Sulfanilamide

ABSTRACT. Background: Trauma increases the enzyme arginase, thus depleting arginine necessary for producing nitric oxide. Arginine and ω-3 fatty acids are components in immune-enhancing diets. These diets decrease infections in surgical patients, perhaps by preventing arginine deficiency. This study examines whether ω-3 fatty acids alter the metabolic fate of arginine. Thus, we hypothesized there could be differential effects of varying prostaglandins on regulation of arginase. Methods: Prostaglandins PGE1, PGE2, and PGE3 were tested using RAW 264.7 cells cultured in the presence of these prostaglandins for 24 hours. IL-13 (10 ng/mL) was added 24 hours later to induce arginase I. NO production was induced by adding LPS (2 µg/mL) to the cultures after another 24 hours. Results: Arginase activity (nmol/min/mg) was induced by all prostaglandins but significantly more by PGE1 (466.05 ± 30.25) and PGE2 (248.45 ± 15.05) than PGE3 (139.87 ± 19.88; p

Trauma and surgical patients are known to have an impaired immune response, including the suppression of T-cell proliferation and function.1-4 In recent years, increasing evidence has shown that the use of immune-enhancing diets (IEDs) has led to reduced infection rates and postoperative morbidity in surgical and trauma patients.5,6 Though the exact mechanism is unclear, IEDs seem to exert their benefits by restoring depressed T-cell proliferation, which increases the production of nitric oxide and modulates the production of certain inflammatory cytokines.7

Varying IED regimens are similar in that they contain high concentrations of arginine and ω-3 fatty acids. To date, we have assumed that ω-3 fatty acids and arginine would exert "beneficial" immune effects independent of each other.8,9 In this paper, we forward the hypothesis that ω-3 fatty acids may play a synergistic role in overcoming impaired immune function after surgery or trauma.

We have previously demonstrated that trauma is associated with increased arginase I expression in myeloid cells, both in humans and in mice.10 Arginase I expressed in myeloid cells is able to deplete arginine and by this mechanism play an immunoregulatory role, depressing T-cell function and the production of nitric oxide.11 Thus, not surprisingly, elevated expression of arginase I and the consequent decrease in arginine correlate with T-cell dysfunction and decreased nitric oxide production in vivo.12

ω-3 Fatty acids, namely, eicosapentaenoic and docosahexaenoic acids (fish oils), metabolically yield series PGE3 prostaglandins, whereas the use of ω-6 linoleic acid (corn oil) preferentially leads to the formation of PGE2 upon induction of cyclo-oxygenase 2 (COX 2) by traumatic stimuli. Borage oil (also an ω-6 fatty acid) leads to the production of PGE1. All of these fatty acids are being used in different diets clinically with the intention to improve immune dysfunction.13-15 However, the use of ω-3 fatty acids and arginine has gained the greatest attention.

Arginase I expression is up-regulated by prostaglandin E2 and by T helper 2 cytokines such as interleukin 13.lb Thus, it would be possible that supplementation with ω-6 fatty acids such as corn oil after surgery or trauma could profoundly affect the metabolism of arginine by leading to the up-regulation of arginase I and the depletion of arginine. Conversely, we hypothesized that ω-3 fatty acids would protect arginine by attenuating the up-regulation of arginase. This paper demonstrates the "proof of concept" that the supplementation of arginine and ω-3 fatty acids may exert biologic functions through interactions with each other. RAW 264.7 cells cultured with PGE1 and PGE2 (ω-6 fatty acids) up-regulate arginase I expression significantly and synergize with IL-13 to even further induce arginase I expression. PGE3 causes only a slight induction in arginase I, even in the presence of IL-13. Conversely, RAW 264.7 cells cultured with PGE3 exhibit the highest production of nitric oxide in response to endotoxin when compared with PGE1 or PGE2. Production of nitric oxide is increased in all cells when the arginase inhibitor arginase inhibitor N-ω-hydroxy-nor-L-arginine (norNOHA) is added to cultures demonstrating the regulatory effect of arginase I on nitric oxide production.

The data presented in this paper demonstrate that there may be significant metabolic interactions when giving 2 nutrients together, in this case ω-3 fatty acids and arginine. Such is the case in IEDs. This paper raises the hypothesis that ω-3 fatty acids help prevent arginine deficiency after trauma by modulating arginase I expression.

MATERIALS AND METHODS

Reagents

PGE1 and PGE2 were purchased from Sigma-Aldrich (St. Louis, MO). PGE3 was obtained from Cayman Chemical (Ann Arbor, MI). PGE1 and PGE2 were reconstituted with 200-proof ethanol (ETOH). The solvent for PGE3 was changed from methylacetate to 200-proof ETOH by evaporating the methyl acetate with nitrogen gas and reconstituting the solute with 200-proof ETOH. The percent of ETOH in cell culture media never exceeded 0.036%. IL-13 was purchased from R&D Systems (Minneapolis, MN). LPS was obtained from Sigma-Aldrich. N-ω-hydroxy-nor-L-arginine (norNOHA) was purchased from Bachern (Torranee, CA). KT5720 was purchased from EMD Biosciences, Inc (San Diego, CA).

Cell Culture and Treatments

RAW 264.7 cells were obtained and were maintained in complete RPMI1640 (C-RPMI), which contains 1140 mmol/LL-Arg (BioWhitaker Walkersville, MD) and supplemented with 10% fetal calf serum, 25 mmol/L HEPES, 4 mmol/L L-glutamine, and 100 units/mL penicillin/streptomycin. The cells were grown to approximately 80% confluence on polystyrene 6-well plates in C-RPMI unless otherwise specified. All experiments were done in triplicate.

Arginase Assay

Arginase activity was measured as arginine conversion to ornithine using a modification of the Konarska and Tomaszewski17 assay. Cells were harvested using a cell scraper and lysed with lysis buffer containing 0.5% Triton solution with Trypsis-Chymotrypsin Inhibitor (Sigma-Aldrich), Leupeptin (Boehringer Ingelheim GmbH, Germany), Aprotonin (Boehringer), and PMSF (Roche Daignostics Corp, Chicago, IL). After lysis, 25 µL of MnCl^sub 2^ was added to 25 µL of sample. Incubation at 55°C for 20 minutes was followed by 150 μL of carbonate buffer to each sample. Using a timer, L-arginine was then added and incubated at 37°C for exactly 10 minutes. The reaction was stopped with 750 mL of glacial acetic acid, 250 mL of ninhydrin solution was added, and the samples were boiled for 1 hour. Ornithine production was measured by spectrophotometry (Molecular Devices, Sunnyvale, CA) using a wavelength of 515 nm. Protein concentration was measured using the procedure described by Bradford18 with reagents from Bio-Rad Laboratories, Hercules, CA. Results of all experiments are the mean values ± SD of triplicate cultures.

Nitrite Production

To examine the activity of iNOS, the amount of NO was estimated as nitrite accumulation in the conditioned medium by means of the Griess assay. Culture supernatant (100 µL) was mixed with 100 µL of 1% sulfanilamide, 0.1% naphtylenediamine dihydrochloride and 2.5% H^sub 3^PO^sub 4^. Absorbance was measured at 550 nm in an ELISA reader. Nitrite concentration was quantitated by reference to NaNO^sub 2^ standards. Results of all experiments are the mean values ± SD of triplicate cultures.

Arginase I Western Blot

Lysates used for the Western blot were the same as those used for the arginase assay. The samples were boiled in sodium dodecylsulfate (SDS) buffer for 7 minutes before they were loaded in s 20% SDS stacking gel. They were subsequently separated in a 12.5% SDS polyacrylamide resolving gel for 1 hour at 200 mV using Invitrogen/Novex Tris-glycine SDS running buffer in a XcellII MiniCell electrophoresis system (Carlsbad, CA). After separation, protein was transferred to a nitrocellulose membrane (Invitrogen) with Tris-glycine SDS transfer buffer for 1.5 hours at 250 mAmp. The membranes were blocked in 5% milk for 1 hour before arginase I primary antibody (mouse IgG1) was added at a dilution of 1:50,000 and the membranes incubated overnight at 4°C. After washing with Tween-TBS solution 5 times, secondary antibody (polyclonal antimouse IgG coupled to horseradish peroxidase) was added at a dilution of 1:5000 (BD PharMingen, San Diego, CA) for 1 hour at room temperature. After two 10-minute washes and one 2-hour wash with Tween-TBS, the membranes were developed with the SuperSignal (Pierce, Rockford, IL) chemiluminescence kit.

Cyclic AMP Assay

A commercial immunoassay was used to measure cAMP accumulation in RAW 264.7 (R & D Systems). Briefly, RAW 264.7 cells were pretreated with 0.5 mmol/L 3-isobutyl-1-methylxanthine (Sigma-Aldrich) to inhibit cAMP destruction by phosphodiesterases. RAW 264.7 cells were then placed in culture for 15 minutes in the presence of 10 µmol/L PGE1, PGE2, or PGE3. After that, cells were harvested, lysed with 0.1 M HCl and cAMP measured after the instructions of the immunoassay kit.

Statistical Analysis

Results are represented as means ± SD. Data analysis was performed by one-way ANOVA. Differences were considered statistically significant when the value was

RESULTS

PGE1, 2, and 3 Differentially Up-Regulate Arginase Activity and Synergize With IL-13 to Up-Regulate Arginase I Expression

Induction of arginase activity with Prostaglandin E2 has been previously been reported by others. The effect of other prostaglandins (ie, PGE1 and PGE3) has not been studied. To determine if all prostaglandins induced arginase activity, RAW 264.7 cells were cultured in the presence of increasing concentrations of PGE1, PGE2 or PGE 3 (0-10 µmol/L) for 24 hours. Cells were then harvested, and arginase activity was measured. Induction of arginase activity was observed with all 3 prostaglandins at all concentrations. However, there was a significant difference between the activity induced by the different prostaglandins, with PGEl inducing the greatest activity (44.7 ± 6.58), whereas PGE3 induced arginase activity poorly (15.71 ± 1.68; Fig. 1).

To determine whether prostaglandins synergized with T helper 2 cytokines for the induction of arginase I expression and activity, we added IL-13 (10 ng/mL) to RAW cells cultured in the presence of the different prostaglandins. Arginase activity was induced by IL-13 alone (31.36 ± 6.9 nmol/min/mg; p

Prostaglandins induce the accumulation of cAMP in myeloid cells. In turn, arginase 1 expression is induced by cAMP. We therefore measured the effect of PGE1, PGE2, and PGE3 on cAMP accumulation in RAW 264.7 cells. A greater accumulation of cAMP was observed when RAW 264.7 cells were cultured in the presence of PGE1 or PGE2 when compared with PGE3 (Fig. 2c). These data correlate with the higher induction of arginase I protein expression observed with the different prostaglandins.

PGE1 Promotes the Arginase Pathway; PGE3 and PGE2 Preferentially Promote the iNOS Pathway

To examine the iNOS and arginase pathways, RAW 264.7 cells were incubated with either PGE1, PGE2, or PGE3 for 24 hours. The cells were then incubated with IL-13 (10 ng/mL) and LPS (2 µ/mL) to induce the arginase and iNOS pathways respectively. After 24 hours, the medium was collected for measurement of nitrite production, and the cells were harvested for measurement of arginase activity.

Figure 3 shows the results for nitrite production. The positive control, LPS, produced nitrite at a concentration of 3.38 (± 0.7) µM. Nitrite produced by cells treated with PGE1, PGE2, or PGE3 in the presence of LPS was 3.89 (± 0.19), 2.75 (± 0.49), and 1.54 (± 0.19), respectively. PGE1 and PGE2 with LPS shows a significant decrease in nitrite production when compared with cells cultured in PGE3 and LPS or LPS alone (p

Several reports have suggested that arginase I successfully competes with iNOS for available arginine. Therefore, we tested the hypothesis by blocking arginase I activity, expecting restoration of nitric oxide production found to be decreased in cells treated with either PGE1 or PGE2 (Fig. 3). RAW 264.7 cells were cultured for 24 hours in the presence of PGE1, PGE2, or PGE3. norNOHA (100 µmol/L) was then added with LPS (2 µg/mL). After 24 hours, cell culture supernatant was collected for measurement of nitrite use. The addition of norNOHA to RAW 264.7 cells increased nitrite production significantly, independent of the type of prostaglandin added (Fig. 4). The difference in nitrite accumulation observed with the different prostaglandins was abrogated with the use of norNOHA. Therefore, these data demonstrate that there is significant interaction between arginase I and nitric oxide synthesis. Inhibition of arginase I results in an increase in arginine availability for the production of nitric oxide.

DISCUSSION

Arginine and co-3 fatty acids are common ingredients in IEDs, but the mechanism of how these ingredients "enhance" the immune system is unknown. Also unknown is the interaction between arginine and ω-3 fatty acids. This study presents a "proof of concept" and is intended to raise the hypothesis that there are significant potential interactions between the 2 different dietary components.

Supplemental dietary fatty acids have multiple possible effects on the immune system, among them, the basic substrate for the production of prostaglandins. There have been numerous studies showing the benefits of dietary ω-3 fatty acids over ω-6 fatty acids in various physiologic conditions, including coronary artery disease; chronic inflammatory conditions such as cancer; and autoimmune disorders, specifically lupus erythematosus, rheumatoid arthritis, and other inflammatory-mediated states.19-24 Most of these studies demonstrate that ω-3 fatty acid diets shift the balance in the type of prostaglandin produced. Diets rich in fish oils are associated with a greater production in PGE3 and a decrease in PGE1 and PGE2.25 Prostaglandins are a product of the metabolism of different fatty acids by COXs. Two COX isoenzymes are described: COX1 and 2. COX2 is grossly considered an inducible enzyme, and its expression is up-regulated in myeloid cells upon immune activation. COX metabolizes arachidonic acid into PGE2, whereas eicosapentaenoic and docosahexaenoic acids (from fish oil) are the substrate for the production of PGE3.26,27

Recently, it has been shown that arginase I, expressed in myeloid cells in the immune system, exerts a regulatory effect on T-cell function because it causes a depletion in arginine.11,28 The idea that the immune system is able to deplete specific nutrients, and, through this mechanism, control its own response has been recently supported in the literature as it is observed in a growing number diseases such as in cancer and in chronic infections.29-32

Arginine is crucial for normal T-cell proliferation and function. A decrease in arginine, whether caused in vitro by the lack of arginine in culture media or the addition of the enzyme arginase or in vivo by trauma or transplantation causes significant alterations in T-cell structure, including a decrease in CDS receptors and a decrease in the ζ-chain subunit of the T-cell receptor.33,34

Other authors have previously suggested that arginase I expression in myeloid cells is induced by prostaglandin E2.16 Arginase I is induced in trauma and surgery and appears to significantly contribute to arginine depletion in this patient population.10,35 Through arginine depletion, arginase I appears to play a major role in T-cell dysfunction after trauma or surgery and in preventing the production of nitric oxide in response to infection.35 Immune dysfunction after trauma leads to increased susceptibility to infection and increased morbidity and mortality.

Our results indicate that PGE1- and PGE2-series prostaglandins significantly induce arginase activity, whereas PGE3 has only a modest effect. Not surprisingly, RAW 264.7 cells grown in the presence of PGE3 are capable of generating nitric oxide in response to endotoxin, probably reflecting better arginine availability. In contrast, nitric oxide production is blunted by induction of arginase by both PGE1 and PGE2. To prove the inhibitory effect of arginase on nitric oxide production, we have used the arginase inhibitor norNOHA. Indeed, the addition of norNOHA increases nitric oxide proportionately so that nitric oxide production is similar in the presence of any of the prostaglandins.

IEDs restore T-cell function after surgery and trauma and result in a significant decrease in infections, but the mechanism of how IEDs work remains elusive. The discovery that arginase I expression is increased by trauma and that arginine depletion occurs in this patient population suggests that arginine supplementations at supraphysiologic levels restore arginine to levels necessary for normal immune function. Furthermore, this paper raises the hypothesis that by shifting the balance in the production of PGE2, ω-3 fatty acids may blunt the up-regulation of arginase and, consequently, arginine deficiency.

This paper lays a foundation to begin both animal and human trials to study whether supplementation with fish oil will decrease the up-regulation of arginase I expression observed with trauma and help restore arginine levels. We look forward to seeing results from these types of studies.

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Vishai Bansal, MD; Kimberly M. Syres, MD; Valeryia Makarenkova, MD, PhD; Ryan Brannon, BS; Benjamin Matta, BS; Brian G. Harbrecht, MD; and Juan B. Ochoa, MD

From the Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Received for publication August 2, 2004.

Accepted for publication August 30, 2004.

Correspondence: Juan B. Ochoa, MD, FACS, F1265 PUH-UPMC, 200 Lothrop St., Pittsburgh, PA 15213. Electronic mail may be sent to ochoajb@upmc.edu.

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