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Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455 and * Ross Products Division/Abbott Laboratories, Columbus, OH 43215
3To whom correspondence should be addressed. E-mail: jyono001{at}tc.umn.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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(IFN-) and
interleukin-5 (IL-5), respectively. Anti-OVA IgG subclass and IgE
Ab levels were determined 3 wk after the first OVA challenge and 5 d and 2 wk after the second OVA challenge. Dietary ribonucleotides
significantly augmented OVA-specific IFN- production by the
regional draining LN cells after the first and second OVA challenges.
The NS diet increased anti-OVA IgG2a Ab levels after the first OVA
challenge and both anti-OVA IgG2a and anti-OVA IgG2b after the
second challenge. OVA-specific IgG1 and IgE Ab levels were lower
(P < 0.05) after the second OVA challenge in mice
fed the NS diet. Dietary ribonucleotides did not affect
production or expression of IL-5. Our findings thus indicate that in
Th2-prone BALB/c J mice, dietary ribonucleotides modulated skewed Th2
responses against OVA toward Th1 as measured by production of IFN-,
a Th1 cytokine, and changes in anti-OVA Ab isotype levels.
KEY WORDS: • cytokine • primary and secondary antibody responses • T-helper cells • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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, Pickering et al. 1998
). Immune responses in humans are biased toward type 2
T-helper cells (Th2) at birth and shift progressively toward Th1
during y 1 of life (Adkins 2000
, Prescott et al. 1998
). Because skewed Th2 responses are implicated in atopic
disorders (Doria and Frasca 1997
, Romagnani 1997 and 2000
), it is important to determine how dietary
ribonucleotides influence T1/T2 balance in a Th2-biased immune system.
There are no reports in the literature describing the effects of
dietary ribonucleotides on Th1/Th2 balance in infants. Our data
(Jyonouchi et al. 2000
) and those of others
(Nagafuchi et al. 2000
) in rodents indicate that dietary
ribonucleotides augment Th1 responses in Th1-biased mice. It is not
clear how dietary nucleotides affect the Th1/Th2 balance in animals
that, like human neonates, have a genetic predisposition toward Th2
responses. BALB/c J mice challenged with ovalbumin (OVA) generate
potent Th2 responses, resulting in high levels of anti-OVA
immunoglobulin (Ig)G1 and IgE Ab (Abbas et al. 1997
,
Adkins and Du. 1998
, Yip et al. 1998
) and
have been used as a murine model of atopic disorders.
Immune responses in mice are significantly diminished by a
ribonucleotide-free (NF) diet (Jyonouchi 1994
,
Van Buren and Rudolph 1997
) despite the presence of
active metabolic pathways for the salvage and de novo
synthesis of nucleotides. Similarly, reduced immune responses occur in
infants fed formulas containing negligible levels of ribonucleotides
compared with infants fed higher levels in a supplemented formula or
human milk (Carver et al. 1991
, Martinez-Augustin et al. 1997a
, Pickering et al. 1998
).
In this study, we sought to determine whether dietary ribonucleotides
added to a NF diet modify the Th1/Th2 balance in BALB/cJ mice, a strain
genetically programmed for skewed T2 responses. OVA, a protein antigen
(Ag), was given with incomplete Freund’s adjuvant (IFA), a combination
that induces predominantly Th2 responses in BALB/cJ mice (Yip et al. 1998
). Administration of OVA with IFA into the footpad of
BALB/cJ mice allowed us to examine both serum anti-OVA IgG
subclass/IgE Ab levels and Th1/Th2 responses in the lymph nodes (LN)
draining the site of OVA injection (Mondino et al. 1996
)
by measuring the production and mRNA/protein expression of Th1/Th2
cytokines.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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BALB/cJ female mice (5–6 wk old, n = 250) were purchased from Jackson Laboratories, Bar Harbor, ME and maintained in the animal facility at the University of Minnesota, Minneapolis, MN. The mice were housed in groups of 4 per cage. Blood samples (1–1.2 mL/mouse) were collected by cardiac puncture after a brief exposure to CO2. Popliteal LN were obtained from mice killed in a CO2 chamber, as approved by the Institutional Animal Care and Use Committee (IACUC), University of Minnesota. Serum and LN were collected from the same mice when sampling time points coincided.
Diet.
Mice were fed either a NF diet or the same diet supplemented with
monomeric ribonucleotides (NS diet; 4.74 g nucleotides/kg diet)
(Table 1
). The concentration of ribonucleotides and energy density of the NS
diet were similar to a typical nonpurified Ralston Purina rodent diet
(# 5002 Mouse Chow, Purina, Richmond, IN). The daily dose of
ribonucleotides [2820 µmol/(kg · d)] in the NS diet
group was therefore similar to that in mice fed a nonpurified rodent
diet.
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Experimental design |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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BALB/cJ female mice were fed a nonpurified rodent diet (Purina) for 2–3 d after being delivered to the animal care facility. Then mice consumed either the NF or NS diet ad libitum and were weighed weekly throughout the experiment. OVA (Sigma, St. Louis, MO; 10 µg/mouse) was given at 3 and 6 wk after the mice began consuming the NF or NS diet. OVA was suspended into one part PBS and 1 part IFA (0.2 mL/dose, Sigma) and injected subcutaneously into the right posterior footpad. The OVA dose (10 µg/dose) was chosen on the basis of preliminary data revealing that dietary nucleotides altered anti-OVA IgG subclass levels at this dosage level but not with a high dose of OVA (100 µg/dose). Anti-OVA IgG Ab levels were not detectable when mice were immunized with a 1 µg dose of OVA (n = 6). Anti-OVA IgG and IgE Ab levels were not detectable in unprimed mice or those challenged with keyhole limpet hemocyanin (KLH) plus IFA.
Serum samples and regional draining LN cells.
Blood samples (1–1.2 mL/mouse) were collected 3 wk after the first OVA
challenge, and 5 and 14 d after the second OVA challenge.
Popliteal LN (regional draining LN) were dissected and cells were
suspended in medium before being passaged through a coarse filter as
previously described (Jyonouchi et al. 1996
). We
measured mRNA expression and protein production of interferon-
(IFN-) and interleukin-5 (IL-5) by LN cells at 5, 10 and 21 d
after the first OVA challenge, then 5 and 14 d after the second
OVA challenge. For in vitro IFN- and IL-5 production, LN cells were
cultured with OVA (100 mg/L) for 4 d; IL-5 and IFN- production
by OVA-primed regional draining LN cells was maximum when cells
were cultured for 4 d in preliminary experiments (data not shown).
We also determined intracellular IFN- and IL-5 expression by LN
cells 5 d after the first and second OVA challenges. IL-5 and
IFN- production was not detectable when LN cells from unprimed mice
or mock-immunized mice were stimulated by OVA in vitro.
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Analytical methods |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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Anti-OVA IgG subclass Ab levels were detected by ELISA. A 96
well-ELISA plate (Nunc, Naperville, IL) was coated with OVA (2.5
mg/L in 0.1 mol/L NaHCO3, pH 9.6) overnight at 4°C. The
plate was then treated with a blocking buffer (PBS with 100 mL/L fetal
calf serum) for 1 h at 37°C, and incubated with diluted serum
samples (Jyonouchi et al. 2000
). The remaining reactions
were performed as previously described (Jyonouchi et al. 1995
), using alkaline phosphatase-conjugated anti-murine
IgG1, IgG2a and IgG2b Ab as the second Ab (PharMingen, San Diego, CA).
For detecting total IgE, unconjugated and biotinylated anti-murine
IgE Ab (PharMingen) were used as the first and second Ab, respectively.
For detection of anti-OVA IgE Ab, the plates were coated with OVA
(0.1 mg/L) in 0.1 mol/L NaHCO3, pH 9.6, incubated with a
blocking buffer at room temperature for 1 h and then incubated
with diluted serum samples. Color was developed by incubating the plate
with biotinylated anti-mouse IgE Ab for 2–3 h and
streptavidin-horseradish peroxidase (PharMingen) for 30 min,
followed by the addition of the substrate buffer
[3-ethylbenzthiazoline-6-sulfonic acid (300 mg/L; Sigma) in 0.1 mol/L
citric acid, pH 4.35, with 0.3 ml/L H2O2].
Color development was stopped by adding sodium dodecyl sulfate (200
g/L) solution with 500 mL/L N,N-dimethyl
formamide. Pooled sera from untreated and KLH-immunized mice were
used as a negative control in each experiment. Quantification of serum
Ab levels was performed using a series of sequentially diluted serum
samples and comparing the data with the standard curves. The standard
curves were generated using unconjugated anti-murine IgG1, IgG2a,
IgG2b and IgE monoclonal Ab as the first Ab (PharMingen) and purified
murine IgG1, IgG2a, IgG2b and IgE (PharMingen) as standards. The limit
of detection for anti-OVA IgG subclass and IgE Ab ELISA was 3.7
µg/L.
ELISA for IFN- and IL-5.
IL-5 and IFN- levels in culture supernatants were measured by ELISA
as previously described (Jyonouchi et al. 1996
).
Unconjugated anti-murine IL-5, or anti-murine IFN- Ab
(Endogen, Cambridge, MA) and biotinylated anti-murine IL-5, or
anti-murine IFN- (Endogen) were used as the first and second Ab,
respectively. The limit of detection for the cytokine ELISA was 3.7
ng/L.
RNA purification and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) for cytokine mRNA expression.
Total RNA and ssDNA were derived from homogenized LN cells as
described (Jyonouchi et al. 1996
). The PCR protocol used
in this study was described previously (Jyonouchi et al. 1996
). Primers of IL-5, IFN- and glyceraldehyde-3-phosphate
dehydrogenase (G3PDH; internal control) were synthesized on the basis
of published DNA sequences (Microchemical Facilities, Genetics
Institute, University of Minnesota). IFN- mRNA was measured by
competitive RT-PCR using cDNA mimics for IFN- and G3PDH
(Clonetech, Palo Alto, CA) (Siebert and Larrick 1993
).
The results were expressed per 106 G3PDH mRNA
transcript. IL-5 mRNA expression was assessed by
semiquantitative RT-PCR (Jyonouchi et al. 1996
).
Intracellular cytokine staining.
LN cells (2–4 x 109 cells/L) were incubated in a
24-well tissue culture plate coated with hamster anti-murine CD3 Ab
(25 mg/L) (Jyonouchi and Sun 1997
) for 6 h in the
presence of monensin (2 µmol/L, Sigma) and hamster
solubilized anti-murine CD28 Ab (10 mg/L) (Jyonouchi and Sun 1997
). Then cells were stained with fluorescein-conjugated
anti-murine CD4 Ab for 30 min at 4°C, washed, fixed and permeabilized
in Cytofix/Cytoperm solution (PharMingen) for 20 min at 4°C. The
cells were then washed with Perm/Wash solution and stained with
phycoerythrin-conjugated anti-murine IFN or anti-murine IL-5
Ab in Perm/Wash solution (PharMingen) for 30 min at 4°C. Finally,
cells were washed, and analyzed by flow cytometry (FACSCalibur 501,
Software: Cell Quest, Becton Dickinson, San Diego, CA). Results
were expressed as x 104 cells in the LN by
multiplying the total number of popliteal LN cells by the percentage of
IL-5+ or IFN-+ cells.
Statistics.
For comparison between the NF and NS diet group values, equality of
means was evaluated by the Mann-Whitney test (Norusis 1993
). Comparisons of multiple values were assessed by
one-way ANOVA and by the Duncan or Kruskal-Wallis tests
(Norusis 1993
). Differences with P
< 0.05 were considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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),
the diet used did not affect weight gain or spleen weight (data not
shown).
Anti-OVA IgG and IgE Ab levels were not detectable in sera obtained
from mice before and 10 d after the primary OVA challenge.
Consistent with the skewed T2 responses characteristic of BALB/cJ mice
(Abbas et al. 1997
, Forsthuber et al. 1996
), anti-OVA IgG1 Ab levels were higher than
anti-OVA IgG2a and IgG2b Ab levels 3 wk after the first OVA
challenge (Table 2
). Dietary nucleotides augmented primary anti-OVA IgG2a Ab levels
(P < 0.05; Table 2
). Anti-OVA IgG subclass levels
were higher 5 and 14 d after the second challenge, indicating a
good secondary response to OVA (Table 2)
. Dietary nucleotides resulted
in lower anti-OVA IgG1 at 5 and 14 d and higher anti-OVA
IgG2a and IgG2b Ab levels at 14 d after the second OVA challenge
compared with controls fed the NF diet (Table 2)
. As a result, the
ratio of anti-OVA IgG2a Ab to anti-OVA IgG1 Ab was greater in
the NS diet group at 5 and 14 d after the second OVA challenge
(Table 2)
. Total IgE levels were not affected by dietary nucleotides
after the first and second OVA challenges (Fig. 1
). In contrast, anti-OVA IgE levels were lower in the NS diet group
than in those fed NF after the second OVA challenge (Fig. 1)
.
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/IL-5 production was undetectable (<3.7 ng/L) in
mice not primed with OVA. After OVA challenges, detectable levels of
IFN-/IL-5 were produced when regional draining LN cells were
restimulated by OVA in vitro. Without OVA, these cells produced little
IFN- or IL-5. OVA-specific IFN- production was higher 5 d after the primary and secondary OVA challenges than that at other
time points tested in the NS diet group (Kruskal-Wallis test,
P < 0.05) (Fig. 2A
). In the NF diet group, IFN- levels produced with OVA were lower
14 d after the secondary OVA challenge than those measured at
other time points (Kruskal-Wallis test, P < 0.05)
(Fig. 2A
). OVA-specific IL-5 production was lower
21 d after the primary OVA challenge and 5 d after the
secondary OVA challenge than that at other time points (Fig. 2B
). Dietary nucleotides augmented in vitro IFN-
production by regional draining LN cells harvested 5 d after the
first OVA challenge and 5 and 14 d after the second OVA challenge
(Fig. 2A
). Dietary nucleotides did not alter IL-5 production
(Fig. 2B
).
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and IL-5 mRNA expression decreased from 5 to 21 d
after the first OVA challenge, then increased to the d 5 levels after
the second OVA challenge. Dietary nucleotides did not alter IFN- or
IL-5 mRNA expression in response to the first OVA challenge. However,
5 d after the secondary OVA challenge, IFN mRNA expression was
augmented by dietary nucleotides (15,350 ± 4200 vs. 8171 ± 2227/106 G3PDH transcripts, P < 0.05)
The number of cells expressing intracellular IFN- was not altered by
dietary nucleotides 5 d after the first OVA challenge. However,
dietary nucleotides increased the number of cells expressing
intracellular IFN- 5 d after the second OVA challenge (2.29
± 0.46 vs. 1.62 ± 0.56 x 104
cells, P < 0.05). Dietary nucleotides did not alter
the number of cells expressing intracellular IL-5 after either OVA
challenge. The number of cells expressing intracellular IFN- or IL-5
was not altered by dietary nucleotides when mice were given IFA
(adjuvant) without OVA (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSExperimental designAnalytical methodsRESULTSDISCUSSIONREFERENCES |
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and 1997b
), healthy term
infants (Pickering et al. 1998
) and rodents
(Jyonouchi 1994
, Van Buren and Rudolph 1997
). Augmented antibody responses could be due to increased
IgG1, Ig2a and IgG2b, or all three simultaneously. In rodents,
antigen-specific IgG2a and IgG2b antibodies are induced by Th1
cells and Th1 cytokines, whereas IgE and IgG1 antibody production and
eosinophil-dominant inflammatory responses are induced by Th2 cells
and cytokines (Abbas et al. 1997
, Adkins and Du 1998
, Forsthuber et al. 1996
). Thus, the effect
of dietary nucleotides on Th1/Th2 balance and the potential for a
ribonucleotide-mediated modulation of Th2 dominant responses have
become important questions. We reported that dietary ribonucleotides
augmented Th1 but not Th2 responses in B6 mice (Jyonouchi et al. 2000
). However, B6 mice are a Th1-prone strain and may not
reflect the effect of dietary ribonucleotides in a Th2-prone system
such as human infants. BALB/c J mice challenged with OVA plus IFA
generate potent Th2 responses, resulting in high levels of anti-OVA
IgG1 and IgE Ab (Abbas et al. 1997
, Adkins and Du 1998
, Yip et al. 1998
) and have been used as a
murine model of atopic disorders. This characteristic of BALB/cJ mice
provided a critical test of the ability of dietary nucleotides to
modify Th1/Th2 balance toward Th2 responses in a Th2-predominant immune
system.
Our data revealed lower anti-OVA IgG1/IgE Ab levels and higher
anti-OVA IgG2a and IgG2b Ab levels in BALB/cJ mice fed the NS diet
compared with those fed the NF diet. These changes were accompanied by
augmented OVA-specific IFN- production by regional draining LN
cells, suggesting that dietary nucleotides may have shifted skewed T2
responses against OVA toward Th1 by augmenting Ag-specific IFN-
production. These results are consistent with and extend our previous
findings in B6 mice (Jyonouchi et al. 2000
). In both
studies, dietary nucleotides increased Th1 Ab responses measured as
antigen-specific IgG2a and IgG2b Ab levels after secondary antigen
challenge. The current data extended our previous report by revealing
decreased Th2 responses as measured by lower anti-OVA IgE and IgG1
Ab levels. Thus, dietary nucleotides augment Th1 but not Th2 responses
in mice strains with Ab responses strongly skewed toward Th1 and Th2.
These observations are consistent with those of Nagafuchi et al. (1997),
indicating a decrease in total IgE in mouse pups born
to dams fed a NS diet. The implications of these observations for
allergy-prone and therefore Th2-prone infants (Doria and Frasca 1997
, Romagnani 1997
and 2000
) remain
speculative.
The effects of dietary nucleotides on Ab responses were observed when
mice were challenged with submaximal Ag doses plus a Th2-polarizing
adjuvant (IFA). No nucleotide action was observed when mice were
challenged with a maximal dose of OVA or with a Th1-polarizing adjuvant
such as a complete Freund’s adjuvant (Jyonouchi et al. 2000
, unpublished observations). Multiple factors, including Ag
dose, are often manipulated in animal models in order to yield the
maximal antibody responses (Mondino et al. 1996
,
Yip et al. 1998
). However, natural antigen exposure
normally occurs at lower Ag doses and leads to effective immune
responses. The experimental system used in this study is closer to a
natural immune response.
Dietary nucleotides likely increased cytokine synthesis in
OVA-specific Th1 cells after the primary Ag challenge. This was
indicated by the increase in OVA-specific IFN- production by
regional draining LN cells after the primary OVA challenge. However, a
booster OVA dose was required for an increase in total IFN- mRNA or
the total number of intracellular IFN-+ cells
to be detected in the LN. These results may indicate that repeated OVA
doses caused sufficient clonal expansion of OVA-specific T1 cells
to change total IFN- expression in the LN. The absence of a
nucleotide-mediated increase in IL-5 production is consistent with
previous findings in B6 mice challenged with KLH (Jyonouchi et al. 2000
), in vitro studies using Th cell clones
(Jyonouchi et al. 1997
) and with OVA-specific T-cell
receptor transgenic mice (Nagafuchi et al. 2000
). The
preferential increase in IFN- production may have resulted in
increased Th1 Ab responses and down-regulated Th2 Ab responses.
Alternatively, decreased production of other Th2 cytokines that we did
not measure could explain this shift in Th1/Th2 balance.
It has been suggested that development of atopic disorders in high risk
infants is attenuated or delayed in breast-fed compared with
formula-fed infants (Chandra 1997
, Isolauri et al. 1999
). Ribonucleotide is one of the components not
contained in standard cow’s milk–based infant formula at human milk
concentrations (Leach et al. 1995
, Thorell et al. 1996
). In that context, the shift toward Th1 in a Th2-prone
(atopic) mouse strain mediated by dietary ribonucleotides is
intriguing. We speculate that our results may support a potential
preventive or therapeutic effect of dietary ribonucleotides in atopic
individuals early in life. In contrast, other researchers have shown
that dietary nucleotides augment toluene diisocyanate–induced rhinitis
and eosinophilic colitis induced by trinitrobenzene sulfonic acid in
rodents (Adjei et al. 1996
, Almansouri et al. 1996
). Because eosinophilic inflammation is one of the
characteristic features of atopic disorders, their results may indicate
the potential for dietary nucleotides to augment allergic inflammation.
However, these studies utilized inflammatory chemicals and provided no
measurement of Th1 and Th2 Ab levels or cytokine balance.
CpG oligodeoxynucleotides from microbial cells augment Th1 responses at
the site of Ag challenge (Davis et al. 1998
,
Krieg 2000
, Van Uden and Raz 1999
).
However, the ribonucleotides used in this study were purified from
yeast RNA containing no measurable DNA. It is unlikely that the effects
we observed were due to the presence of CpG DNA motifs.
It is not clear which components of dietary ribonucleotides or their
metabolites affect Th1/Th2 balance. Dietary purine and pyrimidine
bases, ribonucleosides and ribonucleotides are rapidly absorbed,
extensively catabolized in the gut mucosa and readily excreted
(Boza et al. 1996
, Clifford and Story 1976
, Ho et al. 1979
, Sonada and Tatibana 1978
). Data from infants also indicate extensive catabolism and
excretion of purines as uric acid (Kuchan et al. 2000
).
In rodents, incorporation into peripheral tissues is limited to 2–5%
of an oral dose, with pyrimidines incorporated to a greater extent than
purines (Burridge et al. 1976
, Ho et al. 1979
). Uracil and adenine are the predominant pyrimidines and
purines incorporated into tissue pools (Clifford and Story 1976
, Sonada and Tatibana 1978
). Surprisingly,
addition of uracil, but not adenine, to a NF diet in mice recovered
immune responsiveness measured by a variety of methods (Van Buren et al. 1994
). Thus, the individual ribonucleotides used
in this study may have differential effects on Th1/Th2 balance, and
their effect may not be dependent on incorporation into tissue pools.
To date, the effect of individual ribonucleotides on Th1/Th2 balance
has not been reported.
In summary, this study demonstrated that dietary nucleotides augmented
Th1 responses as measured by anti-OVA IgG subclass and IgE Ab
levels in BALB/cJ mice. These results indicate that genetic
predisposition to Th2 (atopic predisposition) can be modulated by
dietary components in specific strains of mice. Our results also
suggest that the mechanisms of this ribonucleotide action are
associated with preferentially augmented Ag-specific production of
IFN-, a T1 cytokine.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Supported in part by a grant from Ross Products Division/Abbott Laboratories, Columbus, OH.
4 Abbreviations used: Ab, antibody; Ag, antigen; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; IFA, incomplete
Freund’s adjuvant; IFN-, inter-feron-; Ig, immunoglobulin; IL-5, interleukin-5; KLH, keyhole limpet hemocyanin; LN, lymph node; NF, nucleotide-free; NS, nucleotide-supplemented; OVA, ovalbumin; RT-PCR, reverse transcriptase-polymerase chain reaction; Th cells, T-helper cells; Th1 and Th2, type 1 and type 2 Th cell.
Manuscript received August 28, 2000. Initial review completed October 5, 2000. Revision accepted January 16, 2001.
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higher (P < 0.05) than other time points for the N S diet group (Kruskal-Wallis
test); 
lower (P < 0.05) than other
time points for the NF group (Kruskal-Wallis test). Panel
B, §lower (P < 0.05) in both
groups compared with the other time points tested.