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2
Division of Nutritional Sciences,
* Department of Animal Sciences and
** Department of Veterinary Pathobiology, University of Illinois, Urbana, IL 61801 and
Purina Mills, Inc., Gray Summit, MO 63039
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • small intestine • weaning • inflammation • pigs
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kelly et al. 1991a and 1991b
,
Miller et al. 1984a
). The role of specific inflammatory
cell populations in villus atrophy in weanling piglets has not been
determined. Furthermore, only recently has the impact of feed intake on
intestinal morphology been fully appreciated (Kelly et al. 1991a and 1991b
,
McCracken et al. 1995
, Pluske et al. 1996a
).
Weaning-associated intestinal inflammation has been described for
numerous species. For example, activation of the mucosal immune system,
villus atrophy and crypt hyperplasia has been reported for the weanling
rat (Cummins et al. 1988a, 1988b and 1991
).
Also, intestinal histopathological lesions at weaning often resemble
those observed for gastrointestinal diseases. Immune cells,
particularly T lymphocytes, contribute to the pathogenesis of
gastrointestinal diseases such as Crohn's disease, celiac disease, and
intractable diarrhea of infancy (Choy et al. 1990
,
Cuenod et al. 1990
, Lionetti et al. 1993
,
MacDonald 1990
). In addition, activated T cells may
contribute to a reduction in villus height in human intestinal explants
maintained in vitro and in neoplastic human intestine (MacDonald and Spencer 1988 and 1990
). Activation of T lymphocytes in the lamina propria of
intestinal explants was associated with activation of matrix
metalloproteinases
(MMP),3
which are normally involved in turnover of the extracellular matrix
(ECM; Pender et al. 1997
). Because a full understanding
of the cellular mechanisms responsible for weaning-associated changes
in intestinal morphology has yet to be developed, and T cells are
implicated as important contributors of compromising changes to the
intestinal epithelium, perhaps by affecting ECM turnover, this study
evaluated the effect of weaning on local T cell numbers and population
phenotypes in the piglet intestine.
We demonstrated recently that weaning in pigs is associated with
diet-dependent and diet-independent metabolic responses that may
contribute to the characteristic postweaning growth stasis
(McCracken et al. 1995
). From that study, it became
apparent that feed intake was an important consideration in light of
the diet-independent metabolic effects observed. Diets used in that
study were a liquid milk replacer and a pelleted soy-based starter
diet. Therefore, diet form was standardized in this study to facilitate
more equal feed consumption. Additional objectives were to evaluate
small intestinal inflammatory responses and local morphological
alterations by examining T cell numbers, tissue prostaglandin
E2 (PGE2) concentrations and local expression
of major histocompatibility complex (MHC) genes and the MMP
stromelysin. Stromelysin was evaluated because this MMP is responsive
to inflammatory stimuli and increased stromelysin activity results in
the breakdown of the lamina propria ECM as well as laminin and type IV
collagen in the epithelial basement membrane of the intestine
(Pender et al. 1997
).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Crossbred pigs (n = 112, average age = 21 d)
from 19 litters were used to evaluate intestinal inflammatory responses
to weaning. Pigs were either killed at weaning (n = 12)
or weaned to a pelleted milk- or soy-based diet (Table 1
)and killed at 0.5, 1, 2, 4, or 7 d postweaning (n = 10 for each d/diet combination). Pigs were housed individually with
free access to feed, consumption of which was measured daily. Animals
were supplied by Purina Mills (Gray Summit, MO), and published
guidelines for the care and use of animals were followed
(Consortium for Developing a Guide for the Care and Use of
Agricultural Animals in Agricultural Research and Teaching
1988
).
|
Immediately after death by electrical stunning and exsanguination, the
intestinal tract was removed, freed from mesentery and arranged on a
table to allow tissue sampling from the proximal jejunum as described
(McCracken et al. 1995
). Briefly, the intestine was
arranged on a table into six segments of equal length. The ends and
each curve were given a number 1–7 beginning with the proximal
duodenum. Samples of proximal jejunum were taken from the site numbered
3. Tissue samples were flushed immediately with sterile water to remove
luminal contents and were either frozen in liquid nitrogen and stored
at -80°C for later analyses or were fixed in Bouin's solution,
embedded in paraffin and sectioned onto glass slides for
immunocytochemistry. Approximate time from exsanguination to tissue
freezing was 10–15 min.
RNA isolation and Northern blot analysis.
Total RNA was isolated from samples of jejunum as described by
Sprenger et al. (1995)
. Total RNA concentrations were
determined by spectrophotometry and 10 µg was loaded onto
formaldehyde-containing agarose gels for electrophoretic separation.
RNA was then transferred to nylon membranes (Micron Separations,
Westborough, MA) using standard Northern blotting techniques
(Sambrook et al. 1989
). Membranes were probed with
32P-labeled cDNA probes specific for porcine major
histocompatibility complex class I and II (kindly provided by L. B. Schook, University of Minnesota). To verify equivalent RNA loading
in the agarose gels, blots were probed with an
32P-labeled cDNA probe specific for 28S rRNA (L. B.
Schook, University of Minnesota). Bands were visualized by X-ray
autoradiography, and densiometric analysis of autoradiographic images
was performed.
Tissue prostaglandin E2 concentrations.
Tissue PGE2 concentrations were measured in a subset of animals (4 animals per day/diet combination) with an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI) following the manufacturer's protocol. Approximately 1 g of each tissue sample was weighed and homogenized on ice in 2 mL methanol and centrifuged at 3000 x g for 15 min at 4°C. The supernatant was combined with 8 mL of 0.1 mol potassium phosphate buffer/L (pH 4), mixed, loaded onto 5 mL capacity C-18 reverse phase cartridges, and washed sequentially with 5 mL water and 5 mL hexane. PGE2 was eluted with 1 mL ethyl acetate/1% methanol. The eluant was dried in a SpeedVac (Savant Instruments, Farmingdale, NY) and resuspended in 1 mL buffer (provided in the kit). Enzyme immunoassay plates were read on a Thermomax microplate reader (Molecular Devices, Menlo Park, CA). Concentrations of PGE2 were determined against a standard curve. Total protein concentrations were measured by the BioRad DC protein assay (BioRad Laboratories, Hercules, CA), and PGE2 concentrations are expressed as pg PGE2/mg tissue protein.
Histology, immunocytochemistry, and immunofluorescence.
Villus height and crypt depth were measured on jejunal sections stained
for CD8 using a Nikon Diaphote microscope and Image-1 software
(Universal Imaging, West Chester, PA). Goblet cells were counted in 10
villi and 10 crypts on four jejunal sections per day/diet combination
stained with the PAS/alcian blue pH 2.5 procedure (Carson 1990
).
To analyze CD8+ T cell populations by immunocytochemistry,
Bouin's-fixed samples were soaked in four changes of 70% ethanol to
remove excess picric acid, then embedded in paraffin and sectioned onto
glass slides. A biotinylated mouse anti-pig CD8 antibody (F-O-10, 134
clone 76-211; Saalmüller 1996
) was used for
immunocytochemistry. Slides were deparaffinized in xylene then
rehydrated through a series of alcohol, water, citric acid and Tris
buffer washes (Carson 1990
). Jejunum sections were
blocked with 20% mouse serum, then incubated with biotinylated
anti-pig CD8 antibody for 2 h at 37°C followed by
streptavidin/alkaline phosphatase incubation for 30 min at 37°C.
Jejunal CD8+ cells were visualized using Histomark Red
(Kirkegaard & Perry Laboratories, Gaithersburg, MD) following the
manufacturer's protocol. Jejunal CD8+ cells were counted
in five villi and five crypts per section of four animals per day/diet
combination using an Olympus BH-2 microscope (Olympus, Lake Success,
NY) at 400 x magnification. Numbers are expressed as the number
of CD8+ cells per 100 µm villus or crypt.
CD4+ T cell populations were measured by
immunofluorescence. Tissues were embedded, sectioned onto glass slides
and processed as for CD8 staining. Sections were incubated with a 1:200
dilution of an anti-pig CD4 antibody (clone 74-12-4;
Saalmüller 1996
) for 30 min at room temperature in
the dark. Slides were counterstained with hematoxylin, dehydrated and
mounted with Crystal Mount (Biomedia, Foster City, CA). Jejunal
CD4+ cells were counted in stained jejunum sections using
an Olympus BH-2 fluorescence microscope (Olympus) at 1000 x
magnification under oil immersion. Fluorescent cells were counted in
the lamina propria or along the epithelial border of five villi and
along five well oriented crypts from four animals per day/diet
combination. Numbers are expressed as number of CD4+ cells
per 100 µm of villus or crypt.
Western blotting.
For protein isolation, whole frozen jejunal samples (100 mg) were homogenized in sample buffer (10% glycerol, 2% sodium dodecyl sulfate (SDS) and 0.625 mol Tris-HCl/L, pH 6.8) containing 0.83% Triton X-100, 0.003 mmol phenylmethylsulfonylfluoride/L, 0.041 g iodoacetic acid/L, 0.17 g EDTA/L and the protease inhibitors aprotinin (5 mg/L), pepstatin A (5 mg/L) and chymostatin (1 mg/L). Protein concentrations were determined using the BioRad DC Protein Assay as described above. Equal amounts of protein were then loaded in SDS-polyacrylamide gel electrophoresis (PAGE) gels, electrophoresed and blotted to nitrocellulose membranes (Micron Separations). An anti-human stromelysin antibody (The Binding Site, San Diego, CA) was used to identify the inactive (60 and 58 kD) and active (44 and 28 kD) forms of stromelysin.
Statistical analysis.
Data were analyzed using the GLM procedure of SAS (SAS 1985
). Litter of origin and replicate were included in the
model. Significance of treatment differences were calculated using the
LSMEANS statement, and results are presented as least-square means ± SEM. Pearson correlation analysis was performed to evaluate a
possible correlation between intake and morphological or histological
variables by day. Differences were considered significant at
P < 0.05; trends were identified at P < 0.1.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Independent of diet, average cumulative intake was <100 g per pig
during the first 2 d postweaning. By the fourth day postweaning,
average cumulative intake was 250 g per pig (Fig. 1
).Over time, intake increased (P < 0.05) for pigs fed
either diet. A day by diet interaction was not observed. However,
examination of least square means indicated that on Day 7, total
cumulative intake for pigs fed the soy-based diet was greater (0.73 kg
per pig; P < 0.05) than pigs fed the milk-based diet
(0.5 kg per pig).
|
Independent of diet, villus height decreased (P <
0.05) over time postweaning (Fig. 2
).A day by diet interaction was not observed. For pigs fed the milk-based
diet, villus heights were 34 and 59% of Day 0 values on Days 2 and 7,
respectively. Similarly, for pigs fed the soy-based diet villus heights
were 37 and 66% of Day 0 values on Days 2 and 7, respectively. Crypt
depth was reduced to 38% (P < 0.05) independent of
diet on Day 2 compared to Day 0, but was similar to Day 0 by 7 d
postweaning. Pearson correlation analysis indicated a trend for a
positive correlation between intake and villus height
(r = 0.42, P = 0.06) and crypt depth
(r = 0.31, P = 0.09) on Day 7.
|
Overall, the number of goblet cells per 100 µm of villus was not different (P > 0.05) over time postweaning or across diet treatment (data not shown). Furthermore, significant interactions between diet and time postweaning were not observed for goblet cell numbers. However, evaluation of least square means indicated that the number of goblet cells tended to increase per 100 µm of crypt from Day 0 (39 cells /100 µm of crypt) to Day 7 for pigs fed both the milk- (146; P = 0.067) and soy-based diet (138; P = 0.074).
T lymphocytes.
CD8+ T cell numbers per 100 µm of villus tended to
increase through the second day postweaning (P < 0.10)
regardless of diet offered (Fig. 3
).A diet by day interaction was not observed; however, examination of
least square means indicated that pigs fed the soy-based diet had more
(P < 0.05) CD8+ T cells on Day 2 compared
to Day 0. The number of CD8+ T cells per 100 µm of crypt
was higher (P < 0.05) on Day 2 independent of diet,
and a day by diet interaction was not observed.
|
).A significant day by diet interaction was observed, however, for crypt
area lamina propria CD4+ cell numbers. Evaluation of least
square means revealed on Day 7 a greater number of
CD4+ cells in the crypt lamina propria of pigs fed the
soy-based diet compared to pigs fed the milk-based diet.
|
Due to individual animal variation, PGE2 concentrations did not differ across time or between diet groups (data not shown). Jejunal PGE2 concentrations tended to be higher (P < 0.1) early in the postweaning period (Day 1) compared to Days 2 and 7. Jejunal PGE2 concentrations ranged from 5 pg/mg tissue protein for pigs killed on Day 0 to 33 pg/mg protein for pigs fed the soy-based diet and killed on Day 1.
MHC class I and II expression.
Northern blot analysis demonstrated that jejunal MHC class I RNA
expression was lower (P < 0.05) on Day 1 postweaning
compared to the constitutive levels of expression on Days 0, 4 and 7.
Densiometric analysis (MHC class I image/28s rRNA image; Fig. 5A
)revealed an average of 0.2 and 0.1 arbitrary units for pigs fed the
milk- or soy-based diets, respectively, compared to an average of 1.23
arbitrary units on Day 0. Average densitometric values for pigs fed the
milk- or soy-based diets on Day 4 were 0.97, 1.03, respectively, and on
Day 7 were 0.60 and 0.85 arbitrary units, respectively.
|
B) resulted in means of 0.13
arbitrary units for pigs fed either the milk- or soy-based diet on Day
1 compared to 0.35 arbitrary units on Day 0, and 0.2 and 0.2 on Day 4,
and 0.1 and 0.05 arbitrary units on Day 7 for pigs fed the milk- and
soy-based diets, respectively. Stromelysin expression.
Western blot analysis of jejunal samples from pigs fed either the milk-
or soy-based diets demonstrates enhanced expression of the 58 and 60
kDa stromelysin isoforms (Fig. 6A
and
B).These isoforms are two of the inactive isoforms of stromelysin
(Woessner 1991
). A weak band at 44 kDa and a band at 28
kDa representing the active forms of stromelysin (Woessner 1991
) were also observed. The active form (28 kDa) appears to
be upregulated in both groups postweaning (Fig. 6
A and
B). Expression of the 58, 60 and 28 kDa isoforms was
enhanced for pigs fed the soy-based diet on Day 1–4 postweaning
compared to pigs fed the milk-based diet (Fig. 6
C).
|
|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Li et al. 1991
, Miller et al. 1984a
, Miller et al. 1994
). Studies to evaluate
that possibility have demonstrated that pigs weaned at 3 wks of age to
a diet containing soy protein display transient increases in serum
anti-soy IgG antibodies followed by systemic tolerance (Bailey et al. 1993
, Miller et al. 1984a
). However,
mechanistic links between serum anti-soy antibodies and compromised
intestinal structure or function have not been considered either
theoretically or empirically in the literature. Serum anti-soy
antibodies were not measured in this study; however, increased crypt
CD4+ T cell numbers by Day 7 in pigs weaned to the
soy-based diet, but not the milk-based diet, indicate that dietary soy
antigens may cause local inflammatory responses and subsequent immune
responses in weanling pigs. In light of the temporal response pattern
observed, however, it must be concluded that soy-induced inflammation,
if present, is likely secondary to compromised intestinal morphology
due to local inflammation caused by anorexia during the immediate
postweaning period.
A second hypothesis is that lack of luminal stimulation from reduced
intake during the first few days postweaning is responsible for early
alterations in intestinal morphology (Cera et al. 1988
,
Kelly et al. 1991a and 1991b
, McCracken et al. 1995
, Pluske et al. 1996a
and 1996a). A compromised mucosal barrier caused by acute
fasting may allow passage of luminal antigens into the lamina propria
where an inflammatory response can be initiated. Clear evidence that
absence of food in the intestine leads to adverse morphological changes
and even atrophy of the mucosa (Hughes and Dowling 1980
,
McManus and Isselbacher 1970
, Steiner et al. 1968
) indicates the importance of considering the effects of
feed intake on intestinal structure and function in weanling pigs. Our
data demonstrate that inflammatory responses and villus atrophy
correlate with depressed feed consumption and that intestinal
inflammation subsides and epithelial morphology improves when normal
feed intake patterns resume. This observation supports the conclusion
from our earlier study that pigs offered a pelleted soy-based diet
compared to pigs fed a milk-based liquid diet exhibited compromised
jejunal morphology due to a difference in feed intake and not due to
differences in diet composition (McCracken et al. 1995
).
This study also demonstrates for the first time that upregulation of
MMP by activated immune cells in the lamina propria may contribute to
villus atrophy at weaning. We suggest that aberrant upregulation of
stromelysin and other MMP in response to weaning anorexia may increase
ECM breakdown, resulting in compromising changes to the intestinal
epithelium, including a reduction of villus height. Similarly, inflamed
intestinal tissue from patients suffering from inflammatory bowel
diseases displayed enhanced stromelysin expression (Bailey et al. 1994
). Collagenase, stromelysin and matrilysin were also
up-regulated in the colon of ulcerative colitis patients
(Saarialho-Kere et al. 1996
). Because intestinal
epithelial cells express receptors for basement membrane components
that are responsible for cellular attachment to the basal lamina
(Alho and Underhill 1989
) and for migration of
epithelial cells to villi tips (Beaulieu 1992
),
breakdown of the basal lamina may weaken epithelial attachments and
reduce cell migration rates, resulting in a compromised epithelial
layer. Further studies to characterize interactions between intestinal
T cells and ECM in weanling pigs are needed to fully define the
etiology of the observed structural changes. In addition, intestinal
expression of tissue inhibitors of MMP should be evaluated during
weaning anorexia, as these molecules act in concert with MMP to
regulate ECM turnover; a decrease in their expression may be reflected
by the enhanced stromelysin protein expression demonstrated herein.
Generally, MHC class I expression is upregulated during inflammatory
responses (Abbas et al. 1991
), yet we observed a
decrease in MHC class I expression immediately postweaning, possibly
caused by weaning-associated stress. Increased plasma cortisol
concentrations, which inhibit activity of the AP-1 family of
transcription factors (Ponta et al. 1992
), have been
observed postweaning in pigs (Worsae and Schmidt 1980
).
MHC class I genes are among those induced by the AP-1 family
(Yamit-Henzi et al. 1994
). Therefore, stress-associated
changes in plasma cortisol levels may have indirectly influenced MHC
class I RNA expression by inhibiting AP-1-mediated activation of MHC
class I gene expression. Impaired immune responsiveness through
reduction of intestinal MHC class I expression may predispose pigs to
enteric infections, particularly viral infections, as MHC class I
surface expression is required for presentation of viral peptides to
inflammatory T cells (Abbas et al. 1991
). Indeed,
newly-weaned pigs are particularly susceptible to enteric infections
resulting in diarrhea and decreased growth (Blecha et al. 1983
, Cera et al. 1988
, Kenworthy 1976
, Miller et al. 1984b and 1994
, Van Beers-Schreurs et al. 1992
).
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FOOTNOTES |
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1 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
3 Abbreviations used: ECM, extracellular matrix;
MHC, major histocompatibility complex; MMP, matrix metalloproteinase;
PAGE, polyacrylamide gel electrophoresis; PGE2,
prostaglandin E2; SDS, sodium dodecyl sulfate.
Manuscript received May 28, 1998. Initial review completed July 2, 1998. Revision accepted November 19, 1998.
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