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Nutritional Immunology

Arginine Supplementation Enhances Mitogen-Induced Splenocyte Proliferation but Does Not Affect In Vivo Indicators of Antigen-Specific Immunity in Mice^1,2

M. F. Suarez Butler, Bobbi Langkamp-Henken³, Kelli A. Herrlinger-Garcia, Amy E. Klash, Michelle E. Szczepanik, Carmelo Nieves, Jr, Robert J. Cottey^* and Bradley S. Bender

Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611-0370; ^* Research Service, North Florida/South Georgia Veterans Health System, Gainesville, FL 32608-1197; and North Florida/South Georgia Veterans Health System, Gainesville, FL 32608-1197 and Department of Medicine, University of Florida, Gainesville, FL 32610-0277

³To whom correspondence should be addressed. E-mail: henken{at}ufl.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Arginine is a conditionally essential amino acid with many physiologic roles. Its role in immune function has been one of major focus with conflicting results. Early in vitro immune studies demonstrated increased mitogen-induced lymphocyte proliferation with dietary arginine supplementation; however, not all studies confirmed this effect. Even less is known about the effect of arginine supplementation on in vivo immune responses. To test whether arginine supplementation enhances in vivo indicators of immune function, young female BALB/c mice were fed either the AIN-93G rodent diet (6.4 g arginine/kg diet) or the same diet with 20 g total arginine/kg diet for 15 d before delayed-type hypersensitivity (DTH) testing with 2,4-dinitrofluorobenzene (n = 16–18/diet group). The same mice were challenged with influenza virus A/Port Chalmers/1/73 (H3N2) 15 d later. Mice were killed 3, 6, or 31 d postinfluenza challenge (5–6/diet group on each day). Mitogen-induced splenocyte proliferation, body weight, anti-influenza serum antibody, lung viral titers, and serum arginine were measured. DTH did not differ between diet groups. On d 6 and 31 postchallenge, mitogen-induced proliferation of splenocytes from mice fed the arginine diet was >1.5-fold that of mice fed the control diet (P < 0.05). Body weight and influenza lung viral and serum antibody titers did not differ between diet groups. These data suggest that despite significant enhancement of in vitro mitogen-induced splenocyte proliferation, arginine supplementation does not have a biologically significant effect on antigen-specific in vivo indicators of immune function in this model.

KEY WORDS: • arginine • influenza • mice • antibody • delayed-type hypersensitivity

The amino acid arginine plays an important role as an intermediate in the urea cycle and in the synthesis of protein and nonprotein, nitrogen-containing compounds (e.g., nitric oxide, polyamines). Although arginine is synthesized in the body, it must be included in the diet during times of growth, suggesting that arginine is conditionally essential. Additionally, physiologic concentrations of dietary arginine appear to be essential to maintain immune function. Ronnenberg and colleagues (1) showed that in vitro mitogen-induced splenocyte proliferation and interleukin-2 production were significantly lower in splenocytes from young and aged rats fed an arginine-free vs. control diet (11.2 g arginine/kg diet). Kobayashi and colleagues (2) reported a decrease in antigen-specific fecal IgA when mice were fed an arginine-free vs. an arginine-containing liquid diet (8.75 g arginine/L diet). Further evidence of the importance of adequate arginine levels is that physiologic concentrations of arginine are required in culture media to support cell survival and proliferation (1–4).

During times of stress, serum arginine levels decrease (5–7). This decrease is interpreted as another condition in which synthesis does not keep pace with metabolic demands. Consequently, pharmacologic doses (i.e., levels above those found in standard diets) of arginine are added to enteral formulas or experimental animal diets to improve immune outcomes during critical illness. More recently, this practice of pharmacologic arginine supplementation has been called into question because of concern that excess nitric oxide will be produced from the arginine during sepsis.

Although early immune studies demonstrated increased in vitro mitogen-induced lymphocyte proliferation with pharmacologic concentrations of dietary arginine, not all studies confirm this finding (8–10). Even less is known about the role of arginine supplementation on in vivo immune function and in particular, specific immunity. The purpose of this study was to determine whether a diet containing pharmacologic levels of arginine, compared with a standard diet containing physiologic levels of arginine, enhances in vitro mitogen-induced lymphocyte proliferation and in vivo indicators of antigen-specific immunity in mice before and after an acute stress (challenge with the influenza virus).

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Animals and diets. Female BALB/c mice (3–4 wk old; Charles River) were individually housed in polycarbonate shoeboxes with stainless steel lids in a 22°C room with a 12-h light:dark cycle. Mice were allowed to acclimate for 1 wk with standard diet and water freely available. Mice were then fed either an AIN-93G diet (control diet, 6.4 g arginine/kg diet, Harlan Teklad), the same diet supplemented with arginine (arginine diet) to provide a total of 20 g arginine/kg diet, or an AIN-93G diet made isonitrogenous to the arginine diet with the addition of alanine (isonitrogenous diet, preliminary studies only) (11,12). Diet and water were freely available. Mice were weighed at baseline and weekly before the influenza challenge and daily postinfluenza challenge. The University of Florida Institutional Animal Care and Use Committee approved all animal procedures.

    Study design. On d 9 through 15 of the study diets, delayed-type hypersensitivity (DTH)⁴ responses were elicited and measured (Fig. 1). Mice were challenged with influenza virus 2 wk later. Mice were anesthetized with halothane and killed by cardiac puncture and exsanguination on d 3, 6, or 31 postinfluenza challenge (after 33, 36, or 61 d of consuming the diets). On those days, spleens were removed for lymphocyte proliferation, lungs were removed for influenza viral titers, and serum was taken to measure influenza-specific antibody titers and arginine concentrations.

View larger version (15K): [in this window] [in a new window]   FIGURE 1 Study design. Mice consumed the control diet (6.4 g arginine/kg diet) or arginine diet (20 arginine/kg diet). Between d 9 and 15 of the preinfluenza challenge period, mice were sensitized to an antigen (2,4-dinitrofluorobenzene) and DTH was measured (n = 16 to 18/diet group). After 30 d of consuming the diet, mice were challenged with A/Port Chalmers/1/73 (H3N2) and then killed on d 3, 6, or 31 postchallenge to measure in vitro mitogen-induced splenocyte proliferation, influenza antibody and lung viral titers, and serum arginine concentrations (n = 5 to 6/d postinfluenza challenge for each diet group).

    Influenza challenge. On d 30 of the diet, mice were anesthetized and challenged intranasally with a total of 20 µL of influenza A/Port Chalmers/1/73 [H3N2; 50% tissue culture infective dose (TCID₅₀) 10^10.71/L], prepared as described previously (13). Once the mice were challenged, they were placed in microisolator cages and continued to consume the study diets.

    Lung influenza viral titers. The lungs were aseptically removed and homogenized in cold PBS and then centrifuged at 200 x g for 1 min at 5°C. The supernatant was stored at –80°C until assayed. The amount of virus in the lung homogenates (replicates of 5) was determined using a previously published method (14). Modifications to this method include the use of Madin Darby canine kidney cells (American Type Culture Collection) in DMEM complete medium without phenol red containing 5% v:v fetal calf serum at a concentration of 2.5 x 10⁴ cells/well, the addition of 50 µL of a 1% v:v fresh chicken RBC (CRBC, collected by cardiac puncture and mixed 1:1, v:v with an anticoagulant) suspension in PBS to each well on d 5 of incubation, and the determination of the agglutination pattern after 1 h of incubation. The TCID₅₀ was calculated; in samples in which virus was undetectable, a zero was assigned for the log TCID₅₀.

    Serum anti-influenza antibody titers. Blood from cardiac puncture was collected into CAPIJECT T-MG tubes (Terumo Medical Corporation) containing gel silica particles for collection of serum. A traditional hemagglutination inhibition assay with modifications was performed (15). Briefly, 50 µL of serum was incubated with 150 µL of cholera toxin (Accurate Chemicals) for 18–20 h at 37°C. The serum/cholera toxin mixture was then heated to 56°C for 60 min before incubation with the 1% CRBC mixture 1:4 (sera-cholera:CRBC) dilution for 30 min at room temperature with mixing every 10 min. The solution was centrifuged at 12,000 x g for 5 s and 50 µL of the supernatant was plated in triplicate. Serial dilutions (1:2) were performed with PBS containing 0.05% v:v bovine serum albumin down a 96-well V-bottomed plate. A viral antigen solution containing 4 hemagglutination units in 50 µL was added to each well. The plates were incubated for 30 min at room temperature after which 50 µL of a 1% CRBC suspension was added and the plates were incubated for an additional 45 min. Plates were then tilted to read the agglutination patterns. If antibody was undetectable a value of one half of the lowest possible dilution was assigned for calculations.

    Mitogen-induced splenocyte proliferation. Splenocytes were isolated, stimulated with or without 10 mg/L of phytohemagglutinin (Sigma), and incubated for 66 h as previously described (16). Cells were then stimulated with 37 kBq/well of ³H-thymidine (specific activity: 740 GBq/mmol, DuPont NEN) and harvested at 72 h with an automated cell harvester. The amount of ³H-thymidine incorporated into the DNA was counted in a Beckman LS2800 Liquid Scintillation Counter (Beckman Instruments). Unstimulated counts were subtracted from stimulated counts and data are reported as disintegrations per second (Bq).

    Serum arginine concentrations. Serum was isolated from blood samples and arginine concentrations were determined by HPLC. Serum samples were deproteinized with sulfosalicylic acid and derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (Waters). Samples were analyzed with an Alliance 2695 system (Waters) and a RF-10AXL fluorescence detector (Shimadzu) using a reverse-phase Zorbax Extend-C18 5-µm column (250 mm x 4.6 mm, Agilent Technologies) and a Zorbax Extend-C18 5-µm guard column (12.5 mm x 4.6 mm, Agilent Technologies).

    Statistical analysis. The association between diet group and weight as a percentage of baseline was determined using a 2-way univariate repeated-measures test. The effect of diet and day on mitogen-induced splenocyte proliferation, anti-influenza serum antibody, lung influenza viral titers, and serum arginine were analyzed using 2-way ANOVA with a regular factorial design. Preplanned comparisons were made between means using Fisher’s least significant difference tests. The effect of diet on DTH response was analyzed using a 2-tailed Student’s t test. Statistical analyses were performed using SAS (version 9.0, 2002, SAS Institute) or GraphPad InStat (version 3.05, GraphPad Software). Data were graphed using Prism 4 for Windows (version 4.02, GraphPad Software). All data are expressed as means ± SEM, and differences were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Preliminary studies included 3 diet groups (e.g., control, arginine, and isonitrogenous diets); however, these studies were stopped when 4 of 7 mice in the isonitrogenous group died or were euthanized due to excessive (>30%) weight loss postinfluenza challenge. Additionally, DTH data showed a lower antigen-specific response (P < 0.05) in mice fed the isonitrogenous diet compared with the control diet. The challenged ear thickness was 146 ± 8% [n = 6], 139 ± 5% [n = 16], and 129 ± 4% [n = 16] in mice fed the control, arginine, and isonitrogenous diets, respectively. Subsequent studies excluded the isonitrogenous group and these data are presented below.

Baseline weight (d 0 of diet) did not differ between the control and arginine diet groups. The mice gained weight at a similar rate preinfluenza challenge and lost 10% of body weight postinfluenza challenge (Fig. 2). The percentage of prechallenge weight on d 25–31 postchallenge was significantly greater than that on the day of influenza challenge in both diet groups (P < 0.05, Fig. 2).

View larger version (16K): [in this window] [in a new window]   FIGURE 2 The percentage of preinfluenza challenge weight in mice fed either the control or arginine diet and challenged with influenza on d 0. Data presented as means ± SEM. *Different from day of influenza challenge, P < 0.05.

Influenza lung viral and serum anti-influenza antibody titers were measured on d 3, 6, and 31 postinfluenza challenge. Arginine supplementation did not affect lung viral titers or clearance. Lung viral titers were 5.3 ± 0.2 and 5.7 ± 0.4 log₁₀ on d 3 in the control and arginine diet groups, respectively. Viral clearance from the lungs was evident in 1 mouse in the control and in 2 mice in the arginine diet groups on d 6 postchallenge. By d 31 postinfluenza challenge, all mice had cleared the virus. Serum anti-influenza antibody titers were not detectable on d 3 or 6 postinfluenza challenge for either diet group (data not shown). Serum antibody titers to influenza at d 31 postchallenge were detectable but not different between the control (7.2 ± 0.2 log₂) and arginine (7.2 ± 0.3 log₂) diet groups.

Net thymidine incorporation into splenocytes was used as an indicator of proliferation. Splenocyte proliferation from mice fed the arginine diet was greater than that from control mice on d 6 (P < 0.05) and 31 (P < 0.05) postinfluenza challenge (Fig. 3). Splenocyte proliferation did not differ between diet groups on d 3 postinfluenza challenge; however, proliferation for both diet groups was significantly greater (P < 0.001) on d 6 and 31 than on d 3 postinfluenza challenge. Unstimulated counts for d 3 (18 ± 5 Bq, 11 ± 3 Bq) and d 31 (61 ± 13 Bq, 61 ± 8 Bq) for control and arginine groups, respectively, did not differ between diet groups; however, they were significantly greater on d 6 in the control group (110 ± 23 Bq) than in the arginine group (48 ± 13 Bq).

View larger version (19K): [in this window] [in a new window]   FIGURE 3 Mitogen-induced proliferation of splenocytes from mice fed the arginine vs. control diet on d 3 (n = 5/group), 6 (n = 6–7/group) and 31 (n = 6/group) postinfluenza challenge. Data are expressed as mean stimulated Bq minus unstimulated Bq ± SEM. *Different from d 3, P < 0.05; different from control diet group on that day, P < 0.05.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Although arginine is conditionally essential to maintain growth in vivo and in vitro and to maintain immune function during acute stress, there is no consensus on the benefit of supplementing dietary arginine beyond the level found in a standard diet (1,2,8,9). The purpose of this study was to determine whether a diet containing pharmacologic levels of arginine, compared with a standard diet containing physiologic levels of arginine, enhanced in vitro and in vivo immunity in mice before and after an acute stress (i.e., challenge with the influenza virus). In this model, arginine supplementation did not enhance antigen-specific in vivo immune function.

Arginine supplementation had no significant effect on DTH responses. Others have demonstrated an arginine-mediated effect on DTH responses using different models (12,17,18). Saito et al. (17) demonstrated a significantly greater DTH response in burned guinea pigs fed 7.2 g arginine/L vs. 2.4 g arginine/L in an elemental enteral diet. Kennedy et al. (18) showed a significantly greater DTH response in rats with obstructive jaundice fed a diet with 20 g arginine/kg diet with or without 1.8 g arginine/L in the drinking water. Both studies assessed DTH responses after an acute stress during which the animals lost significant weight (17,18). The enhanced DTH responses suggest that arginine supplementation may be necessary to keep pace with metabolic demand during these conditions. In the present study, mice were not acutely stressed during DTH testing. This suggests that supplementing arginine to levels beyond that found in a standard diet is not beneficial in a healthy rodent model.

Although the conclusion above is reasonable considering that in vivo arginine synthesis should meet normal demands in an unstressed animal, this conclusion does not support our previous findings. Earlier we reported an increase in DTH response in healthy young, adult, and aged mice fed an arginine-supplemented diet (20 g arginine/kg diet) vs. an isonitrogenous control diet (6.4 g arginine/kg diet) that was made isonitrogenous with the addition of alanine (12). Our preliminary studies leading to the present work showed that arginine supplementation did not potentiate the DTH response; rather the alanine control diet attenuated the response. The isonitrogenous (alanine) control diet was later dropped from the study when 4 of 7 mice from this group died or were euthanized due to excessive weight loss following influenza challenge. This demonstrates the difficulty in determining the optimal control diet for arginine supplementation studies in which increased nitrogen load (i.e., isonitrogenous control diet) without a similar increase in urea cycle intermediates may result in toxic conditions. On the basis of these data, arginine added to a standard rodent diet does not appear to enhance DTH responses in healthy mice.

Arginine supplementation is more likely to have an effect on in vivo specific immune responses during an acute stress when arginine may become conditionally essential; however, this was not the case. Mice fed the arginine-supplemented diet did not clear influenza virus from the lung to a greater extent nor did they produce more influenza-specific antibodies than mice fed the standard diet. It is possible that the influenza challenge was not a great enough stress to increase the metabolic demand for arginine, that arginine was not supplemented in a quantity great enough to meet conditional needs, or that arginine was not required for influenza-induced immune responses. These scenarios are unlikely considering that postinfluenza challenge, mice lost 10% of body weight and mean serum arginine levels were >40% lower on d 6 postinfluenza challenge vs. d 3 and 31 postchallenge in mice fed the control diet (P < 0.05), whereas serum arginine levels postinfluenza challenge did not differ in mice fed the arginine-supplemented diet. These data suggest that arginine supplementation may have prevented the expected decline in serum arginine after an acute stress.

The influenza model was specifically selected because many of the purported effects of arginine supplementation are T-cell–mediated effects and influenza is cleared via T-cell–mediated immune mechanisms (14). Through a series of in vitro studies Ochoa and colleagues (4) demonstrated that physiologic concentrations of arginine are required for the proliferation of ß T lymphocytes and more specifically, CD8+ (cytotoxic) T lymphocytes. Additional in vitro studies showed that a physiologic concentration of arginine is essential for the expression of the (zeta) chain, the principal signal transduction element of the T-cell antigen-binding receptor (19). Mice infected with influenza virus generate CD8+ cytotoxic T lymphocytes. These lymphocytes, which require arginine, are the primary mechanism responsible for influenza virus clearance and recovery (4,14,20). Influenza infection also elicits both T helper 1 (T_H1) and T_H2 cytokines (21). The production of cytokines from both T-cell phenotypes may promote antibody responses and cell-mediated immunity. Physiologic in vitro concentrations of arginine also support production of T_H1 (e.g., interferon- and interleukin-2) and T_H2 (e.g., interleukin-4 and interleukin-5) cytokines (2). Additionally, physiologic concentrations of dietary arginine (8.75 g arginine/L vs. 0 g arginine) enhanced antigen-specific mucosal IgA and serum IgG titers in mice orally immunized with tetanus toxiod plus cholera toxin (2). In light of the studies discussed above, it is difficult to interpret why in vivo supplementation of arginine resulted in greater in vitro mitogen-induced splenocyte proliferation when arginine was present in the cell culture media (1050 µmol arginine/L) in concentrations > 16 times that found in the serum. It is possible that splenocyte cell populations or cytokine expressions were altered in vivo with arginine supplementation. However, if mitogen-induced in vitro splenocyte proliferation is physiologically relevant, then the differences in proliferation do not appear to be associated with plasma or in vitro arginine concentrations.

The studies discussed above demonstrate the importance of arginine in the diet and cell culture media. The absence of arginine results in impaired immune function, but physiologic concentrations (i.e., arginine added to cell culture media to mimic plasma arginine concentrations or arginine added to the diet in proportions similar to that found in standard diets) maintain immune function. On the basis of data from the present study, supplementing arginine in a standard arginine-containing diet is not of benefit to antigen-specific immune function in health or after an acute infectious challenge. This conclusion is noteworthy because many "immune-enhancing" enteral formulas used in intensive care units today contain pharmacologic concentrations of arginine. However, the extra arginine may not be of benefit to antigen-specific immune function and could potentially become a liability in the patient who suddenly becomes septic, and for whom excessive production of nitric oxide (a by-product of arginine metabolism) can result in cell damage and uncontrolled systemic vasodilation (22). At this time, data do not support arginine supplementation above that found in standard arginine-containing diets to promote antigen-specific in vivo immune function.

	FOOTNOTES

 
¹ Presented in part in abstract form at Experimental Biology ’02, April 2002, New Orleans, LA [Suarez, M. F., Herrlinger-Garcia, K. A., Bender, B. S., Nieves, C., Jr. & Langkamp-Henken, B. (2002) Arginine supplementation enhances in vitro but not in vivo immune responses in mice. FASEB J. 16: A984 (abs.)].

² Supported by the Florida Agricultural Experiment Station Journal series No. R-10633.

⁴ Abbreviations used: CRBC, chicken RBC; DTH, delayed-type hypersensitivity; TCID₅₀, 50% tissue culture infective dose; T_H, T helper.

Manuscript received 6 December 2004. Initial review completed 7 January 2005. Revision accepted 7 February 2005.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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16. Lanningham-Foster, L., Green, C. L., Langkamp-Henken, B., Davis, B. A., Nguyen, K. T., Bender, B. S. & Cousins, R. J. (2002) Overexpression of CRIP in transgenic mice alters cytokine patterns and the immune response. Am. J. Physiol. 282:E1197-E1203.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suarez Butler, M. F. Articles by Bender, B. S. Search for Related Content PubMed PubMed Citation Articles by Suarez Butler, M. F. Articles by Bender, B. S.