of L-glutamine had no apparent effect on lymphoblastic transformation. Furthermore, attempts to supplement L-glutamine-free media with equimolar concentrations of ammonium glutamate, ammonium bicarbonate, or ammonium chloride did not restore 3H-thymidine incorporation. This specific requirement for L-glutamine rather than its components may relate to differential transport of L-glutamine into cells compared to other amino acids (Yamane, 1977) or to a specific intracellular function of L-glutamine per se.

Immunoglobulin synthesis and secretion Figure 1 shows the pattern of immunoglobulin synthe-

sis (ICF, intracellular fluid) and secretion (ECF, extracellular fluid) in day 0 unstimulated and day 7 pokeweed-mitogen-stimulated human peripheral blood mononuclear cell cultures from donor C, as determined by 3H- leucine incorporation into immunoglobulin ) (3H- Leu- Ig cpm x lOP3/lO6 cells). G+ refers to cells cultured in glutamine deficient RPMI-1640 plus 10% heat- inac- tivated, non dialyzed FBS plus 2 mM L-glutamine supplementation. G - cultures were identical except that no glutamine supplement was added to that already present in the FBS. Day 0 cultures demonstrate low, but detectable, levels of immunoglobulin synthesis and secretion in unstimulated peripheral blood lymphocytes in both the G+ and G- cultures. These levels do not change significantly over 7 days in culture in the absence of pokeweed mitogen (data not shown). However, in the pokeweed-mitogen-stimulated G + 7-day cell culture, a dramatic increase in immunoglobulin synthesis and secretion occurs, with synthesis (ICF) reaching a steady state between 3 and 6 hours of cell culture and secretion (ECF) increasing linearly from 3 to 9 hours. Our laboratory has previously reported similar immunoglobulin kinetic curves using mature human plasma cells (Mabry et al., 1977). By contrast, in the G- day 7 culture, despite the presence of pokeweed mitogen stim- ulation, the immunoglobulin synthesis and secretion kinetic curve is essentially unchanged from that of resting nonstimulated lymphocytes. Increment in immunoglobulin synthesis and secretion

with L-glutamine Similar to results obtained with donor C, in 12 differ-

ent donor experiments, striking differences in immunoglobulin synthesis and secretion were seen in 7-day pokeweed-mitogen-stimulated mononuclear cell cultures depending upon the absence (-1 or presence (+) of L-glutamine supplementation at the initiation of culture. Figure 2 emphasizes the differences seen in immunoglobulin synthesis (ICF) and secretion (ECF) after

and PWM and assessed for 3y H thymidine incorporation


D a y 7

G - ---- ECF - G-- - - - E C F o

-

-

I I I

3 6 9 3 6 9 Hours Hours

Fig. 1. Pokeweed-mitogen-stimulated lymphocyte immunoglobulin determined by the immune coprecipitation assay. G+ cells were cul- synthesis and secretion. Immunoglobulin synthesis (ICF) and secretion tured in the presence of 2 mM L-glutamine, while G- cells were not. (ECF) in unstimulated day 0 and pokeweed-mitogen-stimulated day 7 Note the inability of the G- cell culture to develop immunoglobulin normal human peripheral blood mononuclear cells (donor C). 3H-Leu- secretory kinetics characteristics of plasma cells, as shown by the G+ Ig represents specific 3H-leucine incorporation into immunoglobulin as cell cultures.

O I C F 9 h r *ECF 9h r

24 t

L- - + A

1 T

u'""""-- ! ! L j / O ; ~ - + . + - + - + - + - + - + - +

B C D E F G H I

8-w- - + - + - + J K L

7 Day Donor Cultures f. L-Glutornine

Fig. 2. Increment in immunoglobulin synthesis and secretion with L- glutamine. The difference in immunoglobulin synthesis (ICF) and secretion (ECF) in 7day pokeweed-mitogen-stimulated cell cultures without (-) and with (+) 2 mM L-glutarnine supplementation in 12 different normal adult donor experiments. 3H-Leu- Ig CPM for ICF and ECF represent the 9-hour time point as shown on the curves in Figure 2 (donor C).

Comparison of 3H-thymidine incorporation to protein and immunoglobulin synthesis and secretion

studies Table 3 compares the thymidine incorporation at day

3 to the 3H-protein and 3H-immunoglobulin synthesis and secretion studies at day 7 in G- and G+ cell cultures for donor F, as previously described. In addition, in this donor experiment, simultaneous cell cultures were also performed using dialyzed heat-inactivated FBS without and with 2 mM L-glutamine supplementation.

In the G- culture using nondialyzed FBS (line l), maximal 3H-thymidine incorporation was present and was not further enhanced by additional L-glutamine (G+, line 2). In the culture with dialyzed FBS and no glutamine supplementation (line 3), thymidine incorporation was minimal. Despite the difference in 3H-thymidine incorporation between the nondialyzed FBS cell culture without L-glutamine (line 1) and the dialyzed FBS cell culture without L-glutamine (line 3), in both cultures 3H- leu-protein and immunoglobulin synthesis (ICF) and secretion (ECF) at day 7 were similar to values obtained routinely for day 0 unstimulated cell cultures. The addition of 2 mM L- glutamine to either the culture with nondialyzed FBS (line 2) or the culture with dialyzed fetal bovine serum (line 4) resulted in a doubling of the plateau of intracellular immunoglobulin synthesis and a four fold increase in cumulative immunoglobulin secretion, similar to plasma cell kinetics.

Influence of L-glutamine on pokeweed-mitogen- induced plasma cell differentiation 9 hours of leucine incorporation. While the magnitude

of response to L-glutamine varied from donor to donor, Cytoplasmic immunofluorescence was measured in 7- the plateau of intracellular immunoglobulin synthesis day pokeweed-mitogen-stimulated cell cultures in glu- doubled in most cases, while cumulative immunoglobu- tamine-deficient RPMI media with 10% heat-inacti- lin secretion was generally fivefold higher over the 9- vated, nondialyzed FBS without (G- ) or with (G+) 2 hour time period (G+ > G-). mM L-glutamine supplementation (Table 4). Despite the


TABLE 3. The requirement of L-glutamine for pokeweed mitogen-induced blastogenesis and immunoglobulin synthesis

Day 7 Day 7 Day 3 3H-Leu-prot2 3H-Leu-Ig2 Supplemental 3H-Thy'

L.glutamine incorporation (cpm/106 cells) (cpm/106 cells) Fetal bovine serum (mM) (cpm/106cells) ICF ECF ICF ECF

1) Heat-inactivated ( G - ) 0 38,983 27,416 6,084 2,950 3,117

2) Heat-inactivated (G+) 2 39,644 43,356 17,216 7,238 11,899 nondialyzed

nondialyzed 3) Heat-inactivated 0 1,240 25,514 4,369 5,779 448

dialyzed

dialyzed 4) Heat-inactivated 2 25,267 59,083 21,088 12,736 11,711

iResults rrpresent average of duplicater sample CPM of 'H.ttiymidine incorporation at day 3 in single donor experiment (donor I;) with cells initially cultured in RPMI 1640 media plus lOPr heat-indctivaled dialyzed or nondialyzed fetal bovine serum with or without supplemental L-glutamine. 'Fiesul~~ reprrsent average of duplicnte sample C'PM for 'H leucinc incorporation into protein ?II-I.eu-prot and irnmunoglnhulin ("1i.I.eu-lgi. Data displayed is the %hour time point of synthesis rlCF1 and secretion G-;C'I;i wee Fig 2,

TABLE 4. The influence of L-glutamine on cytoplasmic immunofluorescence in dav 7 cell cultures

% Positive cells' Donor G - G +

A 0 6 B 1 3 C F G H

2 1 1 2

12 4 5 9

I 2 10

'Results represent the percent of cytoplasmic fluorescence positive cells in seven different donor cell cultures after 7 days in RPMI-1640 media plus 10% heat- inactivated fetal bovine serum without ( G - ) or with ( G + ) 2 mM L-glutamine supplementation. Slides of cytofuged cell cultures were fixed in acetic acid/ ethanol incubated with fluorescein-conjugated goat polyvalent antihuman immunoglobulin, Fab fragment, and extensively washed prior to examination with a Leitz fluorescent microscope. The percentage of positive cells were determined from a minimum of a 200 cell normal count, utilizing several different areas of the cytofuge preparation.

fact that these cultures had equivalent thymidine incorporation after 3 days in culture, plasma cell differentiation was markedly enhanced only in the cultures that were supplemented with L-glutamine. These changes in cytoplasmic fluorescence correlated with the findings of increased immunoglobulin synthesis and secretion, as shown in Figure 2.

Electron microscopy performed on the day 7 glutamine-supplemented cell cultures from donor A showed development of rough endoplasmic reticulum in approximately 6% of cells, consistent with an active protein synthetic and secretory process (Fig. 3B). No such cells could be seen in the culture without glutamine supplementation (Fig. 3A), correlating well with the findings by cytoplasmic fluorescence. This correlation between the presence of rough endoplasmic reticulum and cytoplasmic fluorescence was also noted in the two other donor cell cultures studied by electron microscopy.

Influence of L-glutamine on other cell culture parameters

Table 5 reviews other parameters that were assessed after 7 days in culture in the G- and G+ cultures. Cell recovery, viability, and transformation were comparable. Analysis of two-donor cell cultures, using an auto- mated Hemolog D, showed no difference in the size

TABLE 5. The influence of L-glutamine on day characteristics'

Cell recovery CSP Viability (%bP Light microscopy Esterase-positive cells (%)

(D) (I)

%Bl%T (A) (B) (HI (1)

G -

70-80 70-85 -

0.9 2.8

12163 6153

18154 20165

7 cell culture

G+

75-85 70-85 -

1.6 3.2

10160 8156

20167 17161

'Morphologic exam of Wright-strained cytofuge preparations a t day 7 in 12 donor culture experiments. Esterase-positive cells in two different donor cell cultures at day 7 determined by Hemalog analysis of 10,000 cells. %B cells determined by surface staining of cells with fluorescein-conjugated fluorescent microscope. %T cells determined by light microscopy of % rosettes in cell cultures after incubation with neuraminidase-treated sheep red blood cells. Results represent four different donor cell cultures at day 7, from at least a 200-cell normal counff cell culture. %suits of 12 donor culture experiments without (G- ) and with (G+) L-glutamine supplementation, determined by day 7 cell counWday 0 cell count x loo%, using a Coulter counter. 3Viability determined by trypan blue exclusion a t day 7, from at least a 200-cell normal count. 4Comparable transformation.

distribution of the cells or in the percent of cells that were esterase positive. The percent B cells and percent T cells were also equivalent after 7 days in culture. Furthermore, in four additional donors, the ratios of T helper to T suppressor cells were not affected by the difference in glutamine content of the two cultures.

DISCUSSION Tissue culture media is a complex system with which

to work, because of the wide variety of amino acids, inorganic salts, vitamins, glucose, and other components present. In addition, most in vitro culture systems, including mitogen-stimulated human lymphocytes, have required the addition of serum for optimal survival and growth, introducing many unknown variables. To begin to dissect the role of individual nutrients in such a complex in vitro system as human lymphocyte transformation and plasma cell differentiation, we studied one variable, L-glutamine. Intracellularly, L-glutamine serves as an amide donor in many amidotransferase

Fig. 3.A. Electron micrograph of day 7 pokeweed-mitogen-stimulated peripheral blood mononuclear cells cultured without L-glutamine supplementation. B. Electron micrograph of day 7 pokeweed-mitogen-

stimulated mononuclear cells from same donor cultured with 2 mM L- glutamine supplementation.


reactions, such as the formation of carbamyl phosphate (pyrimidine biosynthesis), 5-phosphoribosylamine (purine biosynthesis), NAD, GMP, CTP, asparagine, and glucosamine-6-phosphate and also serves as an energy source as a precursor of alpha-ketoglutarate in the tri- carboxylic acid cycle (Kovacevic and McGiran, 1983). Glutamate is also an intermediary in the gamma-gluta- my1 cycle, which is important for amino acid transport and the generation of glutathione (Jensen and Meister, 1983).

In our experiments involving lymphoblastic transformation, L-glutamine could not be replaced by L-glutamic acid, ammonium glutamate, other ammonium salts, or another amide donor, L-asparagine (Table 2). Thus, L-glutamine per se seems essential for lymphoblastic transformaton, as has been previously suggested (Schrek et al., 1973; Baechtel et al., 1976). This specific requirement for L-glutamine for lymphoblastic transformation has also been documented for concanavalin A (Con-A)-stimulated rat lymphocytes (Ardawi and Newsholme, 1983). In their system, maximal 3H-thymidine incorporation occurred at a concentration of .1 mM L-glutamine, similar to our results in Table 1. Our study extends these observations to human lymphocytes and describes a distinct, additional role for L-glutamine in plasma cell differentiation and immunoglobulin synthesis and secretion. As shown in Table 3, despite equivalent lymphoblastic transformation, only cultures further supplemented with L-glutamine underwent plasma cell differentiation by cytoplasmic fluorescence, electron microscopy, and immunoglobulin synthesis and secretion.

This central role we have described for L-glutamine in lymphocyte differentiation is consistent with its role in other in vitro cell systems. In human fibroblasts, L- glutamine accounts for approximately 30% of the total energy requirement of the cells (Zielke et al., 1978) and is the major amino acid in the regulation of DNA replication of these cells (Zetterberg and Engstrom, 1981). In addition, although the mechanism remains unclear, the importance of L-glutamine in protein synthesis and secretion has been demonstrated in several cell types. Collagen synthesis by chick tendon cells requires L- glutamine (Lehtinen et al., 1978). In rat pancreatic islet cells, L-glutamine markedly enhances insulin release (Malaisse et al., 1980). In chondrocytes, L-glutamine is necessary for glycosaminoglycan synthesis (Handley et al., 1980). Of interest, related to our findings, is that electron micrographs of these glutamine-deficient chondrocytes show a disaggregation of polyribosomes that can be reversed by the addition of L-glutamine.

In our in vitro system, the requirement for L-glutamine for lymphoblastic transformation may be due to a role as an energy source, a role in purine or pyrimidine biosynthesis (McKeehan, 19821, and/or by specific effects on lymphocyte subsets and monocytes or lymphokine production (Muraguchi and Fauci, 1982). The additional requirement of L-glutamine for plasma cell differentiation and immunoglobulin synthesis may relate to an effect on the development of rough endoplasmic reticulum or associated polyribosomes, as in chondrocytes; to glycosylation via the formation of glucosamine-6-phosphate; andfor indirectly by activation of accessory cell function.

In vivo, the role of L-glutamine in the immune response has been studied indirectly via L-asparaginase- glutaminase injections in mice (Katkenwitz and Ben-

dick, 1983). A marked decrease in spleen B cell immu- noblasts occurs as a direct correlate of glutamine depletion (Distasio et al., 1982). Furthermore, use of glutaminase-asparaginase in man results in a loss of skin test reactivity in vivo, as well as diminished phy- tohemagglutinin-induced blastogenesis in vitro (Abu- chowski et al., 1981). Studies of leukemic lymphoblasts in vitro suggest that glutamine deprivation profoundly depresses purine metabolism in B cell and null cell lymphoblastic leukemia lines but not in a T cell acute lymphoblastic leukemia line (Raivio and Anderson, 1982). Two other glutamine antagonists, DON and Aciv- icin, inhibit glutamine transport and/or L-glutamine amidotransferases and may be useful agents to study differences in metabolic requirements for L-glutamine in different cell populations (Kovach et al., 1981; Poster et al., 1981).

Further in vitro studies of the effects of L-glutamine on normal and neoplastic lymphocytes may clarify the mechanism of action of L-glutamine in lymphocyte/ plasma cell differentiation as well as the role of glutamine and its antagonists in both immunosuppressive and antineoplastic therapy in man. Furthermore, exper- imental approaches comparable to those reported here for L-glutamine may be useful in understanding the relationship 06 other nutritional variables to the biochemistry of th% immune response.

ACKNOWLEDGMENTS This work was supported in part by General Medical

Research, Durham VAMC, the VA Geriatric Fellowship Program, the Mallinkrodt Foundation, and the Diagnos- tic Electron Microscopy Laboratory, VAMC, Durham, North Carolina. The authors thank Jane Davis and Wal- ter Fennell, Jr., for their technical assistance.

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The essential role of L-glutamine in lymphocyte differentiation in vitro

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