J. Anim. Sci. 2003. 81:2552-2561
© 2003 American Society of Animal Science
Effect of boron supplementation of pig diets on the production of tumor necrosis factor-
and interferon-
1,2
T. A. Armstrong3 and
J. W. Spears4
Department of Animal Science and Interdepartmental Nutrition Program, North Carolina State University, Raleigh 27695-7621
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Abstract
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Two experiments were conducted to determine the effects of dietary B on the production of cytokines following an endotoxin challenge. In both experiments, pigs were obtained from litters generated from sows fed low-B (control) or B-supplemented (5 mg/kg, as-fed basis) diets. In Exp. 1 and 2, 28 and 35 pigs, respectively (21 d old), remained with their littermates throughout a 49-d nursery phase and were fed either a control or B-supplemented diet. In Exp. 1, 12 pigs per treatment were moved to individual pens at the completion of the nursery phase and fed their respective experimental diet. On d 99 of the study, pigs were injected with 150 µg of phytohemagglutinin (PHA) to evaluate a local inflammatory response. Pigs receiving the B-supplemented diet had a decreased (P < 0.01) inflammatory response following PHA injection. Peripheral blood monocytes were isolated from six pigs per treatment on d 103 and cultured in the presence of lipopolysaccharide (LPS) to determine the effect of dietary B on tumor necrosis factor-
(TNF-) production from monocytes. Isolated monocytes from pigs that received the B-supplemented diet had a numerically greater (P = 0.23) production of TNF-. In Exp. 2, pigs were group housed with their littermates following the nursery phase for 43 d, after which 10 pigs per treatment were moved to individual pens. In Exp. 1 and 2, pigs were assigned randomly within dietary treatment to receive either an i.m. injection of saline or LPS on d 117 and d 109, respectively. The dose of LPS in Exp. 1 and 2 was 100 and 25 µg of LPS/kg of BW, respectively. In Exp. 1, serum TNF- was increased (P < 0.01) at 2 h and tended to be increased (P < 0.11) at 6 and 24 h after injection by dietary B; however, only numerical trends existed for a B-induced increase in TNF- in Exp. 2. Serum interferon-gamma (IFN-
) was increased (P < 0.01) at 6 h and tended to be increased (P < 0.08) at 24 h after injection in Exp. 1. In Exp. 2, dietary B also numerically increased IFN-. These data indicate that dietary B supplementation increased the production of cytokines following a stress, which indicates a role of B in the immune system; however, these data do not explain the reduction in localized inflammation following an antigen challenge in pigs.
Key Words: Boron • Cytokines • Inflammation • Pigs
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Introduction
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Boron has been implicated to function in a variety of metabolic and physiological systems in man and animals (Nielsen, 1997
). Recently, our laboratory reported that pigs that consumed B-supplemented diets had a decreased inflammatory response to an intradermal injection of phytohemmaglutinin (PHA; Armstrong et al., 2001a). The mechanism behind the ability of B to reduce inflammation is unclear. Inflammation is characterized by an increase in the production of the proinflammatory cytokines, interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor- (TNF-) from monocytes/macrophages (Akira et al., 1990; Murtaugh, 1994; Johnson, 1997).
Cytokines have a significant role in an animal’s response to and recovery from an immune challenge (Klasing and Johnstone, 1991; Johnson, 1997). An animal’s response to disease stress situations has been studied using injections of lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria (Culbertson and Osburn, 1980; Henderson et al., 1998). Treatment of pigs with LPS can produce an array of metabolic effects, including increased production of TNF- (Webel et al., 1997; Leininger et al., 2000a; Wright et al., 2000), interferon- a (INF-; Zannelli et al., 2000), and IL-6 (Webel et al., 1997; 1998).
We hypothesized that the decrease in inflammation observed in pigs that consumed B-supplemented diets (Armstrong et al., 2001a) might be due to a decrease in cytokine production. Therefore, we designed two experiments to study the effects of dietary B on cytokine production in pigs following an i.m. injection of LPS.
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Materials and Methods
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These experiments utilized offspring from the mating of gilts receiving a low-B basal diet (control) or the control diet supplemented with 5 mg of B/kg, as previously described (Armstrong et al., 2002). All experimental procedures, care, and handling of animals were approved by the North Carolina State University Institutional Animal Care and Use Committee.
Experiment 1
Two litters of pigs from each experimental treatment, for a total of 28 pigs (control, n = 16, average initial weight = 4.8 kg; control + 5 mg B/kg diet, n = 12, average initial weight = 6.1 kg), were weaned at approximately 21 d of age, transferred to a nursery facility, and remained penned with their littermates during the nursery phase. Pigs remained on their preassigned experimental treatments (control or control + 5 mg B/kg diet), and B was supplemented as sodium borate decahydrate (Na2B4O7•10H2O; Sigma Chemical Co., St. Louis, MO). The nursery phase was 49 d, and all animals had ad libitum access to feed and water. The nursery basal diet was formulated to contain low B concentrations by using protein sources of animal origin (Hunt, 1997; Table 1). Nursery diets were offered in meal form, and were formulated to meet or exceed the requirements for all nutrients (NRC, 1998). At the end of the 49-d nursery period, the average weight of the pigs in control and control + 5 mg of B/kg diet treatments were 22.5 and 29.2 kg, respectively.
Following the nursery phase, 12 pigs per treatment were moved to individual pens and maintained on their respective dietary treatment. Ingredient composition of the basal diet fed during the growing phase is shown in Table 2. The basal diet was formulated to meet or exceed the requirements for all nutrients (NRC, 1998). Diets were fed for 75 d, and pigs had ad libitum access to feed and water.
On d 50 following the nursery phase (d 99 of the study), all 12 pigs per treatment were used for the assessment of the inflammatory response using a PHA skin test (Kornegay et al., 1989; Fritz et al., 1990). Pigs were injected intradermally with 0.1 mL of PHA (150 µg/0.1 mL) in the flank region on both the right and left sides. Inflammatory response was measured as a change in skin thickness before and at 6, 12, 24, and 48 h after injection using skinfold calipers. The average of the measurements from the two sites at each time period was used for statistical analyses.
On d 54 postnursery (d 103 of the study), animals within a B treatment were randomly assigned to receive either an i.m. injection of saline or 100 µg of LPS (Escherichia coli 055:B5, Sigma Chemical Co., St. Louis, MO)/kg of BW to create a 2 x 2 factorial arrangement of treatments. Factors were supplemental dietary B (0 or 5 mg of B/kg of diet) and endotoxin challenge (saline or LPS). Pigs that were randomly assigned to receive the saline injection were used for in vitro assessment of the effect of dietary B on cytokine production from isolated peripheral blood monocytes. Approximately 40 mL of blood was collected from the jugular vein of each pig for the isolation of peripheral blood monocytes via buoyant density centrifugation on Ficoll-Hypaque gradients (Histopaque 1077, Sigma Chemical Co.). The isolation procedure yielded a population of cells that was 93.1% monocytes as determined by methylene blue-stained cytospin smears. Monocytes were cultured in triplicate within an animal and at a concentration of 6.0 x 106 monocytes/mL. Monocytes were stimulated with 10 µg of LPS/mL (E. coli 055:B5) to produce cytokines according to Genglebach and Spears (1998). The viability of cultured monocytes was 91.3% as assessed by trypan blue exclusion. After an 18-h incubation, culture supernatants were removed and frozen at -70°C until analysis of TNF- and IFN-.
Beginning at d 68 postnursery (d 117 of the study), all pigs were used to assess the effect of dietary B on cytokine production in vivo. On d 68, pigs weighed 80.3 and 96.9 kg in control and control + 5 mg of B/kg diet treatments, respectively. Pigs received i.m. injections of saline or LPS in the ham according to their preassigned treatment. Before injection, all pigs were weighed and feed intake was determined to assess the effects and interactions of B and LPS on animal weight change and feed consumption. Immediately before injection and at 2, 6, and 24 h after injection, venous blood samples were obtained from each pig and rectal temperatures were determined. Pigs were weighed, feed consumption was calculated, and venous blood samples were obtained at 72 and 168 h after injection. Serum was obtained by centrifugation (1,670 x g) for 30 min at 4°C, separated into approximately 1-mL aliquots, and frozen at -70°C until analysis. Serum was analyzed for concentrations of TNF- and IFN- at 0, 2, 6, and 24 h, and for IGF-1 and cortisol at 0, 2, 6, 24, 72, and 168 h, as described below.
Experiment 2
Two litters of pigs from each experimental treatment, for a total of 35 pigs (control, n = 17, average initial weight = 5.6 kg; control + 5 mg of B/kg diet, n = 18, average initial weight = 4.6 kg), were weaned at approximately 21 d of age, moved to a nursery facility, and remained penned with their littermates during the nursery phase. The nursery phase was 49 d, and all experimental procedures were identical to that described for Exp. 1. At the end of the 49-d nursery period, the average weight of the pigs in control and control + 5 mg of B/kg diet treatments were 26.8 and 23.8 kg, respectively. At the completion of the nursery phase, pigs were transferred to a finishing barn and housed with littermates in pens (four or five pigs per pen) for 43 d. Pigs were switched to a growing diet (Table 2
) but were maintained on their respective dietary B treatment. At the end of this 43-d period, the average weight of pigs in the control and control + 5 mg of B/kg diet treatments were 63.7 and 58.5 kg, respectively. Litters were weaned and moved to nursery and finishing barns at different times, due to a range in farrowing dates of sows (Armstrong et al., 2002). Therefore, animal performance data during the nursery phase and the 43-d, group housed growing period were not calculated, because of low degrees of freedom.
Following this 43-d period, 10 pigs per treatment were moved to individual pens and then randomly assigned to receive either an i.m. injection of saline or 25 µg of LPS (E. coli 055:B5)/kg of BW in a 2 x 2 factorial arrangement of treatments as described for Exp. 1. The second experiment was conducted to investigate the effects of a lower dose of LPS on physiological changes and serum cytokine concentrations in pigs that received either low-B or B-supplemented diets. The 10 pigs per treatment were selected so that initial BW would be similar, in order to remove any confounding effects of differences in BW between treatments. The LPS or saline injection occurred on d 60 following the nursery phase (d 109 of the study), when the average weight of the animals was 72.0 and 73.2 kg for control and control + 5 mg of B/kg diet treatments, respectively. The experimental protocol following either the saline or LPS injection was identical to that described in Exp. 1.
Analytical Procedures
Serum and culture supernatant concentrations of TNF- and IFN- were determined using commercially available porcine specific ELISA kits (Endogen, Inc., Woburn, MA). Serum concentrations of IGF-1 were determined by a double-antibody 125I RIA following extraction with glycylglycine hydrochloride as described by Houseknecht et al. (1988), with modifications (Jones et al., 1991; Stanko et al., 1994). The standard source was recombinant human IGF-1 (Monsanto, St. Louis, MO). The primary antibody was rabbit antiserum to human IGF-1 (UB2-495), which was obtained from the National Hormone and Pituitary Program and the National Institute of Diabetes and Digestive and Kidney Diseases. The secondary antibody was goat anti-rabbit serum obtained from Linco Research, Inc. (catalog No. 5060-20, St. Charles, MO). Average interassay and intraassay CV were 8.4 and 9.3%, respectively. Assay sensitivity, defined as 90% bound, was 42.0 ng/mL. Serum cortisol concentrations were determined by a 125I RIA (Coat-A-Count, Diagnostic Products Corp., Los Angeles, CA). Average interassay and intraassay CV were 8.8 and 11.3%, respectively. Assay sensitivity, defined as 90% bound, was 0.26 µg/dL.
The B concentrations of the basal diet fed during the nursery phase and the growing phase were determined via inductively coupled argon plasma atomic emission spectrophotometry (Varian Liberty II, Varian, Inc., Sugarland, TX). The reference and calibrations standards were previously described (Armstrong et al., 2000).
Statistical Analyses
Data were analyzed as a completely randomized design using the GLM procedure of SAS (SAS Inst., Inc., Cary, NC). Pig performance data in the nursery and growing periods in Exp. 1, before endotoxin challenge, were analyzed with a statistical model that contained dietary treatment. Data for the inflammatory response following an intradermal PHA injection were analyzed by ANOVA for repeated measures (Gill and Hafs, 1971). The statistical model consisted of dietary treatment, time, and dietary treatment x time interaction. Animal within treatment mean square was the error term used to test for treatment effects. Cytokine concentrations within the culture supernatant of isolated peripheral blood monocytes were analyzed by ANOVA using animal as the experimental unit. Data collected following endotoxin challenge were analyzed as a factorial using repeated measures. The statistical model consisted of dietary treatment, endotoxin challenge, time, and all possible interactions. Animal was the experimental unit for in vivo data.
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Results
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The basal diets fed during the nursery phase and the growing phase contained 2.2 mg of B/kg diet for both Exp.1 and 2.
Experiment 1
At the completion of the 49-d nursery phase, pigs that received the control diet supplemented with 5 mg of B/kg had an increased (P < 0.01) ADG compared with pigs that consumed the control diet (0.47 vs. 0.36 kg/d, SEM = 0.02). This increase in ADG was maintained (P < 0.01) throughout the growing phase (1.00 vs. 0.85 kg/d, SEM = 0.03). This increase in ADG during the growing phase may be partially related to an increase (P < 0.05) in ADFI in pigs that consumed the control diet supplemented with 5 mg of B/kg compared with pigs that received the control diet (2.48 vs. 2.16 kg/d, SEM = 0.10). Consequently, feed efficiency (gain:feed) was not affected by dietary B supplementation.
The inflammatory response following an intradermal injection of PHA was decreased (P < 0.01) in pigs that received the B supplemented diet (Figure 1). The treatment x time interaction was significant (P < 0.01). The decreased inflammatory response in pigs that received B-supplemented diets was evident beginning at 6 h after injection and was maintained through 48 h after injection of PHA.
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Figure 1. Effect of dietary B supplementation on the inflammatory response in 12 individually housed pigs per dietary treatment following an intradermal injection of 150 µg of phytohemagglutinin. Skinfold difference is the change in tissue swelling from the baseline following the injection of phytohemagglutinin. Means for each time point for both dietary treatments are composed of 12 observations. Treatment x time interaction was significant (P < 0.05). Treatment means within a specific time point after injection are different (*P < 0.01).
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The administration of LPS resulted in a loss of BW (P < 0.01) in pigs from d 0 to 3 after injection (Figure 2A). There was no effect of dietary B on the change in weight from d 0 to 3 after injection. From d 4 to 7 after injection, LPS did not affect the change in weight; however, a B x LPS interaction (P < 0.05) was present. Pigs that consumed the B-supplemented diet and received LPS did not recover from the weight loss that occurred from d 0 to 3, whereas control pigs that received LPS did recover during d 4 to 7.
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Figure 2. Effect of dietary B supplementation and lipopolysaccharide (LPS) injection (100 µg/kg of BW) on pig performance of 12 individually housed pigs per dietary treatment in Exp. 1. Means for each time point are composed of six observations for both change in weight and feed consumption. A B x LPS x time interaction (P < 0.01) was present for both change in weight and feed consumption. Graph A represents the change in weight, and pooled SEM was 1.27. From d 0 to 3, a LPS effect was present (P < 0.01). From d 4 to 7, a B x LPS interaction was present (P < 0.05). Graph B represents the feed consumption, and pooled SEM was 1.58. From d 0 to 3, a LPS effect was present (P < 0.01).
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Administration of LPS decreased (P < 0.01) feed consumption in pigs regardless of dietary B status from d 0 to 3 after injection (Figure 2B). There was no effect on feed consumption from d 0 to 3 with regard to dietary B supplementation. Between d 4 and 7 after injection, LPS tended to decrease (P < 0.09) feed consumption, but dietary B supplementation did not affect feed consumption.
Core body temperature, measured as rectal temperature, increased (P < 0.01) in LPS-challenged pigs (Figure 3). A B x LPS x time interaction (P < 0.01) was present. Between 2 and 6 h after injection, pigs that consumed the control diet and received LPS had decreasing rectal temperatures, whereas pigs that consumed the B supplemented diet and received LPS had increasing rectal temperatures. At 6 h after injection, pigs that consumed the control diet and received LPS had lower (P < 0.01) body temperatures than pigs that received B-supplemented diets and LPS.
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Figure 3. Effect of dietary B supplementation and lipopolysaccharide (LPS) injection (100 µg/kg of BW) on core body temperature change of 12 individually housed pigs per dietary treatment in Exp. 1. Means for each time point are composed of six observations. Pooled SEM was 0.21. A B x LPS x time interaction (P < 0.01), and a LPS effect (P < 0.01) were present. *P < 0.08 at time 2 h and **P < 0.01 at time 6 h after injection represent B x LPS interactions.
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Serum IGF-1 concentrations were decreased by LPS at 24 (P < 0.08) and 72 h (P < 0.02) after injection (Table 3). Dietary B did not affect serum concentrations of IGF-1. Serum cortisol concentrations were increased (P < 0.01) by LPS at 2, 6, and 24 h after injection (Table 4). No B x LPS interactions were noted, and dietary B did not alter serum concentrations of cortisol.
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Table 3. Effect of dietary B and lipopolysaccharide (LPS) injection on serum concentrations of IGF-1 in pigs following the injection of LPS in Exp. 1 (100 µg of LPS/kg of BW) and Exp. 2 (25 µg of LPS/kg of BW)
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Table 4. Effect of dietary B and lipopolysaccharide (LPS) injection on serum concentrations of cortisol in pigs following the injection of LPS in Exp. 1 (100 µg of LPS/kg of BW) and Exp. 2 (25 µg of LPS/kg of BW)
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Before LPS or saline injection, pigs that consumed diets supplemented with B had slightly higher (P < 0.07) serum concentrations of TNF- (Table 5). Dietary B supplementation increased (P < 0.01) TNF- concentrations at 2 h after injection, and tended to increase TNF- concentrations at 6 (P < 0.10) and 24 h (P < 0.11) after injection. Injection of LPS increased (P < 0.01) serum TNF- at 2 h after injection, and a B x LPS interaction (P < 0.01) was present at 2 h after injection. Dietary B supplementation resulted in a much larger increase in TNF- in LPS-injected pigs than in saline-injected pigs.
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Table 5. Effect of dietary B and lipopolysaccharide (LPS) injection on serum concentrations of tumor necrosis factor- (TNF-) in pigs following the injection of LPS in Exp. 1 (100 µg of LPS/kg of BW) and Exp. 2 (25 µg of LPS/kg of BW)
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Serum concentrations of IFN- were not affected by treatment at 0 h; however, LPS increased (P < 0.01) serum IFN- at 2, 6, and 24 h after injection (Table 6). A B x LPS interaction (P < 0.05) was observed at 6 and 24 h after injection. Dietary B supplementation increased serum IFN- concentrations at 6 h (P < 0.01) and tended to increase (P < 0.08) serum IFN- concentrations at 24 h after injection in LPS, but not saline-injected, pigs.
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Table 6. Effect of dietary B and lipopolysaccharide (LPS) injection on serum concentrations of interferon-gamma (IFN-) in pigs following the injection of LPS in Exp. 1 (100 µg of LPS/kg of BW) and Exp. 2 (25 µg of LPS/kg of BW)
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Release of TNF- from isolated peripheral blood monocytes in response to in vitro LPS stimulation was numerically higher (P = 0.23) in monocytes isolated from pigs that received B-supplemented diets (control = 52.4 pg/mL, control + 5 mg of B/kg diet = 90.3 pg/mL; SEM = 20.0). The release of IFN- from isolated monocytes into the culture supernatant was undetectable or below the sensitivity of the ELISA (2.0 pg/mL).
Experiment 2
Administration of LPS resulted in a decrease (P < 0.02) in BW gain, regardless of dietary B from d 0 to 3 after injection (Figure 4A). Dietary B or LPS did not affect the change in weight between d 4 and 7 after injection. Injection of LPS decreased (P < 0.01) feed consumption from d 0 to 3 after injection (Figure 4B). Dietary B did not affect feed consumption from d 0 to 3 after injection. Between d 4 and 7 after injection, neither dietary B nor LPS injection affected feed consumption.
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Figure 4. Effect of dietary B supplementation and lipopolysaccharide (LPS) injection (25 µg/kg of BW) on pig performance of 10 individually housed pigs per dietary treatment in Exp. 2. Means for each time point are composed of five observations for both change in weight and feed consumption. A B x LPS x time interaction (P < 0.01) was present for both change in weight and feed consumption. Graph A represents the change in weight, and pooled SEM was 0.56. From d 0 to 3, a LPS effect was present (P < 0.02). Graph B represents the feed consumption, and pooled SEM was 0.88. From d 0 to 3, a LPS effect was present (P < 0.01).
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Core body temperature increased (P < 0.01) following LPS administration (Figure 5). Dietary B did not affect rectal temperature. After LPS injection, rectal temperatures peaked at 2 h regardless of dietary B concentration.
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Figure 5. Effect of dietary B supplementation and lipopolysaccharide (LPS) injection (25 µg/kg of BW) on core body temperature change of 10 individually housed pigs per dietary treatment in Exp. 2. Means for each time point are composed of five observations. Pooled SEM was 0.21. A B x LPS x time interaction (P < 0.01) and a LPS effect (P < 0.01) were present.
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Serum IGF-1 concentrations were reduced (P < 0.02) in pigs that received diets supplemented with B at 0, 2, 6 and 72 h and tended to be reduced (P < 0.06) at 24 and 168 h after challenge (Table 3). Administration of LPS resulted in decreased (P < 0.02) serum IGF-1 concentrations at 24 and 72 h after injection. Serum cortisol concentrations tended to be higher (P < 0.08) in pigs fed the B-supplemented diet immediately before the challenge regimen (Table 4); however, this tendency was not evident at later time points measured. Before injection of LPS, a tendency was also present for pigs randomly assigned to receive LPS to have lower (P < 0.09) cortisol concentrations. At 2 and 6 h after challenge, LPS administration increased (P < 0.01) serum cortisol concentrations.
Serum TNF- concentrations were increased (P < 0.01) at 2 h after challenge in pigs that received LPS (Table 5). No statistical effect of dietary B supplementation was present due to a high standard error; however, there were numerical tendencies for pigs that received diets supplemented with B to have higher serum TNF- concentrations. Serum IFN- concentrations were increased (P < 0.01) by LPS at 2, 6, and 24 h after injection (Table 6). Pigs that received diets supplemented with B had higher (P < 0.01) IFN- concentrations in the serum immediately before challenge than unsupplemented pigs. At 2, 6, and 24 h after challenge, no statistical differences were attributed to dietary B, but as with TNF-, there were numerical tendencies for IFN- to be higher in pigs that consumed the B-supplemented diet.
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Discussion
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Previous research in our laboratory indicated that supplemental dietary B decreased the inflammatory response to an intradermal injection of PHA in pigs (Armstrong et al., 2001a). Data from the current study confirm this finding. In addition, Hunt and Idso (1999) reported that paw swelling was reduced in adjuvant-induced arthritic rats that received supplemental B. We hypothesized that B may decrease the inflammatory response by decreasing the production of proinflammatory cytokines by the monocyte/macrophage lineage. Cytokines are known to mediate and function in the inflammatory response (Dinarello, 1988; Akira et al., 1990; Murtaugh, 1994). Our hypothesis of B decreasing cytokine production was proven incorrect. In the current study, dietary B supplementation increased serum concentrations of TNF- and IFN- after LPS challenge in Exp. 1. Also, monocytes isolated from animals that received B-supplemented diets tended to have higher TNF- concentrations in the culture supernatant following in vitro stimulation with LPS. The monocyte culture was not analyzed for B concentration in the current study; however, previous data indicate that dietary B supplementation increased serum B concentrations (Armstrong et al., 2000) and tissue B concentrations (Armstrong et al., 2002) in pigs. Therefore, an increase in circulating B concentrations in the serum may expose blood monocytes to an increased concentration of B. In Exp. 2, when a lower dose of LPS was administered, no statistical differences were noted with respect to dietary B and serum TNF- and IFN- concentrations; however, numerical tendencies existed for animals that received B-supplemented diets to have increased serum concentrations of these cytokines. Boron increased TNF- release by cultured human fibroblasts and chick embryo cartilage (Benderdour et al., 1997Benderdour et al., 1998). In addition, B increased total messenger RNA for TNF- in cultured human fibroblasts (Benderdour et al., 1998). Collectively these results indicate that B may act to increase cytokine release and/or synthesis. However, it was not possible, from the current study, to separate possible prenatal effects of dietary B supplementation to the dams (Armstrong et al., 2002) from the possible effects of dietary B supplementation to the offspring.
Cytokines are necessary for the priming of the immune response and are directly or indirectly responsible for increases in hepatic acute phase protein synthesis, anorexia, fever, and lethargy (Klasing and Johnstone, 1991; Johnson, 1997) following a disease challenge. Nonetheless, it is unclear if excessive cytokine production during stress or disease situations results in improved immune system efficiency or has a detrimental effect on the immune system. From these current data, it appears that models of localized tissue inflammation (e.g., PHA injection) are not equivalent to a whole-body inflammatory disease model, such as LPS.
Data that indicate a decreased localized inflammatory response following B supplementation (Bai and Hunt, 1995; Hunt and Idso, 1999; Armstrong et al., 2001a) are not explained by decreased cytokine production by B in the current and previous studies (Benderdour et al., 1997Benderdour et al., 1998). Therefore, a mechanism other than decreased cytokine production by B must explain decreased local tissue swelling following an intradermal injection of PHA. Hunt and Idso (1999) suggested that the reduced inflammation in rats that received B-supplemented diets may be explained by B-induced down-regulation of certain enzymes involved in the respiratory burst cascade. This would result in a decrease in the production of hydrogen peroxide. Activities of copper- and zinc-dependent superoxide dismutase and selenium-dependent glutathione peroxidase have been increased by B supplementation (Griffith et al., 1978; Nielsen, 1994; 1997). In addition, differential display PCR has indicated that B can increase the messenger RNA for superoxide dismutase (Luo and Eckhert, 2000). These increases in enzymatic activity could lead to a reduction in the amount of hydrogen peroxide generated in the respiratory burst cascade. Hydrogen peroxide suppressed the cytotoxic and proliferative activities of human natural killer cells in vitro (Hellstrand et al., 1994), which would decrease the production of TNF- and IFN- from natural killer cells (Jewett et al., 1996). These data form the hypothesis that may explain the increases in TNF- and IFN- in the current study observed with dietary B supplementation. Boron may act through enzymes in the respiratory burst cascade to decrease the production of hydrogen peroxide, thereby leading to an increase in the production of TNF- and IFN-. However, the exact function of B within these enzyme systems is unclear.
The increase in ADG and ADFI in pigs that received B-supplemented diets in Exp. 1 is in agreement with previous work in weaned pigs (Armstrong et al., 2001b), growing-finishing pigs (Armstrong et al., 2001a,b), and suckling piglets (Armstrong et al., 2002). Due to the effect of B to increase growth rate, it is not possible to separate any direct effect of B on cytokine release and/or synthesis from the effect of B on growth rate.
Lipopolysaccharide is a component of the cell wall of Gram-negative bacteria that induces an immune response, which is believed to be a result of lipid A, the biologically active component of LPS (Henderson et al., 1998). Administration of LPS results in a multitude of metabolic effects; however, of interest to this study was the effect of LPS on cytokine production. Injection of LPS in the current study did result in acute increases in the serum concentrations of TNF- and IFN-, which is consistent with previous studies (Webel et al., 1997; Leininger et al., 2000b; Zannelli et al., 2000).
The increased core body temperature resulting from LPS administration in Exp. 1 and 2 is consistent with previous studies using LPS in pigs (Johnson and von Borell, 1994; Wright et al., 2000). In Exp. 1, rectal temperature remained elevated until 6 h after injection in pigs that consumed B-supplemented diets and received LPS. This effect may be related to the increase in serum TNF- concentrations by dietary B in this experiment. Proinflammatory cytokines may manifest their actions through arachadonic acid metabolism (Tizard, 1987; Persico, 1991). Cytokines can activate phospholipase A2, which deacylates membrane phospholipids. Phospholipids can be converted to arachadonic acid, which can follow the cyclooxygenase pathway to produce prostaglandins. Prostaglandins may be responsible for increased body temperatures. However, some studies have not demonstrated a relationship between circulating TNF- and prostaglandin concentrations with an increase in body temperature (Arthington et al., 1997; Balaji et al., 2000).
The decrease in serum IGF-1 and increase in serum cortisol concentrations are in agreement with previous studies investigating physiological changes following LPS administration (Richards and Almond, 1994; Hevener et al., 1997; Webel et al., 1997). The LPS-associated depression in weight gain and feed consumption are in agreement with animals under social or disease stress situations, which is mediated through the actions of cytokines (Klasing and Johnstone, 1991; Johnson, 1997; Spurlock, 1997).
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Implications
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These studies indicate that boron can affect the production of cytokines by increasing the production of tumor necrosis factor- and interferon- after a stress or disease challenge. Increases in proinflammatory cytokines are not consistent with a decrease in localized inflammatory response. Other physiological changes must occur for boron to decrease local inflammatory response to an antigen challenge; however, an increased cytokine production by boron implies a role of boron within the immune system. Further research is necessary to elucidate the potential benefits or harmful effects of boron-induced cytokine release.
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Footnotes
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1 Use of trade names in this publication does not imply endorsement by the North Carolina ARS or criticism of similar products not mentioned. 
2 Appreciation is extended to M. E. Tiffany, S. L. Archibeque, C. L. Wright, K. E. Lloyd, M. Corns, T. Steffel, S. Beasley, and M. Brown for technical assistance and animal care.
3 Present address: Elanco Animal Health, A Division of Eli Lilly and Company, 2001 W. Main St., P.O. Box 708, Greenfield, IN 46140.
4 Correspondence: Box 7621 (phone: 919-515-4008; fax: 919-515-4463; E-mail: Jerry_Spears{at}ncsu.edu).
Received for publication February 12, 2003.
Accepted for publication June 13, 2003.
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Abstract
Introduction
