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© 2005 The American Society for Nutritional Sciences J. Nutr. 135:1752-1756, July 2005

Nutritional Immunology

Alive and Dead Lactobacillus rhamnosus GG Decrease Tumor Necrosis Factor-{alpha}–Induced Interleukin-8 Production in Caco-2 Cells

Liyan Zhang1,*, Nan Li, Ricardo Caicedo and Josef Neu2

Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL 32610-0296 and * Fujian Provincial Maternity and Child Health Hospital, Neonatology, Fuzhou, Fujian, China 350001

2To whom correspondence should be addressed. E-mail: neuj{at}peds.ufl.edu.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
Certain probiotic bacteria show anti-inflammatory activity after being heat killed, whereas others do not, suggesting that the gastrointestinal environment may alter the activity of probiotic bacteria. Occasionally, probiotics are provided when a patient is also being treated with oral antibiotics; this may have an effect on the probiotic activity. We hypothesized that Lactobacillus rhamnosus GG (LGG) are capable of downregulating tumor necrosis factor-{alpha} (TNF{alpha})-induced interleukin (IL)-8 production under all 3 of these conditions, and that LGG act through the nuclear factor {kappa}B (NF{kappa}B)/inhibitor of {kappa}B (I{kappa}B) pathway. Caco-2 cells were treated with live or heat-killed LGG in doses ranging from 104 to 1010 cfu/L, in the presence or absence of antibiotics and TNF{alpha} in the media. TNF{alpha}-induced production of IL-8 by Caco-2 cells was modulated by LGG under all 3 conditions. However, higher doses of live LGG without TNF{alpha} in the presence or absence of antibiotics in vitro induced the production of IL-8 (P = 0.001). Heat-killed LGG also blunted the TNF{alpha}-induced IL-8 production (P < 0.01), but by itself did not increase IL-8 production at higher doses as markedly as live LGG (P < 0.05). LGG reduced the TNF{alpha}-induced NF{kappa}B translocation to the nucleus and lessened the decrease in I{kappa}B in the cytoplasm (P < 0.05). LGG reduced TNF{alpha}-induced IL-8 production by affecting the NF{kappa}B/I{kappa}B pathway in Caco-2 cells. High doses of live LGG markedly increased IL-8 production, but heat-killed LGG caused only a slight increase in IL-8. Thus, heat-killed LGG may effectively ameliorate inflammation with a lower potential than live LGG at high doses to cause inflammation.

KEY WORDS: • probiotic bacteria • intestinal epithelial cells • interleukin-8 • nuclear factor {kappa}B/inhibitor of {kappa}B

The microflora of adult humans is found primarily in the colon and distal small intestine and consists of >1013 microorganisms, comprising nearly 500 species and nearly 2 million genes (the "microbiome") (1). For the most part, the relation between the microflora and the intestine is mutually beneficial or "commensal." The commensal bacteria regulate intestinal development and play critical roles in nutrition, while receiving a place of residence from the host. Evidence is emerging for interactions, termed "cross talk," between the intestinal microflora and the diverse population of cells in the intestinal mucosa that result in several physiological and immunologic responses (2–4,5). The intestinal epithelium synthesizes many mediators that are involved in the innate immune response. Whether the response is pro- or anti-inflammatory may depend on the balance of individual microorganisms that colonize the intestinal lumen at any particular time (6). For example, nonpathogenic Salmonella are able to inhibit tumor necrosis factor-{alpha} (TNF{alpha})3-induced synthesis of interleukin (IL)-8, a potent neutrophil attractant chemokine. Similar mechanisms are involved with flagellin-induced stimulation of IL-8 in both the immature and mature intestinal epithelium (7).

Probiotics are living microorganisms with low or no pathogenicity that exert beneficial effects on the health of the host (8). Modification of gut microflora by probiotic therapy has therapeutic potential in clinical conditions associated with gut barrier dysfunction and inflamed mucosa (9). Probiotic bacteria consist mainly of strains of Lactobacillus, Bifidobacterium, and Streptococcus. Lactobacilli are often part of the human intestinal ecosystem, but highly variable amounts are found. Bifidobacteria are also part of the human microflora, but species differ according to age: newborns are readily colonized by Bifidobacterium breve and B. infantis, and colonization is favored in breast-fed compared with bottle-fed infants. Lactobacillus rhamnosus GG (LGG), a bacterium used in the production of yogurt, is one of the best-studied probiotic bacteria in clinical trials. It is effective in preventing and treating diarrhea, primary rotavirus infection (10), and atopic dermatitis (11).

Putative mechanisms of the action of probiotics include regulation of cytokine and chemokine production (12–15). IL-8 is produced through cell receptors that interact with the nuclear factor {kappa}B (NF{kappa}B) pathway under the action of several proinflammatory mediators such as Escherichia coli lipopolysaccharide (LPS), flagellin, and TNF{alpha}. High concentrations of IL-8 are thought to be involved in the pathogenesis of several diseases including neonatal necrotizing enterocolitis (16,17). Recent studies suggested that certain probiotic bacteria have to be alive to elicit an anti-inflammatory response, (15), whereas others can elicit these responses if they are heat killed (12). Furthermore, it is not known whether probiotic bacteria still function after exposure to antibiotics (18). This may be important in situations in which patients may be receiving oral or systemic antibiotics.

In this study, we tested in Caco-2 intestinal epithelial cells whether LGG prevents TNF{alpha}-induced IL-8 production through the NF{kappa}B/inhibitor of {kappa}B (I{kappa}B) pathway and whether this effect depends on the dose of LGG and the presence or absence of antibiotics in the media.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Reagents. MEM (glutamine-free), fetal bovine serum (FBS), antibiotic antimycotic solution, and trypsin were purchased from GIBCO BRL. TNF{alpha} and lipopolysaccharide (E. coli O127: B8) were obtained from Sigma Chemical. Trizol reagent and a first-strand synthesis system for RT-PCR were purchased from GIBCO BRL, Life Technologies. The oligonucleotide primers were from GenoMechanix. Glutamine, EDTA, and all other chemical reagents were obtained from Sigma Chemical. Antibodies to I{kappa}B{alpha}, NF{kappa}B p65, and ß-actin were obtained from Santa Cruz Biotechnology.

    Cell lines and media. Caco-2 cells were obtained from American Type Culture Collection and grown in a humidified incubator at 37°C under 5% CO2/95% air. For each experiment, Caco-2 cells (passage 15–28) were collected by dissociation of a confluent stock culture with 0.25% trypsin and 1 mmol/L EDTA. Full culture media consisted of glutamine-free MEM supplemented with 20% FBS, 4 mmol/L glutamine, 100 kU/L penicillin, 100 mg/L streptomycin, 0.25 mg/L amphotericin B, 1 mmol/L sodium pyruvate, and 0.15% sodium bicarbonate. Media were changed 3 times/wk. Experiments were initiated on d 14–15 after seeding. Previous studies in our laboratory showed that this is a time at which the cells begin to express alkaline phosphatase activity and are in an early stage of differentiation, corresponding to the upper crypt-lower villus stage of differentiation. The experiments were continued for 24–72 h, as the cells progressed through more mature stages of differentiation.

    Bacteria and related preparations. LGG powder was a gift from Mead-Johnson Nutritionals. Live LGG was stored at 4°C. The activity of this powder is ~3.6 x 1011 cfu/g. Live LGG were suspended at different concentrations in cell culture media with or without antibiotics (penicillin and streptomycin in doses used to inhibit bacterial growth in culture). Heat-killed LGG was prepared by heating bacteria at 80°C for 20 min at a concentration of 1010 cfu/L in cell culture media, and then diluted at different concentrations in cell culture media without antibiotics, and used immediately. Effectiveness of the heat-killing method was confirmed as follows: Samples of LGG before and after heat-killing were diluted in PBS and plated on LB agar; 0.2 mL of the heat-killed sample was plated on LB agar. The plates were incubated under anaerobic conditions for 72 h at 37°C. The untreated sample was at 5.4 x 1010 cells/L. Because no colonies arose from the 0.2-mL heat-killed sample, the minimum detection is 5000 cells/L.

    IL-8 ELISA. Cells were seeded onto 12-well plates (Costar®, Corning) at a density of 2–3 x 105 cells/cm2. Monolayers were rinsed with PBS once, and then pretreated with live or heat-killed LGG at concentrations of 104, 106, 108, and 1010 cfu/L in media with or without antibiotics for 12 h, and then treated with TNF{alpha} (0–100 µg/L) in the presence of LGG for another 24 h. Cell culture media were collected and frozen at –20°C for determination of the concentrations of IL-8 by ELISA (OptEIATM, PharMingen). High Binding Extra 96-well microtiter plates (IMMULON®4HBX, Dynex Technologies) were used to quantify cytokines as described by the manufacturer. Plates were read at 450 nm using a microplate reader (PowerWaveX Bio-TEK Instruments). Protein concentrations of whole-cell lysates were measured using the BioRad Dc protein Assay (BioRad). IL-8 levels were normalized to standard protein concentrations.

    IL-8 mRNA isolation and RT-PCR. Total RNA from Caco-2 cells was isolated using the Trizol reagent (GIBCO) according to the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a SuperScript First-Strand Synthesis System kit (Invitrogen Life Technologies). PCR amplification was performed in a total volume of 50 µL, which included Taq DNA polymerase and specific primers. After initial incubation at 94°C for 5 min, PCR was performed with 30 cycles consisting of denaturation (94°C, 1 min), annealing (60°C, 1 min), and extension (72°C, 1 min), followed by an extension at 72°C, 10 min (using DNA Thermal Cycler 480). The oligonucleotide primers used were as follows (19) (from GenoMechanix, L.L.C. FL): IL-8 (forward) 5''-CTG GCC GTG GCT CTC TTG GCA G-CCT TCC TG-3' (reverse) 5'-GGC AAC CCT ACA ACA GAC CCA CAC AAT A-CA-3' (395 bp); and human glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (forward) 5'-CGA GAT CCC TCC AAA ATC AAG-3' (reverse) 5'-GAG CTT GAC AAA GTG GTC G-3' (693 bp). Human GAPDH was used as an internal control.

    Nuclear protein extraction. Cells (6–8 x 104 cells/cm2) were cultured in 100 mm diameter dishes for 14 d. After incubation with LGG (108 cfu/L) for 12 h, cells were stimulated in the presence of LGG with 50 mg/L TNF{alpha} for 1 h. This dose and time were chosen to ensure maximum IL-8 production, as determined by preliminary dose and time-response studies. All nuclear extraction procedures were performed on ice with ice-cold reagents. Cells were harvested after washing with 5 mL ice-cold PBS twice by centrifuging at 450 x g for 5 min. The cells were resuspended with lysis buffer [10 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 10 mmol/L KCl, and 1 mmol/L dithiothreitol (DTT) and protease inhibitor cocktail]. The disrupted cell suspension was then centrifuged at 11,000 x g for 20 min, the supernatant removed, and the nuclear pellet resuspended with extraction buffer [20 mmol/L HEPES (pH 7.9), 1.5 mmol/L MgCl2, 0.42 mol/L NaCl, 0.2 mmol/L EDTA, 25% (v/v) glycerol, 1 mmol/L DTT, and protease inhibitor cocktail]. Protein concentrations of nuclear extract and cytosolic fraction were measured using the Dc protein assay (BioRad).

    Immunoblotting. Equal amounts of proteins were separated by 12.5% SDS-PAGE. Electrophoresed proteins were transferred from the gel to a polyvinylidene fluoride membrane, blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20, incubated with the primary antibody (anti-I{kappa}B{alpha} or anti-NF{kappa}B p65) and then with the appropriate HRP-conjugated secondary antibody. The blot was developed using ECL (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. The membranes were then stripped and reprobed with antibody to ß-actin to confirm equal loading of lanes.

    Statistical analysis. Values are given as means ± SD of triplicate measurements. ANOVA (1 or 2-way) was performed to determine whether LGG and TNF{alpha} affected the variables measured. Pairwise multiple comparisons were conducted following significant ANOVAs by the Holm-Sidak method. All analyses were performed using Sigma Stat software (SPSS Science) and differences among means were considered significant at P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
TNF induced IL-8 production

    Time course. Caco-2 cells were incubated with TNF{alpha} (at concentrations of 10 µg/L for 0, 3, 6, 12, 24, and 48 h). Significant increases in IL-8 production were first detected at 12 h with further increases (P < 0.05) at 24 and 48 h (Fig. 1A). We chose the 24-h incubation time for additional studies of IL-8 induction.



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  		FIGURE 1 The time course (A) and dose response (B) of TNF{alpha}-induced IL-8 production in Caco-2 cells. Cells were incubated with TNF{alpha} (10 µg/L) for 0, 3, 6, 12, 24, and 48 h and with TNF{alpha} (5, 10, 50, and 100 µg/L) for 24 h. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

 
    TNF dose response. At concentrations of 10, 50, and 100 µg/L, TNF{alpha} dose dependently induced IL-8 production (P < 0.01; Fig. 1B). TNF{alpha} concentrations of 10 and 50 mg/L were used in subsequent experiments.

LGG dose responses

Different concentrations of live or heat-killed LGG (0, 1 x 104, 1 x 106, 1 x 108, and 1 x 1010 cfu/L) were used to determine the dose response when given alone or with TNF{alpha} (10 µg/L) on IL-8 production in media with or without antibiotics.

    Live LGG in media without antibiotics. TNF{alpha} upregulated (P < 0.01) IL-8 production compared with media alone (Fig. 2A) At a concentration of 1 x 106 cfu/L, live LGG inhibited (P < 0.01) TNF{alpha}-induced IL-8 secretion, without reducing it to baseline (media control). At concentrations of 1 x 104 (data not shown) and 1 x 108 cfu/L, live LGG did not inhibit TNF{alpha}-induced IL-8 secretion. A dose of 1 x 1010 cfu/L LGG increased IL-8 production compared with media alone (P < 0.01). Interestingly, the higher concentration of LGG with or without TNF{alpha} resulted in IL-8 concentrations that were greater than those due to TNF{alpha} alone (P = 0.001).



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  		FIGURE 2 Dose-response of live LGG in media without antibiotics (A), LGG in media with antibiotics (B), and heat-killed LGG in media without antibiotics (C) on TNF{alpha}-induced IL 8 production. Different concentrations of LGG (0, 1 x 104, 1 x 106, 1 x 108, and 1 x 1010 cfu/L) were used to determine the dose response of live or heat-killed LGG when given alone on a concentration of 10 µg/L TNF{alpha}-induced IL-8 production in media with or without antibiotics. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

 
    LGG in media with antibiotics. At concentrations of 106 and 108 cfu/L, live LGG inhibited (P < 0.01) TNF{alpha}-induced IL-8 secretion, although it did not reduce this to baseline (Fig. 2B). At a concentration of 1 x 1010 cfu/L, LGG with or without TNF{alpha} increased (P < 0.01) IL-8 production.

    Heat-killed LGG in media without antibiotics. At concentrations of 1 x 106 and 1 x 108 cfu/L, heat-killed LGG inhibited (P < 0.01) TNF{alpha}-induced IL-8 production without reducing it to baseline (Fig. 2C). At a concentration of 1 x 1010 cfu/L, heat-killed LGG only slightly increased IL-8 production in media (P < 0.05).

LGG inhibited TNF{alpha}-induced upregulation of IL-8 and this effect was dose dependent. The TNF{alpha}-induced IL-8 production in Caco-2 cells was modulated by LGG under all 3 conditions. However, higher doses of live LGG without TNF{alpha} in the presence or absence of antibiotics induced the production of IL-8. Heat-killed LGG also blunted the TNF{alpha}-induced IL-8 production, but by itself did not increase IL-8 production at higher doses as markedly as live LGG.

    LGG and TNF{alpha}-induced upregulation of IL-8 mRNA. Representative agarose gels (Fig. 3) show that preincubation for 12 h with live LGG (1 x 106 cfu/L) in media without antibiotics reduced (P < 0.01) TNF{alpha}-induced IL-8 mRNA at 3 h. In comparison with the control, the IL-8 band increased with TNF{alpha} but did not increase when TNF{alpha} was added in the presence of LGG.



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  		FIGURE 3 LGG inhibits TNF{alpha}-induced upregulation of IL-8 mRNA. Representative agarose gels of IL-8 mRNA from Caco-2 cells in the presence or absence of TNF{alpha} (10 µg/L) and LGG (106 cfu/L) are shown. IL-8 mRNA was quantified by comparing the intensity of the IL-8 band with that of GAPDH RT-PCR product from the same multiplex reaction. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

 
LGG and the NF{kappa}B/I{kappa}B system

    Time course of TNF{alpha}-induced NF{kappa}B p65 level in the nucleus. Caco-2 cells were incubated with TNF{alpha} (10 µg/L) for 0, 15, 30, 60, 120, and 1440 min. The effect of TNF{alpha} on NF{kappa}B p65 expression in the nucleus was measured using Western blot analyses on nuclear extracts. NF{kappa}B p65 expression in the nucleus increased (P < 0.01) between 30 and 120 min of incubation with TNF{alpha} compared with media control (Fig. 4A). The highest level occurred at the 60-min incubation; therefore, this time was used in all experiments evaluating NF{kappa}B p65.



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  		FIGURE 4 Effect of LGG on the NF{kappa}B/I{kappa}B system. (A) Time course of TNF{alpha}-induced NF{kappa}B translocation to the nucleus (p65 level). Caco-2 cells were incubated with TNF{alpha} (10 µg/L) for 0, 15, 30, 60, 120, and 1440 min, and Western blot analysis of NF{kappa}B p65 subunit was performed on nuclear extracts. (B) Effect of LGG on NF-{kappa}B p65 level. LGG or media was incubated with Caco-2 cells for 12 h, followed by TNF-{alpha} challenge for 1 h, and nuclear NF{kappa}B p65 was measured by Western blot. (C) Effect of LGG on I{kappa}B degradation. LGG or media was incubated with Caco-2 cells for 12 h, followed by TNF{alpha} challenge for 1 h, and I{kappa}B-{alpha} was quantified by Western blot analysis and densitometry. Values are means ± SD, n = 3. Means without a common letter differ, P < 0.05.

 
    LGG and TNF{alpha}-induced nuclear NF{kappa}B p65 expression. Incubation of Caco-2 cells with TNF{alpha} for 60 min increased NF{kappa}B p65 compared with the media control without TNF{alpha} (P < 0.05; Fig. 4B). NF{kappa}B p65 did not differ between LGG-pretreated Caco-2 cells and media controls. Preincubation with LGG (1 x 106 cfu/L) in media without antibiotics for 12 h reduced (P < 0.01) the TNF{alpha}-induced NF{kappa}B p65 level in the nucleus at 1 h (3rd vs. 4th band).

    LGG and TNF{alpha}-induced I{kappa}B-{alpha} degradation. To study the mechanism of the inhibitory effect of LGG on the TNF{alpha}-induced nuclear translocation of NF{kappa}B p65 and increased IL-8, a Western blot analysis of I{kappa}B-{alpha} was conducted on the cell supernatant (cytosolic fraction) after nuclear extraction. Incubation of TNF{alpha} with Caco-2 cells for 60 min reduced I{kappa}B-{alpha} compared with the media control. (P < 0.05; Fig. 4.C). I{kappa}B-{alpha} did not differ between LGG-pretreated Caco-2 cells and media controls. Preincubation of Caco-2 cells with LGG (1 x 106 cfu/L) in media without antibiotics for 12 h lessened (P < 0.05) the TNF{alpha}- induced I{kappa}B-{alpha} reduction.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
The results of this study demonstrate that the TNF{alpha}-induced release of the proinflammatory mediator IL-8 from Caco-2 cells is modulated by the probiotic LGG. However, in cells dose dependently administered LGG without TNF{alpha}, higher doses of live LGG in the presence or absence of antibiotics in vitro actually induced the production of IL-8. Similar to live and antibiotic-treated LGG, heat-killed LGG also reduced TNF{alpha}-induced IL-8 production; however, by itself, it caused only a relatively small increase in IL-8 production. This study also demonstrates that LGG modulates TNF{alpha}-induced IL-8 production by reversing TNF{alpha}-induced changes in the I{kappa}B/NF{kappa}B system.

Over the past few years, probiotics have been used to treat a variety of human intestinal conditions including diarrhea, (10) pouchitis (20) and atopic eczema (11). These effects have not been clearly explained, but several in vitro investigations have provided some information. Neish and colleagues (6,21) demonstrated that nonpathogenic Salmonella inhibited the synthesis of IL-8 induced by TNF{alpha} in a human intestinal epithelial cell line (T84) by decreasing I{kappa}B/NF{kappa}B binding. Ma and colleagues (15) demonstrated that the probiotic bacteria L. reuteri inhibited mRNA upregulation, cellular accumulation, and secretion of IL-8 induced by TNF{alpha} in both T84 and HT29 intestinal epithelial cells. This occurred only with live organisms in contact with the epithelial cells and was associated with translocation of NF{kappa}B to the nucleus and degradation of I{kappa}B. Similar effects were demonstrated by another group using the probiotics B. longum and L. longum (14). Others demonstrated increased transepithelial resistance, decreased IL-8 secretion, and increased mucin gene and tight junction protein expression in T84 and HT29 cells with heat-killed E. coli Nissle 1917 and the probiotic mixture VSL #3 (12).

The blunting of TNF{alpha}-induced IL-8 production appears to be a general response to probiotic bacteria. Whether a response occurs after treatment with only live (15) or DNA from heat-killed probiotics (12,22,23) appears to depend on the strain of probiotic bacteria used and the condition being evaluated. Differential IL-8 responses of intestinal epithelium under conditions employing heat-killed bacteria and antibiotic incubation were not evaluated previously with LGG; they were chosen for evaluation because the response was considered to be both nonpathogenic and clinically useful.

Our results are of interest in that the antibiotic- and nonantibiotic-treated cells had similar anti-IL-8 responses to TNF{alpha}. The high-dose of LGG without TNF{alpha} increased IL-8 production. However, when heat-killed bacteria were used, the high dose (1010 cfu/L) of LGG alone did not markedly increase IL-8 production. Nevertheless, the heat-killed bacteria at the other doses (108 and 106 cfu/L) still diminished the TNF{alpha}-induced IL-8 response, as did the live and antibiotic-treated bacteria. This suggests that heat-killed probiotics may be able to prevent intestinal inflammation without the potential proinflammatory effect exhibited when the intestinal epithelium is exposed to high quantities of LGG.

At present, we cannot explain why the high-dose probiotic (1010 cfu/L) treatments increased IL-8. This finding has important implications in that it is possible that very high doses of LGG or bacterial overgrowth with LGG may actually exacerbate inflammation, whereas lower quantities can reduce inflammation. Additional studies will be required to determine whether these effects are important in vivo. Immunostimulatory bacterial DNA (unmethylated CpG oligonucleotides) alone can stimulate Caco-2 cells to produce IL-8 (24). {gamma}-Irradiated and live VSL #3 equally suppressed experimental colitis in a mouse model, suggesting that CpG DNA, through toll-like receptor 9 (TLR9) signaling, mediates the anti-inflammatory effect of the probiotic (25). However, the heat-killing method used in this study denatures DNA; thus, an alternative mechanism for the protective effect of the heat-killed LGG is probably independent of the TLR9 pathway.

In summary, these data show that live, antibiotic exposed, and heat-killed LGG decrease IL-8 production in TNF{alpha}-induced Caco-2 cells. However, when exposed to a high dose of LGG, the live and antibiotic-treated cells also markedly increased IL-8 production, suggesting that exposure of the intestinal epithelium to high quantities of LGG may actually increase IL-8 production. This effect still occurred but was much less pronounced with heat-killed LGG, suggesting that this form may actually be safer than live LGG in certain situations. The mechanism of the LGG-related decrease in TNF{alpha}-induced IL-8 production is via inhibition of NF{kappa}B translocation to the nucleus and the associated decrease in I{kappa}B. Whether downregulation of I{kappa}B is due to decreased synthesis, increased ubiquitin-mediated degradation, or another mechanism remains to be determined. Probiotic-conditioned extracts inhibited the NF{kappa}B response by blocking proteasomal degradation of I{kappa}B in mouse colon cells (26). Although it is possible that LGG shifted the timing of NF{kappa}B translocation in our study, this is unlikely given the close correlation between the time courses of IL-8 production and NF{kappa}B activation observed in this and in previous studies (27,28). Electrophoretic mobility shift assays may be a useful technique for measuring NF{kappa}B activation in future experiments. Future studies may include a comparison between the effects on the same cell line of LGG and another probiotic such as L. reuterii. Similarly, whether live or heat-killed, LGG is a safer or more efficacious form of probiotic in the prevention of inflammation remains to be determined with in vivo studies.


   ACKNOWLEDGMENTS
 
The authors acknowledge the Mead Johnson company for the contribution of the LGG.


   FOOTNOTES
 
1 Nestle Nutrition Scholar. Back

3 Abbreviations used: DTT, dithiothreitol; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; I{kappa}B, inhibitor of {kappa}B; IL, interleukin; LGG, Lactobacillus rhamnosus GG; NF{kappa}B, nuclear factor {kappa}B; TNF{alpha}, tumor necrosis factor-{alpha}. Back

Manuscript received 19 November 2004. Initial review completed 24 January 2005. Revision accepted 29 April 2005.


   LITERATURE CITED
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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