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Originally published online as doi:10.1189/jlb.0203064 on August 21, 2003

Published online before print August 21, 2003
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(Journal of Leukocyte Biology. 2003;74:982-991.)
© 2003 by Society for Leukocyte Biology

Multinutrient undernutrition dysregulates the resident macrophage proinflammatory cytokine network, nuclear factor-{kappa}B activation, and nitric oxide production

Gregory M. Anstead*,{dagger},1, Bysani Chandrasekar{dagger}, Qiong Zhang{dagger} and Peter C. Melby*,{dagger}

* Medical Service, Department of Veterans Affairs Medical Center, South Texas Veterans Health Care System and
{dagger} Department of Medicine, University of Texas Health Science Center at San Antonio San Antonio, TX

1 Correspondence: Division of Infectious Diseases, Department of Medicine, University of Texas Health Science Center at San Antonio 7703 Floyd Curl Drive San Antonio, TX 78229-3900. E-mail: anstead{at}uthscsa.edu


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ABSTRACT
 
We have described previously a murine model of multinutrient undernutrition that reproduced the features of moderate human malnutrition and led to increased early dissemination of Leishmania donovani. Peritoneal cells from these malnourished mice produced decreased NO after stimulation with IFN-{gamma}/LPS. We hypothesized that malnutrition may cause a deficit in NF-{kappa}B activation, a principal transcription pathway for inducible NO synthase and proinflammatory cytokines. Macrophages from malnourished mice, stimulated with IFN-{gamma}/LPS, showed increased IL-6 production and decreased IL-10 and TNF-{alpha} production. Neutralization of TNF-{alpha} in macrophage cultures from the control mice mimicked the effect of malnutrition on NO and IL-10 production, whereas supplemental TNF-{alpha} added to cultures of macrophages from malnourished mice increased NO secretion. NF-{kappa}B nuclear binding activity in macrophages from the malnourished mice was reduced early after stimulation, but increased to supranormal values by 16- or 24-h poststimulation. Blocking NO production in the macrophages from the control mice reproduced the effect of malnutrition on the late activation of NF-{kappa}B, whereas supplemental NO decreased the late NF-{kappa}B activation in the malnourished mice. Thus, in macrophages from the malnourished mice, initial deficits in NF-{kappa}B activity probably lead to decreased TNF-{alpha}, which results in decreased NO; however, IL-6 is regulated independently from NF-{kappa}B and TNF-{alpha}. The late activation of NF-{kappa}B in the macrophages from malnourished mice is due to absence of negative feedback from NO.

Key Words: transcription factor • immunodeficiency • tumor necrosis factor-{alpha} • interleukin-6 • interleukin-10


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INTRODUCTION
 
In synergy with infection, malnutrition contributes to 56% of all childhood deaths worldwide [1 ]. However, the molecular and cellular bases for the immunosuppression produced by malnutrition are not well understood [2 ]. In particular, the impact of malnutrition on innate immunity has not been adequately addressed [3 ]. Innate immunity refers to host defense mediated by macrophages, monocytes, neutrophils, and NK cells, which recognize pathogens using receptors that bind to broadly expressed microbial products, such as LPS [4 ]. Macrophages are central to the innate and acquired immune responses; they are activated as part of the innate immune response by a variety of stimuli. IFN-{gamma} is the most potent activating cytokine of macrophages [5 ] and LPS is the best-studied microbial stimulus [6 ]. After treatment with IFN-{gamma} and LPS, macrophages release TNF-{alpha}, IL-1ß, and IL-6, the proinflammatory cytokines [7 ]. Preceding proinflammatory cytokine production is the activation of the transcription factor NF-{kappa}B, which promotes the expression of these cytokines, chemokines, adhesion molecules, and inducible nitric oxide synthase (NOS2) [8 , 9 ]. The effects of malnutrition on macrophage function have been addressed in several studies [10 11 12 13 ]. However, these papers have not assessed the influence of malnutrition on the proinflammatory cytokine network and NF-{kappa}B regulation.

Previously, we developed a mouse model of multinutrient undernutrition (protein-energy, zinc, and iron deficiency) that mimicked the growth characteristics of human weanling malnutrition and predisposed to increased dissemination of the parasite Leishmania donovani [3 ]. In this study, we use this model to demonstrate that multinutrient undernutrition (henceforth referred to as malnutrition) leads to extensive dysfunction of the macrophage proinflammatory cytokine network and NF-{kappa}B regulation.


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MATERIALS AND METHODS
 
Mice
Weanling (3-wk old) female nu/+ BALB/c mice (heterozygous at the nu locus, functionally normal) were obtained from the breeding colony of the South Texas Veterans Health Care System, San Antonio, TX.

Diets and Feeding
Mice received a 3-day acclimation on standard chow after weaning and prior to the change to the two experimental diets. The control group (well-nourished (WN)) mice received a diet that contained 17% protein, 100 ppm iron, 30 ppm zinc, which was provided ad libitum. The mice in the malnourished (MN) group received a diet with 3% protein, 10 ppm iron, 1 ppm zinc; these mice received 12% less food by weight per day compared with the mice in the control group [3 ]. Both diets provided 3.9 kcal/g of chow. Prior to the experimental feeding, the mice were weight-matched and both groups had free access to water. The mice were housed in groups of four with low trace element bedding (Apha-Dri; Shepard Specialty Papers, Kalamazoo, MI).

Culture of resident peritoneal macrophages
Peritoneal cells were collected from mice in both diet groups by lavage using DMEM with 2% FBS. Typically, 8-12 mice from each diet group were used in each experiment. The cells were plated at 1 x 106 cells per mL in 10% FBS in DMEM with 1% (vol/vol) 1 M HEPES, 1% (vol/vol) penicillin-streptomycin (each at 10,000 IU/mL), and 0.1 mM 2-mercaptomethanol. The cells were allowed to adhere for 3 h and then washed 3 times with PBS to remove nonadherent cells. Fresh media (DMEM with 10% FBS) was added. The adherent cells were treated with IFN-{gamma} (PharMingen, San Diego, CA; 80 U/mL). After 1 h, LPS (Escherichia coli 0111:B4; Sigma, St. Louis, MO) (20 ng/mL)) was added. Supernatants and/or cells were collected at the times indicated in the results section. In specific experiments (see below), the stimulated macrophages were treated with a NOS2 inhibitor, an NO donor, a mAb against TNF-{alpha}, or supplemental TNF-{alpha}.

NOS2 was inhibited in macrophage cultures using L-N6-(1-iminoethyl)lysine (L-NIL) (Calbiochem Novabopchem Corp., San Diego, CA); L-NIL was added to the cultures from the WN mice 15-min after the addition of LPS to a concentration of 1 mM. After 24 h, the supernatants were analyzed for TNF-{alpha}, IL-6, IL-10, and NO and the cells were harvested for NF-{kappa}B determination.

Supplemental NO was provided to the IFN-{gamma}/LPS-stimulated macrophage cultures from the MN mice by adding sodium nitroprusside (SNP; Calbiochem) 15-min after addition of LPS to a concentration of 0.1 mM. After 24 h, the supernatants were assayed for TNF-{alpha}, IL-6, IL-10, and NO, and the cells were harvested for NF-{kappa}B determination.

TNF-{alpha} was neutralized in the macrophage cultures using a rat anti-mouse IgG1 mAb (PharMingen). The mAb was added to macrophage cultures from the WN mice 15-min after the addition of LPS, to give concentrations of anti-TNF-{alpha} mAb of either 20 µg/mL or 50 ng/mL. After 24 h, the supernatants were analyzed for TNF-{alpha}, IL-6, IL-10, and NO.

Supplemental TNF-{alpha} was provided to the IFN-{gamma}/LPS-stimulated macrophage cultures from the MN mice by adding murine TNF-{alpha} (PharMingen) 60-min after addition of LPS to a concentration of 500 U/mL. After 24 h, the supernatants were assayed for TNF-{alpha}, IL-6, IL-10, and NO.

NO production by macrophages
Supernatants from the macrophage cultures were tested for nitrite by the Griess reaction, after conversion of nitrate to nitrite with nitrate reductase (colorimetric assay; Cayman Chemical Co., Ann Arbor, MI).

Cytokine assays
Cytokine levels were determined by ELISA, using the following kits: IL-6, IL-10, TNF-{alpha} (eBioscience, San Diego, CA), and IL-1ß (R&D Systems, Minneapolis, MN).

NF-{kappa}B DNA binding activity and dimer composition
Isolation of nuclear protein extracts and EMSA were performed as described previously [14 ]. A ds oligonucleotide (Santa Cruz Biotechnology, Santa Cruz, CA) containing a tandem repeat of the decameric consensus sequence (5'-GGGACTTTCC-3') was used as a probe. For the competition assay, the protein extract (20 µg) was preincubated with homologous unlabeled oligonucleotide for 5 min on ice, followed by the addition of labeled probe. Absence of protein extract, competition with 100-fold molar excess unlabeled NF-{kappa}B, and mutant NF-{kappa}B oligo (5'-AGT TGA GGC GAC TTT CCC AGG C-3'; Santa Cruz Biotechnology) served as controls. In the gel supershift assay to determine the dimeric composition of NF-{kappa}B, the protein extract (20 µg) was preincubated for 40 min on ice with either anti-p50 or -p65 subunit-specific polyclonal antibodies (1 µg; Santa Cruz Biotechnology) prior to the addition of 32P-labeled NF-{kappa}B consensus oligo.

Proinflammatory cytokine and NOS2 mRNA expression by Northern blot analysis
RNA extraction, Northern blotting, autoradiography, and densitometry were performed as described previously [15 ]. Total RNA was extracted from the cultured macrophages using acid-guanidium isothiocyanate-phenol-chloroform. The RNA was denatured in 2.2 M formaldehyde, size fractionated on 0.8% agarose gels containing 0.5 g/ml ethidium bromide to check RNA integrity and loading equivalency, and electroblotted at 4°C onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH) in 0.025 M phosphate buffer, pH 6.5. Ribonucleic acids were UV cross-linked to the membrane. The blot was prehybridized for 4 h at 42°C in a prehybridization buffer that contained 50% formamide, 0.1% SDS, 5 x SSC, 2.5 x Denhardt’s, 250 µg/ml denatured sonicated salmon sperm DNA (Stratagene, La Jolla, CA), 50 mM Na2PO4, pH 6.5. The blots were then hybridized at 42°C for 16 h with 32P-labeled probes (6 x 105 cpm/ml), washed twice at 23°C in 6 x SSC/0.5% SDS, twice at 37°C in 1 x SSC/0.5% SDS, and once at 57°C in 0.l x SSC/0.5% SDS. All blots were then exposed at -80°C to Kodak XAR-5 film with Kodak-intensifying screens, and the intensities of the autoradiographic bands were quantified by videoimaging. The same membrane was reprobed after stripping off its previous label. mRNA sizes were determined in relation to the mobility of 28S and 18S rRNA and an mRNA ladder (GibcoBRL, Grand Island, NY). The cDNA probes (NOS2, IL-6, and TNF-{alpha}; American Type Culture Collection, Manassas, VA) were labeled with [{alpha}-32P]dCTP (3000 Ci/mmol; Amersham, Piscataway, NJ) using random hexanucleotide primers (Boehringer-Mannheim, Indianapolis, IN), and the oligonucleotide probe (h28S rRNA; 40 base ss oligo; Oncogene Science, Uniondale, NY) was 5' end-labeled with ({alpha}-32P)ATP using T4 polynucleotide kinase.

Statistics
Results are expressed as the mean and standard error of the mean. Comparisons between group means were performed using Student’s two-tailed t test.


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RESULTS
 
Malnutrition produced decreased weight-for-age
Mice on the control diet received chow ad libitum and consumed an average of 3.2 g/d. Mice in the deficient diet group were provided 2.8 g/d. At the end of 6 wks of feeding, mice in the deficient diet group showed decreased weight-for-age and an altered growth curve (Fig. 1 ). The MN mice had a slowly declining growth curve (12.5% weight loss) analogous to human weanling malnutrition and were an average of 69% of weight-for-age compared with the controls (P < 0.001), which corresponds to moderate malnutrition [3 ].



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  		Figure 1. Growth characteristics of weanling mice fed the two experimental diets for 6 wks. A composite growth curve of 6 independent experiments is shown. The diet for the well-nourished control (WN) mice contained 17% protein, was zinc and iron sufficient, and was provided ad libitum. The diet for the malnourished (MN) mice contained 3% protein, was zinc and iron deficient, and was 12% calorie deficient. There were 8 mice in the well-nourished group and 8-12 mice in the malnourished group in each of the 6 independent experiments.

Macrophages from malnourished mice produced less NO after stimulation
As described previously with resident peritoneal cells [3 ], resident peritoneal macrophages (isolated by adherence to plastic) from the MN mice also produced less NO after stimulation with IFN-{gamma} and LPS (Fig. 2A ). At 1 h after stimulation, there were equivalent low levels of NO produced by both groups of macrophages. However, at 16 and 24 h, NO production by macrophages from the MN mice was reduced 54 %and 49%, respectively, compared with those from the WN mice (P <= 0.04).



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  		Figure 2. The effect of malnutrition on the secretion of (A) NO, (B) TNF-{alpha}, (C) IL-6, (D) IL-10, and (E) IL-1ß by resident peritoneal macrophages stimulated with IFN-{gamma}/LPS. The graphs show the findings at 3 or 4 time points in 3 or 4 independent experiments. These results were obtained from 8 samples of macrophages from each of 8 well-nourished mice and 8 samples of macrophages from 8-12 malnourished mice (pooling macrophages from the malnourished mice was necessary in some cases).

Macrophages from malnourished mice had an altered proinflammatory response to IFN-{gamma}/LPS
At four time points (1-, 4-, 16-, and 24 h), resident peritoneal macrophages from the MN mice produced decreased levels of TNF-{alpha} after stimulation with IFN-{gamma}/LPS (Fig. 2B) . At 1 h, there was low-level production in macrophages from both MN and WN mice, but the latter secreted a significantly higher amount. At 4-, 16-, and 24 h after stimulation, macrophages from the MN mice produced significantly less TNF-{alpha} (5.7-, 2.7-, and 3.6-fold reductions, respectively; P<= 0.05). Barely detectable levels of IL-10 were observed at 1 h after stimulation; at this time point, IL-10 production was higher from macrophages from the MN mice. However, at the 16- and 24 h time points, macrophages from the MN mice produced 74% and 30% less IL-10, respectively, than those from the WN mice (Fig. 2C ; P <= 0.005).

At 3 time points (1-, 16-, and 24 h), IFN-{gamma}/LPS-stimulated macrophages from MN mice produced increased levels of IL-6 compared with macrophages from the WN mice (Fig. 2D) . At 1 h after stimulation, IL-6 levels were low in both groups, but they were higher in the macrophages from the MN mice. At 16 and 24 h after stimulation, macrophages from the MN mice produced 53% and 76% higher levels of IL-6, respectively, than their WN counterparts (P <= 0.01). At four time points (1-, 4-, 16-, and 24 h after stimulation), stimulated peritoneal macrophages from both groups of mice produced equivalent levels of IL-1ß (Fig. 2E) .

Macrophages from malnourished mice showed decreased transcription of TNF-{alpha} and NOS2 and increased transcription of IL-6
Cytokine production is predominately regulated by the transcription rate of cytokine genes [8 ]. To determine if the alterations in TNF-{alpha} and IL-6 protein and NO were due to changes in gene transcription, RNA was isolated from macrophages from the MN and WN mice after stimulation with IFN-{gamma}/LPS for 4 h. This time point was selected because it has been shown previously that mRNA for TNF-{alpha} and IL-6 is maximal at 2-6 h and that NOS2 mRNA is detectable 3-8 h after macrophage stimulation [16 , 17 ]. The mRNA from the macrophages of five mice from each diet group was pooled and analyzed by Northern blot (Fig. 3 ). On the basis of densitometric analysis of the hybridization signal, levels of TNF-{alpha} and NOS2 mRNA were decreased 59% and 68%, respectively, in the macrophages from the MN mice, and IL-6 mRNA was increased 87%. These results are consistent with the decreased production of TNF-{alpha} and NO and increased IL-6 observed in the cultures of stimulated macrophages from the MN mice.



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  		Figure 3. TNF-{alpha}, IL-6, and NOS2 mRNA levels from macrophages stimulated with IFN-{gamma}/LPS from control (well-nourished (WN)) and malnourished (MN) mice. Each lane represents the total RNA pooled from the resident peritoneal macrophages from 5 mice.

Neutralization of TNF-{alpha} decreased macrophage secretion of IL-6, IL-10, and NO
The decrease in TNF-{alpha} secretion observed in the macrophages from the MN mice suggested that the initial deficits in TNF-{alpha} secretion may influence the subsequent cytokine profile. Thus, to mimic the effect of low TNF-{alpha} production by the macrophages from the MN mice, we either completely or partially (31%) neutralized the TNF-{alpha} produced by the activated macrophages from the WN mice, using either a high (10 µg/mL) or a low (50 ng/mL) concentration of anti-TNF-{alpha} mAb, respectively (Fig. 4A ). After macrophage stimulation, in the presence of 10 µg/mL anti-TNF-{alpha}mAb, IL-10 was reduced 68%; IL-6, 14%; and NO, 40% (Fig. 4) . At a lower concentration of anti-TNF-{alpha} mAb (50 ng/mL), there were lesser, but still significant decreases in IL-10 and NO production, but the effect on IL-6 was lost. Thus, endogenous TNF-{alpha} had a dose-dependent effect on the production of IL-6, IL-10, and NO.



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  		Figure 4. The effect of neutralization of TNF-{alpha} on levels of TNF-{alpha}, IL-10, IL-6, and NO. Peritoneal cells from the WN mice were collected and stimulated with IFN-{gamma}/LPS as described in Materials and Methods. A monoclonal antibody against TNF-{alpha} was added to macrophage cultures 15 min after the addition of LPS to final concentrations of either 10 µg/mL or 50 ng/mL. After 24 h, the culture supernatants were analyzed for TNF-{alpha}, IL-10, IL-6, and nitrate. Six to eight samples of macrophages were obtained from 8 mice for each determination.

Exogenous TNF-{alpha} increased NO secretion by macrophages from the malnourished mice
To determine if correcting the deficit in TNF-{alpha} production by macrophages from the MN mice would rectify the aberrant production of IL-6, IL-10, and NO, exogenous TNF-{alpha} was added to cultures of IFN-{gamma}/LPS-treated macrophages. The addition of 500 U of TNF-{alpha} increased the TNF-{alpha} detected by ELISA by 37.7%. This level of additional TNF-{alpha} increased NO production by 30.8%; IL-6 and IL-10 production were not affected (Fig. 5 ).



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  		Figure 5. The effect of supplementation of TNF-{alpha} on levels of TNF-{alpha}, IL-10, IL-6, and NO. Peritoneal cells from the WN mice were collected and stimulated with IFN-{gamma}/LPS as described in Materials and Methods. TNF-{alpha} was added to macrophage cultures 60 min after the addition of LPS to final concentrations of 500 U/mL. After 24 h, the culture supernatants were analyzed for TNF-{alpha}, IL-10, IL-6, and nitrate. There were 8 macrophage samples from 8 mice for each determination.

Macrophages from malnourished mice had delayed NF-{kappa}B activation
Because NOS2 activity and proinflammatory cytokine transcription are critically dependent on NF-{kappa}B activation, we reasoned that dysregulation of these inflammatory mediators might be determined by malnutrition-induced changes in NF-{kappa}B activation. At four different time points (0.5, 1, 16, and 24 h after IFN-{gamma}/LPS stimulation), NF-{kappa}B DNA binding activity was measured in macrophages from the MN and WN mice. Early after IFN-{gamma}/LPS-stimulation (0.5 and 1 h), macrophages from the MN mice showed significantly decreased NF-{kappa}B DNA binding compared with controls (Fig. 6A 6B ). However, at 16 and 24 h, this pattern reversed, such that NF-{kappa}B binding activity in the macrophages from MN mice exceeded that of the control group (Fig. 6C 6D) .



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  		Figure 6. NF-{kappa}B DNA binding activity. Analysis of macrophage protein extracts from malnourished (MN) and well-nourished (WN; control) mice for NF-{kappa}B DNA binding activity by EMSA; macrophages were activated by IFN-{gamma}/LPS. Each lane represents the protein extracts pooled from two mice. Arrowhead: specific DNA–protein complexes. Solid circle: unincorporated labeled probe. (A) 0.5 h of culture. (B) 1 h. (C) 16 h. (D). 24 h. (E) Supershift assay showing p65p65, p50p65, and p50p50 subunits of NF-{kappa}B at 16 h after IFN-{gamma}/LPS stimulation. Arrow: unincorporated probe. (F) Densitometric analysis. The autoradiographic signals obtained in EMSA (A-D) were quantified by video-image analysis, and the results were presented as mean ± SEM of the arbitrary numbers obtained. * P < 0.05. **P < 0.01.

The specific subunit composition of NF-{kappa}B determines if the complex will stimulate or inhibit cytokine/enzyme mRNA transcription. The p65/p50 heterodimer is the best described stimulatory transcription factor, whereas certain other dimers are inhibitory [8 ]. Using the supershift assay on the activated NF-{kappa}B/DNA complex present at 16 h after stimulation, the macrophages from the mice of both diet groups had similar compositions with respect to the p65/p65, p50/p65, and p50/p50 dimers (Fig. 6E) .

Inhibition of NOS2 increased late NF-{kappa}B activation and increased IL-6 and TNF-{alpha} production in macrophages from well-nourished mice
NO acts as a negative feedback regulator of NF-{kappa}B activation [18 ]. We hypothesized that the decreased NO production from macrophages from the MN mice may result in high levels of NF-{kappa}B activation at 24 h due to the absence of appropriate negative feedback regulation. To test this hypothesis, the production of NO was inhibited in the macrophages from the WN mice and the level of NF-{kappa}B-binding activity was determined. After addition of L-NIL [19 ] to the media of the IFN-{gamma}/LPS-stimulated macrophages from the WN mice, NO accumulation at 24 h decreased 4.3-fold. The level of activated NF-{kappa}B at this time point increased 2.7-fold (Fig. 7 ), reminiscent of the increase in NF-{kappa}B binding in macrophages from MN compared with WN mice after 24 h of stimulation (Fig. 6D) .



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  		Figure 7. The effect of the inhibition of NOS2 and NO supplementation on NF-{kappa}B DNA binding activity. Macrophage protein extracts were analyzed for NF-{kappa}B DNA binding activity by EMSA; macrophages were activated by IFN-{gamma}/LPS. Lane 1 is from macrophages from well-nourished mice without the addition of an NOS2 inhibitor (well-nourished control, WN-C). Lane 2 is from macrophages from well-nourished mice treated with the NOS2 inhibitor L-NIL (WN-L-NIL). Lane 3 is from macrophages from malnourished mice without supplemental NO (malnourished-control, MN-C). Lane 4 is from macrophages from MN mice treated with the NO donor sodium nitroprusside (MN-SNP). Each lane represents the pooled protein extracts of the peritoneal macrophages from 5 mice. Arrowhead: specific DNA–protein complexes.

Cytokine accumulation at 24 h was also examined in the macrophage cultures in which NOS2 was inhibited. Blocking NO production (Fig. 8A ) increased IL-6 production by 65% (Fig. 8C) . There was a trend (p = 0.08) toward increased levels of TNF-{alpha} (Fig. 8B ) and no effect on IL-10 levels (Fig. 8D) .



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  		Figure 8. The effect of inhibition of NOS2 on the levels of nitrate, TNF-{alpha}, IL-6, and IL-10. NOS2 was inhibited using L-N6-(1-iminoethyl)lysine (L-NIL). Peritoneal cells from the WN mice were collected by lavage and stimulated with IFN-{gamma}/LPS as described in Materials and Methods. L-NIL was added to macrophage cultures 15 min after the addition of LPS to a concentration of 1 mM. After 24 h, the culture supernatants were assayed for nitrate, TNF-{alpha}, IL-6, and IL-10. The cells were analyzed for activated NF-{kappa}B (Fig. 7 ). Eight macrophage samples were obtained from 8 mice in both groups (with and without L-NIL).

Exogenous NO reversed the late NF-{kappa}B activation in macrophages from malnourished mice but did not influence cytokine production
When an exogenous source of NO (SNP) was added to cultures of macrophages from the MN mice, NO levels increased by 63% (Fig. 9A ), and NF-{kappa}B activation at 24 h after stimulation decreased 68% compared with macrophages from MN mice in which no SNP was added (Fig. 7) . These levels were similar to those of NF-{kappa}B binding observed in the 24 h-stimulated macrophages from WN mice (compare Fig. 6D with Fig. 7 ). Collectively, these data indicate that when NO production is low (malnutrition or the well-nourished state with NOS2 inhibition), NF-{kappa}B activation is high at 24 h; when the NO concentration is high (the well-nourished state or malnourished state with exogenous NO), the NF-{kappa}B activation at 24 h is low. Despite the effect of exogenous NO on late NF-{kappa}B activation in stimulated macrophages from the MN mice, there was no effect on the production of TNF-{alpha}, IL-6, or IL-10 (Fig. 9B 9C 9D ).



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  		Figure 9. The effect of the addition of exogenous NO on nitrate, TNF-{alpha}, IL-6, and IL-10. Cultures of macrophages from the malnourished mice were supplemented with NO by using sodium nitroprusside (SNP). Peritoneal cells from the MN mice were collected and stimulated with IFN-{gamma}/LPS as described in Materials and Methods. Sodium nitroprusside was added to macrophage cultures 15 min after the addition of LPS to a final concentration of 0.1 mM. After 24 h, the culture supernatants were assayed for nitrate, TNF-{alpha}, IL-6, and IL-10 and the cells were analyzed for activated NF-{kappa}B (Fig. 7 ). Four samples of macrophages were obtained from four mice for each determination (with and without SNP).


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DISCUSSION
 
In this study, we investigated the proinflammatory cytokine network of resident peritoneal macrophages in a malnourished mouse model. After stimulation with IFN-{gamma}/LPS, macrophages from MN mice, as compared with their WN counterparts, produced: (1) less TNF-{alpha}, IL-10, and NO; (2) increased levels of IL-6; and (3) equivalent levels of IL-1ß. These changes were evident at the protein and mRNA levels, indicating the aberrant mediator levels arose, at least in part, from abnormal transcriptional activity. We used cells relevant to innate immunity (resident macrophages) and selected a well-described sequence of activating stimuli: priming with IFN-{gamma} and triggering with LPS [20 ].

IFN-{gamma} and LPS signal through different, but synergistic, pathways. LPS binding results in the activation of NF-{kappa}B, which promotes the transcription of TNF-{alpha}, IL-1ß, IL-6, and NOS2 [21 ]. TNF-{alpha} increases rapidly after stimulation, and it stimulates the release of IL-1ß, IL-6, and IL-10 [22 , 23 ]. IFN-{gamma} activates the transcription factors STAT1 and IRF-1. The latter, in synergy with NF-{kappa}B, is important in the stimulation of NOS2 transcription [24 ]. IFN-{gamma} priming increases TNF-{alpha} transcription and mRNA stability [25 ] and NF-{kappa}B activity [17 ]. IFN-{gamma} synergizes with TNF-{alpha} in the up-regulation of IL-6 and NOS2 production [17 , 26 ].

Altered inflammatory mediator response has been noted in other malnutrition models. Previously, we observed decreased splenocyte production of NO and TNF-{alpha} after L. donovani infection in MN mice [3 ]. Decreased NO production has been reported in other rodent models of protein-energy malnutrition [27 , 28 ]. Macrophages from protein-malnourished animals produce less TNF-{alpha} in response to infection or LPS [29 30 31 ]. TNF-{alpha} levels were reduced in LPS-stimulated mononuclear cells from malnourished children [32 ]. The effect of protein malnutrition on IL-6 and IL-1 production in experimental animals has been variable [10 , 30 , 31 , 33 ]. However, increased levels of IL-6 have been observed in multiple studies of malnourished patients [34 35 36 37 ].

The cytokine profile that observed in the stimulated macrophages from the MN mice could be related to several processes. The diminution of TNF-{alpha} and NO, the inhibition of NF-{kappa}B activation, and the augmentation of IL-6 also occur in endotoxin tolerance [38 , 39 ]. The peritoneal macrophages from the MN mice may show endotoxin tolerance because of bacterial intestinal translocation, which occurs more readily in the immunocompromised host [40 ]. Alternatively, the cytokine profile displayed by the macrophages from the MN mice is analogous to the secretory behavior of macrophages at a low level of differentiation [41 , 42 ]. Previously, malnutrition has been shown to cause retarded macrophage differentiation [33 ].

The deficits in TNF-{alpha} expression observed in the stimulated macrophages from the MN mice may have significant consequences in host defense [43 44 45 46 ]. TNF-{alpha} is the apex of the proinflammatory cytokine cascade because its early production stimulates additional proinflammatory and counter-regulatory mediators [47 ]. TNF-{alpha} and IL-10 form a feedback loop, in which TNF-{alpha} induces IL-10 secretion and IL-10 negatively regulates TNF-{alpha} synthesis [22 ]. In this study, macrophages from the MN mice showed low levels of both TNF-{alpha} and IL-10 production. IL-10 is classically considered an anti-inflammatory mediator [48 , 49 ]. However, IL-10 displays certain proinflammatory activities as well [45 , 48 , 50 ]. Thus, the low levels of IL-10 produced by the macrophages from the malnourished mice may have adverse effects on the innate immune response. Antibody neutralization of TNF-{alpha} from the macrophages from the WN mice decreased IL-10 levels in proportion to the degree of neutralization, as expected based on the potent effect of TNF-{alpha} on IL-10 synthesis. However, TNF-{alpha} supplementation of macrophages from the malnourished mice did not correct the deficit in IL-10, suggesting that factors in addition to TNF-{alpha} deficiency play a role in the IL-10 deficit of malnutrition.

Although IL-6 is essential in host defense [51 ], the overexpression of IL-6 observed in malnutrition may be detrimental to host defense [52 53 54 ]. IL-6 has a number of activities that may be immunosuppressive; it modulates IL-2 reactivity [55 ], inhibits Th1 differentiation [56 ], and impairs early release of TNF-{alpha} [57 ]. IL-6 production is inhibited by IL-10 and NO [18 , 58 ]; thus, the deficiencies of IL-10 and NO production observed from the macrophages from the MN mice may promote excessive IL-6 levels. IL-6 transcription is regulated by multiple transcription factors that either activate (NF-{kappa}B) or suppress its expression [59 ]. Although NF-{kappa}B binding activity was decreased in macrophages from the MN mice, apparently other transcription factors stimulate IL-6 transcription. Dysregulated production of IL-6 also occurs in several pathologic conditions [14 , 60 61 62 ]. IL-6 production is enhanced by TNF-{alpha} [23 ]. However, IL-6 secretion by macrophages from the MN mice was high, despite the lower levels of TNF-{alpha}. Treatment of macrophages from the WN mice with anti-TNF-{alpha} antibodies did not completely suppress IL-6 production, so other factors in addition to TNF-{alpha} must influence its expression [55 ].

In this study, there were decreased levels of NO produced by the macrophages from the MN mice. NOS2 is regulated transcriptionally [18 ], and the expression of the macrophage NOS2 gene is induced synergistically by IFN-{gamma} and LPS [16 ]. The decreased NOS2 transcription observed in macrophages from the MN mice may be due to deficits in early NF-{kappa}B activation and perhaps other defects in LPS and IFN-{gamma} signal transduction. In our system, antibody neutralization of TNF-{alpha} production by macrophages from the WN mice diminished NO production, as described previously [26 ]. By contrast, TNF-{alpha} supplementation of the macrophages from the MN mice increased NO production (Fig. 5) . Thus, the attenuated TNF-{alpha} production observed in macrophages from the MN animals may contribute to the decreased NOS2 mRNA transcription and NO synthesis.

To determine the role of NO in the aberrant cytokine responses of macrophages from the MN mice, cytokine production was examined during NOS2 inhibition (Fig. 8) or NO supplementation (Fig. 9) . Blocking NO production in macrophages from the WN mice increased IL-6 and TNF-{alpha} production [18 , 63 ] but had no effect on IL-10 levels. Previously, inhibition of NO synthesis has been noted to increase IL-10 [64 ].

Supplementation of stimulated macrophages from MN mice with exogenous NO could not reverse the malnutrition-related effects on TNF-{alpha}, IL-6, or IL-10 (Fig. 9) . Previous studies indicate that NO may stimulate or inhibit IL-6 and IL-10 production [58 , 65 , 66 ]. Based on these studies of the manipulation of the NO level in both the MN and WN mice, the altered levels of TNF-{alpha} and IL-10 observed in malnutrition do not arise from feedback regulation due to the lower expression of NO, but result from processes independent from NO production.

Because NF-{kappa}B is a critical transcription factor for genes that control the production of IL-6, TNF-{alpha}, and NO [67 ], we postulated that it could play a role in the dysregulated proinflammatory response in the malnourished host. Previously, Yaman et al. observed that NF-{kappa}B expression was decreased in LPS-stimulated peritoneal macrophages from protein-malnourished rats [68 ].

NF-{kappa}B is the most important transcription factor for the expression of TNF-{alpha} [69 ]. The delayed induction of NF-{kappa}B observed at 0.5 and 1 h after stimulation in the macrophages from the MN mice probably contributes significantly to the decreased levels of TNF-{alpha} and NOS2 transcription seen after 4 h. In contrast, the high levels of activated NF-{kappa}B observed 24 h after stimulation of the macrophages from the MN mice was due to a lack of appropriate negative feedback by NO. The addition of supplemental NO to the macrophage cultures from the MN mice decreased this late NF-{kappa}B activation, whereas inhibiting NO production in the macrophages from the WN mice increased NF-{kappa}B at 24 h, mimicking the effect of malnutrition.

Another possibility to explain defective TNF-{alpha} and NOS2 transcription was that the NF-{kappa}B that reached the nucleus was dysfunctional. NF-{kappa}B attaches to DNA in the promoter regions of target genes as a dimer composed of two Rel proteins. The p50/p65, p50/c-Rel, p65/p65, and p65/c-Rel dimers stimulate inflammatory mediator transcription, whereas the p50 homodimer is inhibitory [67 ]. To investigate the possibility that the NF-{kappa}B subunit composition in the macrophages from MN mice may be predominantly the inhibitory p50 homodimer, a supershift assay was performed. This revealed that the NF-{kappa}B from macrophages of both WN and MN mice both had similar subunit composition.

Because of the integrated network of cytokine regulation, dysregulated expression of even a single crucial cytokine gene can result in immunologic consequences [47 ]. In this model of malnutrition, delayed NF-{kappa}B activation and the resulting early deficits of TNF-{alpha} had a significant effect on subsequent NO production. Furthermore, there was autonomous hyper-expression of IL-6, a potentially immunosuppressive mediator, which was disconnected from early NF-{kappa}B activation and TNF-{alpha} production. These defects in the innate immune response produced by multinutrient undernutrition may contribute to the susceptibility of the malnourished patient to infection.


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ACKNOWLEDGEMENTS
 
The authors thank Wei-guo Zhao for technical assistance and Gabriel Fernandes, Sunil K. Ahuja, Seema S. Ahuja, and Robert A. Clark for helpful discussions. This work was supported by funding from the U.S. Department of Veterans Affairs (GMA, PCM), the Howard Hughes Medical Institute (GMA), a Grant-in-Aid (0150105N) from the American Heart Association (BC), and the National Institutes of Health (NIH HL-68020) (BC).

Received February 10, 2003; revised June 18, 2003; accepted June 19, 2003.


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