Zinc Deficiency Induces Membrane Barrier Damage and Increases Neutrophil Transmigration in Caco-2 Cells

Article (PDF Available)inJournal of Nutrition 138(9):1664-70 · October 2008with 207 Reads 
How we measure 'reads'
A 'read' is counted each time someone views a publication summary (such as the title, abstract, and list of authors), clicks on a figure, or views or downloads the full-text. Learn more
DOI: 10.1093/jn/138.9.1664 · Source: PubMed
Cite this publication
Abstract
Zinc may contribute to the host defense by maintaining the membrane barrier. In this study, we questioned whether zinc deficiency affects the membrane function and junctional structure of intestinal epithelial cells, causing increased neutrophil migration. We used the Caco-2 cell line grown in control (C), zinc-deficient, or zinc-replete medium until differentiation. Zinc deprivation induced a decrease of transepithelial electrical resistance and alterations to tight and adherens junctions, with delocalization of zonula occludens (ZO-1), occludin, beta-catenin, and E-cadherin. Disorganization of F-actin and beta-tubulin was also found in zinc deficiency. These changes were associated with a loss of the amounts of ZO-1, occluding, and beta-tubulin. In addition, zinc deficiency caused a dephosphorylation of occludin and hyperphosphorylation of beta-catenin and ZO-1. Disruption of membrane barrier integrity led to increased migration of neutrophils. In addition, zinc deficiency induced an increase in the secretion of interleukin-8, epithelial neutrophil activating peptide-78, and growth-regulated oncogene-alpha, alterations that were not found when culture medium was replete with zinc. These results provide new information on the critical role played by dietary zinc in the maintenance of membrane barrier integrity and in controlling inflammatory cell infiltration.
Advertisement
The Journal of Nutrition
Nutrition and Disease
Zinc Deficiency Induces Membrane Barrier
Damage and Increases Neutrophil
Transmigration in Caco-2 Cells
1,2
Alberto Finamore,
3
Mara Massimi,
4
Laura Conti Devirgiliis,
4
and Elena Mengheri
3
*
3
Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, 00178 Rome, Italy and
4
Dipartimento Biologia di Base ed Applicata,
Universita
`de L’Aquila, 67100 Italy
Abstract
Zinc may contribute to the host defense by maintaining the membrane barrier. In this study, we questioned whether zinc
deficiency affects the membrane function and junctional structure of intestinal epithelial cells, causing increased neutrophil
migration. We used the Caco-2 cell line grown in control (C), zinc-deficient, or zinc-replete medium until differentiation. Zinc
deprivation induced a decrease of transepithelial electrical resistance and alterations to tight and adherens junctions, with
delocalization of zonula occludens (ZO-1), occludin, b-catenin, and E-cadherin. Disorganization of F-actin and b-tubulin was also
found in zinc deficiency. These changes were associated with a loss of the amounts of ZO-1, occluding, and b-tubulin. In addition,
zinc deficiency caused a dephosphorylation of occludinand hyperphosphorylation of b-catenin and ZO-1. Disrup tion of membrane
barrier integrity led to increased migration of neutrophils. In addition, zinc deficiency induced an increase in the secretion of
interleukin-8, epithelial ne utrophil activating peptide-78, and growth-regulated oncogene-a, alterations that were not found when
culture medium was replete with zinc. These results provide new information on the critical role played by dietary zinc in the
maintenance of membrane barrier integrity and in controlling inflammatory cell infiltration. J. Nutr. 138: 1664–1670, 2008.
Introduction
Zinc, a trace element present in all body tissues and fluids, is
essential for the survival and function of cells. Its importance is
emphasized in conditions of zinc deficiency, when the gut, brain,
and immune system may be impaired, depending on the severity of
the deficiency (1,2). Zinc depletion may contribute to the severity of
infectious diseases and mortality in malnourished children and zinc
supplementation has been shown to reduce the severity of diarrhea
and the incidence of infections (3). Zinc deficiency is estimated not
only to be present in developing countries but also to occur
frequently throughout the world as mild to moderate deficiency in
the elderly, in subjects suffering from cancer or sickle cell anemia, in
patients during the acute-phase response to infection and inflam-
mation, and in some chronic diseases such as asthma and diabetes
(4–6). In addition, low zinc concentrations have frequently been
associated with inflammatory bowel disease (IBD)
5
and in Helico-
bacter pylori-induced gastric mucosa inflammation (7,8).
Zinc may contribute to the host defense by maintaining the
structure and function of the membrane barrier (7,9–11) and
this is particularly important in the intestine, which is contin-
uously exposed to a myriad of pathogens and noxious agents.
The manner in which the intestinal epithelium constitutes a
barrier involves intercellular junctional complexes between
neighboring cells that provide a continuous seal around the
apical region of the cells (12,13). These complexes are composed
of several units, including the tight junctions (TJ) and adherens
junctions (AJ) that form circumferential zones of contact between
adjacent cells. Zonula occludens (ZO) proteins are the major TJ
plaque proteins that bind to the transmembrane protein occludin
and these interactions are crucial for maintaining TJ structure
(14–16). E-cadherin is the main transmembrane adhesion mole-
cule localized at the AJ and its binding to b-catenin is funda-
mental for appropriate AJ organization (17,18). Bundles of actin
form a ring in the apical zone of the cell and link with the TJ and
AJ (19). Previous studies have shown that zinc deficiency alters
the membrane barrier permeability of endothelial and lung
epithelial cells (9) and causes ulcerations of the small intestine
(20,21). A recent study has shown that zinc depletion in com-
bination with proinflammatory cytokines enhances degradation
of E-cadherin and b-catenin proteins of lung epithelial cells (9).
However, it isnot clear whether zinc is crucial for the preservation
of junctional complexes.
Other than constituting the epithelium barrier, TJ may
regulate the passage of polymorphomononuclear cells (PMN),
consisting essentially of neutrophils, which are immune cells for
the protection against pathogen infection. These cells are
1
Supported by a European Community grant, project Feed for Pig Health,
contract number FOOD-CT-2004-506144. The authors are solely responsible for
this publication, and the manuscript does not represent the opinion of the
European Community, which is not responsible for information delivered.
2
Author disclosures: A. Finamore, M. Massimi, L. Conti Devirgiliis, and
E. Mengheri, no conflicts of interest.
* To whom correspondence should be addressed. E-mail: mengheri@inran.it.
5
Abbreviations used: AJ, adherent junction; C, control; ENA-78, epithelial
neutrophil activating peptide-78; FBS, fetal bovine serum; FITC, fluorescein
isothiocyanate; fMLP, N-formyl-methionyl-leucyl-phenyl-alanine peptide; GRO-a,
growth-regulated oncogene-a; IBD, inflammatory bowel disease; IL, interleukin;
PMN, polymorphomononuclear cells; RIPA, cold radio-immune precipitation
buffer; TEER, transepithelial electrical resistance; TJ, tight junctions; ZD,
zinc-deficient cells; ZD-R, zinc-replete cells; ZO, Zonula occludens.
1664 0022-3166/08 $8.00 ª2008 American Society for Nutrition.
Manuscript received 21 March 2008. Initial review completed 18 June 2008. Revision accepted 2 July 2008.
by guest on May 30, 2013jn.nutrition.orgDownloaded from
recruited at sites of injury or inflammation and transmigrate
across mucosal epithelia by a process involving remodeling of TJ
structure and/or localization of its proteins (19,22). Although
the migration of neutrophils represents a first line of defense, a
massive and prolonged infiltration of these cells may perpetuate
inflammation and ultimately lead to cell damage by releasing
mediators such as proteases and cytokines (23,24). Indeed,
clinical studies have shown a neutrophil accumulation within
epithelial crypts and in the intestinal lumen associated with
intestinal disease and epithelial injury (25,26). In previous
studies, we have shown that gut membrane damage caused by
zinc deficiency is associated with inflammatory cell infiltration
(20,21). Interestingly, patients with chronic intestinal perme-
ability disturbances have been shown to have a reduced level of
mucosal zinc (27).
Based on these observations, we hypothesized that zinc
deficiency may affect the TJ structure of intestinal epithelial cells
and consequently allow a more extensive migration of neutro-
phils. By using an in vitro model of intestinal cells, Caco2 cells,
grown in a zinc-deficient (ZD) medium, we found that depletion
of zinc strongly affects membrane barrier function and integrity
and induces an increase in neutrophil transmigration and an
upregulation of chemokines that plays a role in neutrophil
migration and inflammatory development.
Materials and Methods
ZD and zinc-replete culture media. To prepare a ZD serum, fetal
bovine serum (FBS) was stirred with 10% (wt:v) Chelex-100 resin
(Sigma) overnight at 4C, according to Tate et al. (28). An aliquot of the
chelated serum was analyzed for zinc, copper, iron, magnesium, and
calcium content by flame atomic absorption spectrometry. Three
different media were prepared and analyzed for mineral content. The
control (C) medium was the DMEM containing 10% heat-inactivated
FBS, 3.7 g NaHCO3/L, 4 mmol glutamine/L, 10 g/L nonessential amino
acids, 10
5
U/L penicillin, and 100 mg/L streptomycin). The ZD medium
was the C medium in which ZD serum substituted FBS and contained
0.070 60.01 mg/L zinc. The CuCl
2
, FeSO
4,
CaCl
2
, and MgSO
4
were
added to adjust the mineral concentration to that of the C medium (Cu,
0.02 60.004 mg/L; Fe, 0.26 60.025 mg/L; Ca, 64.5 67 mg/L; Mg,
14.1 61 mg/L). The zinc-replete (ZD-R) medium was the ZD medium in
which ZnSO
4
was added to adjust the zinc concentration to that present
in the C medium (0.92 60.21 mg/L). All reagents were from Biochrom.
Epithelial cell culture. The human intestinal Caco-2 cells were
routinely grown in the C medium and maintained at 37Cinan
atmosphere of 5% CO
2
:95% air at 90% relative humidity. In all ex-
periments, Caco-2 cells were seeded (1.5 310
5
cells) on Transwell filters
(polyethylene terephtalate filter inserts for cell culture, 3.0-mm pore
diameter; Becton Dickinson) and grown in C, ZD, or ZD-R medium.
They were named C, ZD, or ZD-R cells, respectively. After confluency,
cells were maintained for 18 d to allow differentiation.
Membrane permeability. Membrane barrier permeability of cells
grown in the C, ZD, or ZD-R medium was tested by measuring the
transepithelial electrical resistance (TEER), according to Ferruzza et al.
(29). TEER was monitored every day until differentiation to test the
effect of the different media using Millicell Electrical Resistance System
(Millipore). TEER was expressed as Ohm (resistance) 3cm
2
(surface
area of the monolayer) after subtracting the filter resistance value.
Neutrophil transmigration. Caco-2 cells were differentiated as
inverted monolayer on Transwell filters to allow the physiological
transmigration of neutrophils from basolateral to apical compartment.
We measured neutrophil transmigration as previously described (30).
Briefly, neutrophils were isolated from whole blood of healthy volunteers
by Ficoll gradient centrifugation, added (1 310
6
cells/well) to the
basolateral compartment (upper reservoir) of the Transwell filters, and
induced to transmigrate by the addition of 1 310
25
mol/L bacterial
peptide N-formyl-methionyl-leucyl-phenyl-alanine (fMLP; Sigma) for
1.5 h. All transmigrated (within the monolayer and apical compartment)
and nontransmigrated (basolateral compartment) neutrophils were
measured by myeloperoxidase activity. All experiments were performed
in HBSS, which eliminates the induction of neutrophil transmigration by
the eventual chemokines secreted in the medium. Experiments on human
volunteers were approved by the National Ethics Committee. Informed
consent was obtained from all participants.
Immunolocalization. Proteins of TJ (ZO-1, occludin), AJ (b-catenin
and E-cadherin), and cytoskeleton (F-actin and b-tubulin) were analyzed
by immunofluorescence analysis as described previously (31) using a
laser scan confocal microscope. Briefly, for junctional proteins, cells were
fixed in ice-cold absolute methanol and incubated for 1 h with rabbit
anti-ZO-1, mouse anti-occludin, mouse anti-b-catenin, or rabbit anti-E-
cadherin antibodies (Zymed Laboratories). For secondary detection, the
cells were incubated with fluorescein isothiocyanate (FITC) or rodamine
conjugated secondary antibodies (Jackson Immunoresearch) added to
the cells for 1 h. For b-tubulin immunolocalization, cells were fixed with
1,4-piperazinediethanesulfonic acid buffer (10 mmol/L 1,4-piperazine-
diethanesulfonic acid, 5 mmol/L EGTA, 1% paraformaldehyde, 0.2%
Triton, 2 mmol/L MgCl
2
) for 30 min and then with cold ethanol for
3 min. Cells were incubated with mouse anti-human b-tubulin (1 mg/L,
Zymed Laboratories) for 1 h followed by FITC-conjugated secondary
antibody incubation for 1 h. For F-actin localization, cells were fixed
with 4% paraformaldehyde-0.2% Triton for 30 min and incubated with
0.4 mg/L FITC-conjugated phalloidin (Sigma). Nuclei were labeled with
propidium iodide (1 mg/L, Sigma) after digestion of cytoplasmatic RNA
with 50 mg/L RNAse (Roche Diagnostics) at 37C for 30 min. Stained
monolayers were mounted on glass slides in vectashields (Vector
Laboratories). The slides were examined under a confocal scanning
laser microscope (Sarastro 2000, Molecular Dynamics) using an argon
ion laser as light source. Negative controls were set by exposing the serial
sections under similar conditions omitting the primary antibody.
Western blot. Caco-2 cells were analyzed for ZO-1, occludin,
b-catenin, E-cadherin, and b-tubulin amounts according to Roselli
et al. (32). Cells were washed with ice-cold PBS and lysed in 0.5 mL of
cold radio-immune precipitation buffer (RIPA) containing 1 mmol/L of
phenylmethylsulphonyl fluoride and protease inhibitor cocktail (Com-
plete Mini, Roche). Equal amount of proteins, measured by Bradford
assay (Bio-Rad), were analyzed by SDS-PAGE (4–20% precast gel,
Cambrex) and electrophoretically transferred to nitrocellulose sheets
(Schleicher and Schuell, Bioscience) using transfer buffer (25 mmol/L
Tris-192 mmol/L glycin, pH 8.3, 20% methanol, or 5% in the case of
ZO-1) at 4C for 1 h. The membranes were incubated with primary
antibodies (2 mg/L in 3% bovine serum albumin, Zymed Laboratories)
for 1 h. Anti-b-actin (Sigma) was also used as loading control.
Preliminary experiments showed that b-actin was not affected by zinc
deficiency (data not shown). After incubation with appropriate horse-
radish peroxidase secondary antibody (1:10,000), blots were incubated
with Luminol Reagent (Tebu-bio) for 1 min to visualize the immuno-
reactive protein bands and exposed to Hyperfilm ECL (Amersham). The
band intensity was measured by Scion image software.
Phosphorylation assay. Tyrosine phosphorylation of ZO-1, occluding,
and b-catenin was analyzed by Western blot of immunoprecipitated
proteins, as previously described (32). Cells were lysed in 0.5 mL of RIPA
containing 1 mmol/L of phenylmethylsulphonyl fluoride and protease
inhibitor cocktail (Complete Mini, Roche). Proteins were immunopre-
cipitated by adding 3 mg of anti-occludin, anti-b-catenin, or anti-ZO-1
antibodies to total proteins (300 mg protein) diluted in 1 mL of RIPA
using an ExactaCruz F kit (Tebu-bio) according to the company in-
structions. Supernatants were used to detect b-actin level as control for
sample loading and the immunoprecipitates were divided into 2 aliquots
for detection of protein and phosphotyrosine level. Samples were
analyzed by Western blot as described above. We reported the results
as the ratio of protein:b-actin and phosphorylated protein:protein.
Zinc deficiency, membrane barrier, and neutrophils 1665
by guest on May 30, 2013jn.nutrition.orgDownloaded from
Chemokine measurements. Interleukin (IL)-8, epithelial neutrophil
activating peptide-78 (ENA-78), and growth-regulated oncogene-a
(GRO-a) levels were assayed in culture medium of Caco-2 cells by
ELISA using a commercial kit (R&D System). The culture medium was
collected at the end of the differentiation time before the neutrophil
transmigration experiments and stored at 280C until usage.
Statistical analysis. The significance of the differences was evaluated
by 1-way ANOVA followed by Tukey’s post hoc test. Significance was set
at P,0.05. All statistical analyses were performed with SPSS software
program (version 8.0).
Results
Zinc deficiency affects membrane function. The TEER of
the ZD cells was significantly lower than that of C cells after 18 d
of culture. The supplementation of zinc in the ZD medium
prevented the increase in TJ permeability (Fig. 1).
Zinc deficiency causes alterations to TJ, AJ, and cytoskel-
eton protein localization. Immunolocalization of junctional
proteins was performed to investigate whether zinc deficiency
affected the correct distribution of these proteins. Immunofluo-
rescence of TJ proteins (Fig. 2) shows a uniform distribution of
ZO-1 and occludin in the C cells almost exclusively at the level
of cell boundaries. On the contrary, the ZO-1 and occludin
immunostaining of the ZD cells was less homogeneous, with
numerous fluorescence interruptions in the plasma membrane.
The AJ proteins b-catenin and E-cadherin (Fig. 3) were properly
localized on the periphery of C cells, whereas a delocalization of
these proteins was detected in the ZD cells as indicated by a
diffuse staining inside the cells and loss of continuous staining
around cell boundaries. The immunolocalization of both TJ and
AJ proteins in the ZD-R cells did not differ from that in C cells,
indicating that the alterations were strictly associated with zinc
deprivation. The staining of cytoskeleton proteins (Fig. 4) shows
a bright and dense fluorescence of F-actin in the C cells at the
apical level, corresponding to microvilli, and a rich network of
actin filaments on the inner surface of the cell membrane close to
the junctional belt. In contrast, a reduction in subcortical and
perijunctional F-actin staining was visible in the ZD cells,
whereas the organization of F-actin at the microvilli level appears
unaltered, as shown by vertical sections. In addition, the
propidium iodine staining of nuclei showed an irregular align-
ment in the ZD cells compared with C cells. There were no
alterations in the organization of F-actin in the ZD-R cells. In C
cells, the immunofluorescence of b-tubulin shows a normal
localization of microtubules, which are organized into a fine
lattice network spanning the cell from the center to the periphery,
as highlighted by the vertical sections. A dramatic disorganiza-
tion of themicrotubular cytoskeleton was caused by zinc
deficiency, together with a strong decrease in b-tubulin labeling.
The ZD-R cells showed characteristics similar to those of C cells.
Zinc deficiency affects the amounts of junctional and
cytoskeleton proteins and phosphorylation level. To inves-
tigate whether the altered junctional and cytoskeletal protein
localization was associated with alterations in their levels, we
performed Western blot experiments. Occludin, ZO-1, and
b-tubulin levels were lower in ZD cells compared with C cells,
whereas those of E-cadherin and b-catenin did not differ. The
levels of all these proteins in ZD-R cells did not differ from those
of C cells (Fig. 5). In addition, the level of phosphorylation was
FIGURE 1 Zinc deficiency induces increased TJ permeability in
Caco-2 cells. The TEER was measured in cells grown in C, ZD, or ZD-R
medium for 18 d. The results are expressed as Ohm (resistance) 3
cm
2
(surface area of the monolayer). Values are means 6SD of at
least 5 experiments. Means without a common letter differ, P,0.01.
FIGURE 2 Zinc deficiency induces alterations of TJ protein locali-
zation in Caco-2 cells. Immunofluorescence of occludin and ZO-1 was
analyzed in cells grown in C, ZD, or ZD-R medium by confocal
microscope. The figure is representative of at least 6 independent
experiments (bar ¼10 mm).
1666 Finamore et al.
by guest on May 30, 2013jn.nutrition.orgDownloaded from
evaluated for those proteins whose activity and/or localization
are known to be deeply affected by changes in phosphorylation
state. The tyrosine residue phosphorylation of b-catenin and
ZO-1 was strongly increased in ZD cells compared with C and
ZD-R cells. In contrast, the phosphorylation of occludin was
lower in ZD cells than in C and ZD-R cells (Fig. 6).
Zinc deficiency induces increased neutrophil migration.
We then assessed whether disruption of membrane barrier
integrity caused by zinc deficiency led to an increase in
neutrophil migration (Fig. 7). At 60 min after treatment with
the chemoattractant, most of the neutrophils in C and ZD-R
cells were located in the basolateral compartment and in the
filter. At the same time after fMLP addition, a higher transmi-
gration of neutrophils occurred in ZD cells than in C and ZD-R
cells, as indicated by the higher number of neutrophils in the
apical compartment.
Increased chemokine secretion induced by zinc depletion.
We further investigated whether the increased passage of
neutrophils was associated with a higher level of chemo-
attractant cytokines by assaying the amounts of IL-8, ENA-78,
and GRO-asecreted in the culture media. Higher quantities of
IL-8, ENA-78, and GRO-awere secreted by ZD cells than by C
cells (Fig. 8). The amount of chemokines released by ZD-R cells
did not differ from that of C cells.
Discussion
In physiological conditions, the small intestine represents an
efficient barrier to noxious antigens and pathogens and disrup-
tion of the intestinal barrier has been considered a major factor
in several inflammatory intestinal diseases (33). We and other
authors have previously shown that zinc deficiency induces
intestinal membrane damage and inflammatory cell infiltration
(9,21). Here, we provide new evidence on the role of zinc in the
maintenance of membrane barrier integrity and prevention of
massive neutrophil infiltration, showing that zinc deficiency
impairs the membrane permeability, the integrity of the apical
junction complexes, and the cytoskeleton organization of in-
testinal cells, favoring neutrophil migration through the para-
cellular space. These effects were caused by the deficiency of
zinc, because zinc supplementation in the ZD culture medium
was highly effective in preserving the membrane barrier. This
finding is in agreement with previous studies reporting that the
addition of zinc to endothelial or lung epithelial cells restores
membrane barrier integrity (9,34). Moreover, Sturniolo et al. (7)
FIGURE 3 Zinc deficiency induced alterations in AJ protein locali-
zation in Caco-2 cells. Immunofluorescence of E-cadherin and
b-catenin was analyzed in cells grown in C, ZD, or ZD-R medium by
confocal microscope. The figure is representative of at least of 6
independent experiments (bar ¼10 mm).
FIGURE 4 Zinc deficiency induces a rearrangement of cytoskeleton
proteins in Caco-2 cells. Immunofluorescence of F-actin and b-tubulin
was analyzed in cells grown in C, ZD, or ZD-R medium by confocal
microscope. Vertical sections are reported at the bottom of each
panel. The images are representative of at least 6 independent
experiments (bar ¼5mm).
Zinc deficiency, membrane barrier, and neutrophils 1667
by guest on May 30, 2013jn.nutrition.orgDownloaded from
showed that individuals with chronic intestinal permeability
disturbances had low plasma zinc concentrations and that zinc
therapy restored the intestinal permeability associated with
increased plasma zinc levels. The importance of zinc for mem-
brane barrier function has also been highlighted by previous
studies showing that zinc-deprived airway epithelial cells
undergo proteolysis to E-cadherin and b-catenin (9). However,
in this study, the ZD culture medium was combined with tumor
necrosis factor-a, interferon-g, and Fas receptor ligand, making
it impossible to separate precisely the effects of zinc alone. Here,
we show that zinc depletion caused disruption of TJ and AJ, as
indicated by the rearrangement throughout the cell of occludin,
ZO-1, E-cadherin, and b-catenin as well as by the dramatic
disorganization of the cytoskeletal tubulin and actin. Because
the association of occludin and ZO-1 is critical for the integrity
of the TJ (13), the decrease in these proteins indicated by
Western blot analysis is further evidence of TJ disruption. In
agreement with these findings, an altered expression of occludin
in enterocytes of patients with IBD, such as Crohn’s disease and
ulcerative colitis, has been shown to coexist with altered
expression of ZO-1. Interestingly, only the expression of ZO-1,
and not of ZO-2, was affected in inflamed mucosal tissues,
suggesting a specialized function of ZO-1 in the rearrangement
of TJ structure in an inflamed milieu (35).
Tyrosine phosphorylation of occludin is necessary for the
stabilization of this protein on the TJ (36). A recent study has
FIGURE 6 Zinc deficiency causes changes in phosphorylation level
of TJ and AJ proteins in Caco-2 cells. The level of occludin, ZO-1, and
b-catenin phosphotyrosine (P-Y) was analyzed in cells grown in C, ZD,
or ZD-R medium by Western blot (top) and the densitometric values of
P-Y-proteins were normalized to their correspondent protein (bottom).
The figure is representative of 4 independent assays. Values are
means 6SD. Within each ratio, values without a common letter differ,
P,0.01.
FIGURE 7 Zinc deficiency induces increased neutrophil transmigra-
tion in Caco-2 cells. The peptide fMLP (1 310–5 mol/L) was used to
induce neutrophil transmigration. The number of transmigrated
neutrophils was measured in cells grown in C, ZD, or ZD-R medium
and quantified by myeloperoxidase assay. Values are means 6SD of 4
independent experiments. Within each column, values without a
common letter differ, P,0.01.
FIGURE 5 Zinc deficiency causes a reduction in TJ, AJ, and
cytoskeleton protein level in Caco-2 cells. The level of occludin, ZO-1,
E-cadherin, b-catenin, and b-tubulin was analyzed in cells grown in C,
ZD, or ZD-R medium by Western blot (top) and the densitometric values
of bands were normalized to b-actin (bottom). The figure is represen-
tative of at least 4 independent assays. Values are means 6SD. Within
each ratio, values without a common letter differ, P,0.01.
FIGURE 8 Zinc deficiency induced an increase in chemokyne se-
cretion in Caco-2 cells. The amount of IL-8, ENA-78, and GRO-a
secreted in culture medium of cells grown in C, ZD, or ZD-R medium
was analyzed by ELISA. Values are means 6SD of 4 independent
assays. Within each column, values without a common letter differ,
P,0.01.
1668 Finamore et al.
by guest on May 30, 2013jn.nutrition.orgDownloaded from
shown that the protein phosphatase 2A interacts with occludin
and modulates its phosphorylation status, inducing a strong
reduction in tyrosine residue phosphorylation during the disas-
sembly of TJ and an increase in phosphorylation during the
reassembly (37). On the other hand, hyperphosphorylation of
ZO-1 was associated with alterations of ZO-1 localization in
Caco 2 cells (38). The interaction of b-catenin with the in-
tracellular domain of E-cadherin is also regulated by tyrosine
phosphorylation of b-catenin and hyperphosphorylation of
b-catenin results in the loss of cadherin-based cell-cell adhesion
(39–41). A recent study has further highlighted that the
dissociation of protein tyrosine phosphatase 1B from the
E-cadherin-b-catenin complex is accompanied by an increase
in tyrosine phosphorylation of b-catenin and by a loss of its
interactions with E-cadherin (37). Consistent with these data,
we report a decrease in tyrosine residue phosphorylation of
occludin and an increase in phosphorylation of ZO-1 and
b-catenin associated with zinc deficiency, suggesting that the
phosphorylation state of these proteins might play a pivotal role
in their dissociation and translocation from the junctional
complexes to intracellular compartments, giving rise to the
disruption of barrier integrity.
In this study, we report that the alterations to the structure
of TJ and AJ favor the passage of neutrophils. In agreement
with our results, Kucharzik et al. (26) have shown a down-
regulation of occludin in regions of actively transmigrating
PMN, together with a decrease in ZO-1, claudin-1, b-catenin,
and E-cadherin in epithelial cells immediately adjacent to
transmigrating neutrophils in colonic mucosa of IBD patients.
The importance of occludin in modulating the migration of
neutrophils has also been shown by Huber et al. (42). Other
authors have demonstrated that hyperpermeability associated
with PMN transmigration occurs concomitantly with tyrosine
hyperphosphorylation of b-catenin and loss of this protein at the
cell membrane (43). In addition, dephosphorylation of occludin
and degradation of ZO-1 have been shown to facilitate the
transepithelial passage of neutrophils in intestinal cells infected
with a pathogen (44).
It could be argued that the enhanced neutrophil migration was
due to an increase in chemokines rather than the leaking barrier,
because we show that depletion of zinc induces an increased
secretion of IL-8, ENA78, and Gro-a, which are known to induce
neutrophil migration (45). However, this increase was not
responsible for the observed enhanced neutrophil migration,
because the neutrophil migration assay was performed in a
chemokine-free culture medium and there was a very low level of
transmigration without the addition of the chemoattractant
fMLP in both the C and ZD cells. Nevertheless, our results
suggests that a condition of hypozincemia may cause uncon-
trolled migration of neutrophils through both the disruption of
junctional complexes and the induction of chemokines, which
may lead to the development or exacerbation of inflammation
and mucosal damage by releasing factors such as inflammatory
cytokines or proteases that contribute further to intestinal
damage. Indeed, an extensive neutrophil migration across the
epithelium has been shown to be associated with epithelial injury
and intestinal disease (46,47). An additional contribution to the
neutrophil migration in hypozincemia may derive from an
increased apoptosis, because previous studies have shown that
zinc deprivation increases susceptibility to apoptosis and facil-
itates barrier disruption in epithelial cells (9,48).
In conclusion, our results provide new information on the
critical role played by dietary zinc in the maintenance of mem-
brane barrier function and in controlling inflammatory reactions
by showing that the depletion of zinc causes phosphorylation-
mediated disruption of junctional complexes and cytoskeleton
disorganization, thus promoting the migration of neutrophils.
Although the present study was conducted in vitro, our
results may provide an explanation of the findings that patients
with IBD and low mucosal zinc concentration typically present
an accumulation of neutrophils in epithelial crypts and intestinal
lumen resulting in the formation of crypt abscesses (25–27).
Literature Cited
1. Aggett PJ. Acrodermatitis enteropathica. J Inherit Metab Dis. 1983;6
Suppl 1:39–43.
2. Prasad AS. Zinc deficiency. BMJ. 2003;326:409–10.
3. Haase H, Mocchegiani E, Rink L. Correlation between zinc status and
immune function in the elderly. Biogerontology. 2006;7:421–8.
4. Prasad AS. Zinc and immunity. Mol Cell Biochem. 1998;188:63–9.
5. Moshage H. Cytokines and the hepatic acute phase response. J Pathol.
1997;181:257–66.
6. Devirgiliis C, Zalewski PD, Perozzi G, Murgia C. Zinc fluxes and zinc
transporter genes in chronic diseases. Mutat Res. 2007;622:84–93.
7. Sturniolo GC, Di Leo V, Ferronato A, D’Odorico A, D’Inca R. Zinc
supplementation tightens ‘‘leaky gut’’ in Crohn’s disease. Inflamm Bowel
Dis. 2001;7:94–8.
8. Sempe
´rtegui F, Dı
´az M, Mejı
´a R, Rodrı
´guez-Mora OG, Renterı
´a E,
Guarderas C, Estrella B, Recalde R, Hamer DH, et al. Low concentra-
tions of zinc in gastric mucosa are associated with increased severity of
Helicobacter pylori-induced inflammation. Helicobacter. 2007;12:43–8.
9. Bao S, Knoell DL. Zinc modulates cytokine-induced lung epithelial cell
barrier permeability. Am J Physiol Lung Cell Mol Physiol. 2006;
291:L1132–41.
10. Lambert JC, Zhou Z, Wang L, Song Z, McClain CJ, Kang YJ.
Preservation of intestinal structural integrity by zinc is independent of
metallothionein in alcohol-intoxicated mice. Am J Pathol. 2004;164:
1959–66.
11. Rodriguez P, Darmon N, Chappuis P, Candalh C, Blaton MA, Bouchaud
C, Heyman M. Intestinal paracellular permeability during malnutrition
in guinea pigs: effect of high dietary zinc. Gut. 1996;39:416–22.
12. Berkes J, Viswanathan VK, Savkovic SD, Hecht G. Intestinal epithelial
responses to enteric pathogens: effects on the tight junction barrier, ion
transport, and inflammation. Gut. 2003;52:439–51.
13. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, Tsukita S.
Direct association of occludin with ZO-1 and its possible involvement
in the localization of occludin at tight junctions. J Cell Biol. 1994;
127:1617–26.
14. Salama NN, Eddington ND, Fasano A. Tight junction modulation and
its relationship to drug delivery. Adv Drug Deliv Rev. 2006;20:15–28.
15. Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions.
Nat Rev Mol Cell Biol. 2001;2:285–93.
16. Denker BM, Nigam SK. Molecular structure and assembly of the tight
junction. Am J Physiol. 1998;274:F1–9.
17. Scott JA, Yap AS. Cinderella no longer: alpha-catenin steps out of
cadherin’s shadow. J Cell Sci. 2006;15:4599–605.
18. Weis WI, Nelson WJ. Re-solving the cadherin-catenin-actin conundrum.
J Biol Chem. 2006;281:35593–7.
19. Edens HA, Levi BP, Jaye DL, Walsh S, Reaves TA, Turner JR, Nusrat A,
Parkos CA. Neutrophil transepithelial migration: evidence for sequen-
tial, contact-dependent signaling events and enhanced paracellular per-
meability independent of transjunctional migration. J Immunol. 2002;
169:476–86.
20. Mengheri E, Nobili F, Vignolini F, Pesenti M, Brandi G, Biavati B.
Bifidobacterium animalis protects intestine from damage induced by
zinc deficiency in rats. J Nutr. 1999;129:2251–7.
21. Canali R, Vignolini F, Nobili F, Mengheri E. Reduction of oxidative
stress and cytokine-induced neutrophil chemoattractant (CINC) ex-
pression by red wine polyphenols in zinc deficiency induced intestinal
damage of rat. Free Radic Biol Med. 2000;28:1661–70.
22. Dejana E, Zanetti A, Del Maschio A. Adhesive proteins at endothelial
cell-to-cell junctions and leukocyte extravasation. Haemostasis. 1996;
26:210–9.
Zinc deficiency, membrane barrier, and neutrophils 1669
by guest on May 30, 2013jn.nutrition.orgDownloaded from
23. Elliott SN, Wallace JL. Neutrophil-mediated gastrointestinal injury. Can
J Gastroenterol. 1998;12:559–68.
24. Gewirtz AT, Liu Y, Sitaraman SV, Madara JL. Intestinal epithelial
pathobiology: past, present and future. Best Pract Res Clin Gastro-
enterol. 2002;16:851–67.
25. Chin AC, Parkos CA. Neutrophil transepithelial migration and epithe-
lial barrier function in IBD: potential targets for inhibiting neutrophil
trafficking. Ann N Y Acad Sci. 2006;1072:276–87.
26. Kucharzik T, Walsh SV, Chen J, Parkos CA, Nusrat A. Neutrophil
transmigration in inflammatory bowel disease is associated with
differential expression of epithelial intercellular junction proteins. Am
J Pathol. 2001;159:2001–9.
27. Sturniolo GC, Mestriner C, Lecis PE, D’Odorico A, Venturi C, Irato P,
Cecchetto A, Tropea A, Longo G, et al. Altered plasma and mucosal
concentrations of trace elements and antioxidants in active ulcerative
colitis. Scand J Gastroenterol. 1998;33:644–9.
28. Tate DJ, Miceli MV, Newsome DA. Zinc protects against oxidative
damage in cultured human retinal pigment epithelial cells. Free Radic
Biol Med. 1999;26:704–13.
29. Ferruzza S, Scacchi M, Scarino ML, Sambuy Y. Iron and copper alter
tight junction permeability in human intestinal Caco-2 cells by distinct
mechanisms. Toxicol In Vitro. 2002;16:399–404.
30. Roselli M, Finamore A, Britti MS, Mengheri E. Probiotic bacteria
Bifidobacterium animalis MB5 and Lactobacillus rhamnosus GG protect
intestinal Caco-2 cells from the inflammation-associated response induced
by enterotoxigenic Escherichia coli K88. Br J Nutr. 2006;95:1177–84.
31. Ara C, Conti Devirgiliis L, Massimi M. Influence of retinoic acid on
adhesion complexes in human hepatoma cells: a clue to its antiprolifer-
ative effects. Cell Commun Adhes. 2004;11:13–23.
32. Roselli M, Finamore A, Britti MS, Konstantinov SR, Smidt H, de Vos
WM, Mengheri E. The novel porcine Lactobacillus sobrius strain
protects intestinal cells from enterotoxigenic Escherichia coli K88
infection and prevents membrane barrier damage. J Nutr. 2007;
137:2709–16.
33. Fasano A, Shea-Donohue T. Mechanisms of disease: the role of intestinal
barrier function in the pathogenesis of gastrointestinal autoimmune
diseases. Nat Clin Pract Gastroenterol Hepatol. 2005;2:416–22.
34. Hennig B, Wang Y, Ramasamy S, McClain CJ. Zinc deficiency alters
barrier function of cultured porcine endothelial cells. J Nutr. 1992;
122:1242–7.
35. Gassler N, Rohr C, Schneider A, Kartenbeck J, Bach A, Obermuller N,
Otto HF, Autschbach F. Inflammatory bowel disease is associated with
changes of enterocytic junctions. Am J Physiol Gastrointest Liver
Physiol. 2001;281:G216–28.
36. Tsukamoto T, Nigam SK. Role of tyrosine phosphorylation in the
reassembly of occludin and other tight junction proteins. Am J Physiol.
1999;276:F737–50.
37. Sheth P, Seth A, Atkinson KJ, Gheyi T, Kale G, Giorgianni F, Desiderio
DM, Li C, Naren A, et al. Acetaldehyde dissociates the PTP1B-E-
cadherin-beta-catenin complex in Caco-2 cell monolayers by a phos-
phorylation-dependent mechanism. Biochem J. 2007;402:291–300.
38. Katsube T, Tsuji H, Onoda M. Nitric oxide attenuates hydrogen
peroxide-induced barrier disruption and protein tyrosine phosphoryla-
tion in monolayers of intestinal epithelial cell. Biochim Biophys Acta.
2007;1773:794–803.
39. Van Buul JD, Anthony EC, Fernandez-Borja M, Burridge K, Hordijk
PL. Proline-rich tyrosine kinase 2 (Pyk2) mediates vascular endothelial-
cadherin-based cell-cell adhesion by regulating beta-catenin tyrosine
phosphorylation. J Biol Chem. 2005;280:21129–36.
40. Basuroy S, Sheth P, Mansbach CM, Rao RK. Acetaldehyde disrupts
tight junctions and adherens junctions in human colonic mucosa:
protection by EGF and L-glutamine. Am J Physiol Gastrointest Liver
Physiol. 2005;289:G367–75.
41. Xu G, Craig AW, Greer P, Miller M, Anastasiadis PZ, Lilien J, Balsamo
J. Continuous association of cadherin with beta-catenin requires the
non-receptor tyrosine-kinase Fer. J Cell Sci. 2004;117:3207–19.
42. Huber D, Balda MS, Matter K. Occludin modulates transepithelial
migration of neutrophils. J Biol Chem. 2000;275:5773–8.
43. Tinsley JH, Ustinova EE, Xu W, Yuan SY. Src-dependent, neutrophil-
mediated vascular hyperpermeability and beta-catenin modification.
Am J Physiol Cell Physiol. 2002;283:C1745–51.
44. Ko
¨hler H, Sakaguchi T, Hurley BP, Kase BA, Reinecker HC,
McCormick BA. Salmonella enterica serovar Typhimurium regulates
intercellular junction proteins and facilitates transepithelial neutrophil
and bacterial passage. Am J Physiol Gastrointest Liver Physiol. 2007;
293:G178–87.
45. Sachse F, Ahlers F, Stoll W, Rudack C. Neutrophil chemokines in
epithelial inflammatory processes of human tonsils. Clin Exp Immunol.
2005;140:293–300.
46. Jaye DL, Parkos CA. Neutrophil migration across intestinal epithelium.
Ann N Y Acad Sci. 2000;915:151–61.
47. Ginzberg HH, Cherapanov V, Dong Q, Cantin A, McCulloch CA,
Shannon PT, Downey GP. Neutrophil-mediated epithelial injury during
transmigration: role of elastase. Am J Physiol Gastrointest Liver Physiol.
2001;281:G705–17.
48. Truong-Tran AQ, Grosser D, Ruffin RE, Murgia C, Zalewski PD.
Apoptosis in the normal and inflamed airway epithelium: role of zinc in
epithelial protection and procaspase-3 regulation. Am J Respir Cell Mol
Biol. 2002;27:286–96.
1670 Finamore et al.
by guest on May 30, 2013jn.nutrition.orgDownloaded from
  • ... A 70 kg adult contains about 2-3 g zinc, about 0.1% of which are replenished daily. The daily amount of zinc that is needed is relatively small, about 2-3 mg in adults to compensate for the relatively small loss of zinc in urine, stool, and sweat [33][34]. Intake recommendations for zinc and other nutrients are provided in the Dietary Reference Intakes (DRIs) developed by the Food and Nutrition Board (FNB) at the Institute of Medicine of the National Academies (formerly National Academy of Sciences) [35]. ...
    Article
    Full-text available
    Zinc is an essential trace element of all highly proliferating cells in the human body. It is essential to the development and growth of all organisms. Zinc plays a critical role in modulating resistance to infectious agents and reduces the duration, severity, and risk of diarrheal disease via improved regeneration of intestinal epithelium, improved absorption of water and electrolytes, increased levels of brush border enzymes, and, possibly, an enhancement in the immune response allowing better clearance of pathogens. On the cellular level, zinc finger motifs play various roles including diverse functions that involve specific gene expression for ion channels throughout the body. It maintains the function and the structure of the membrane barrier by contributing to host defense, which is particularly crucial in the intestines due to the continuous exposure to noxious agents and pathogens. Zinc deficiency is characterized by impaired immune function, loss of appetite, and growth retardation. More severe cases cause diarrhea, delayed sexual maturation, hair loss, eye and skin lesions, impotence and hypogonadism in males, as well as weight loss, taste abnormalities, delayed healing of wounds, and mental lethargy. The objective of this study is a critical review of the molecular and genetic regulation of zinc in various cellular processes and organs, the association between zinc and diarrheal disease, the recommended dietary zinc intake, and the effects of zinc deficiency on the human body.
  • ... So far, medical studies have demonstrated the role of zinc in maintaining the integrity and permeability of the cell membrane, the integrity of intercellular junctions and of the physiological organization of the intestinal cells' cytoskeleton, associated with the role of preventing migration for polymorphonuclear cells [1]. At the level of epithelial barriers, the role of zinc in the nonspecific immune response has been demonstrated as breaking down the intercellular junctional complexes and disorganizing the cytoskeleton, following the zinc deprivation of the organism [2]. Also, low concentrations of zinc were determined at the mucous membranes level in the case of patients with intestinal permeability chronic disorders. ...
    Article
    Full-text available
    Zinc modulates the human body defence against infections. Mild and medium deficiency in this mineral appears usually sub-clinically, being mistaken for other diseases, but the severe form can be fatal. The purpose of the study was to determine the plasma zinc concentration (PZC) for the most common infectious pathology in children. Zinc was measured in plasma using direct colorimetric assay based on the 5-Br-PAPS method (CV% 0.98-4.64%). In the paediatric patients, 0-3 years old, the PZC values were 15.20±1.37 μmol/L, with limits ranging between 13.05-20.6 μmol/L, the values falling within normal limits and proving the absence of zinc deficiency in the investigated population. During 3 years of follow up, 137 healthy children presented low values of plasma zinc concentration if they had acute lower respiratory infections, acute otitis media or giardiasis in past medical history. There were not found significant differences in case of children with viral or bacterial acute diarrheal diseases or viral exanthemas. In the present study, the children exposed to severe, complicated or chronic forms of infectious diseases were predisposed to low plasma zinc concentrations.
  • ... 58 Similarly, disruption of the intestinal barrier by a deficiency of vitamins and trace minerals, as well as alcohol consumption, involve a combination of multiple events, including tight junction, mucus layer and microbial alterations. [59][60][61][62] Environmental factors, such as medication or stress, also induce intestinal barrier dysfunction via multiple mechanisms. Non-steroidal anti-inflammatory drugs are well known for their damaging effects on the gut. ...
    Article
    Full-text available
    Dietary, environmental or genetic changes contribute to the development of several metabolic and neurological diseases. Extensive research efforts have been directed towards examining how these factors modulate gut bacterial composition. However, the mechanisms by which changes in gut bacterial diversity affects host function are not completely defined. Intestinal barrier dysfunction is being increasingly recognized as an early or initiating step in communicating bacterial diversity-dependent changes to the host. Here, we review the functional composition of the intestinal barrier and describe the consequences of the breach of this barrier. The functional layers of the intestinal barrier provide an initial defense and prevent the infiltration of bacteria or bacterial products from the gut lumen to the underlying lamina propria. Mesenteric lymph nodes subsequently act as the first firewall where dendritic cells from the lamina propria transport the infiltrated bacterial or bacterial products during the early stages of barrier dysfunction. When overwhelmed, the liver functions as the second firewall to detoxify bacterial endotoxins, such as lipopolysaccharide (LPS), and limit systemic involvement. Continuous translocation of LPS from the leaky gut to the liver results in liver damage or dysfunction that leads to the development of type 2 diabetes, atherosclerosis, heart disease, heart failure and also neurological diseases, such as Alzheimer’s and Parkinson’s disease. Thus, targeted restoration of intestinal barrier function represents a novel strategy for the attenuation of multiple diseases.
  • ... Inflammatory processes, hyperresponsiveness, antioxidants and zinc deficiency may affect the disruption of bronchial epithelium. (2) Zinc is a vital factor in the lung epithelium, protecting against barrier dysfunction. Zinc deprivation would induce caspase-3, enhancing apoptosis, leading to the degradation of junction proteins, loss of cell-to-cell contact, and increased permeability of the epithelium. ...
    Article
    Full-text available
    BACKGROUND: Airway epithelium is the first line of defense against a variety of exposures. Inflammatory processes, hyperresponsiveness and zinc deficiency cause epithelial damage. Zinc is involved in apoptosis and microtubule formation. However, its role in the integrity of bronchial mucosa and cilia is unclear.METHODS: To assess the effect of zinc on the integrity of the bronchial epithelium, 24 male Rattus norvegicus strain Wistar rats were randomized into four experimental groups: normal zinc diet group without zinc supplementation, normal zinc diet group with 60 ppm zinc supplementation, zinc deficient diet group without zinc supplementation, and zinc deficient diet group with 120 ppm zinc supplementation. Bronchial mucosal integrity was measured with the number of epithelial cells, and the number and length of cilia.RESULTS: Number of cell in normal zinc diet group was 8.8±1.82, while it was only 8.1±1.08 in zinc deficient diet group (p<0.001). Number of cilia per cell was 4.6±1.08 in normal zinc diet group, compared to 4.0±0.79 in zinc deficient diet group (p<0.001). Ciliary length also differ by 7.68±0.66 μm in normal zinc diet group and only 5.16±0.91 μm in zinc deficient diet group (p<0.001).CONCLUSION: Zinc supplementation of the normal zinc diet group affected the length of bronchial cilia. Zinc supplementation of the zinc deficient diet group affected the integrity of the bronchial epithelium, which was shown by the number and length of cilia, and the number of epithelial cells.KEYWORDS: zinc, bronchial epithelial integrity, cilia length, number of cilia, epithelial cell
  • ... The exact basolateral zinc concentrations in these studies are unknown. FCS generally contains higher amounts of zinc than cell culture medium [293], but its total zinc content varies significantly, which leads to final zinc concentrations between 3 [74] and 14 µM [294] in complete media. The addition of albumin, apart from the amount already present in FCS, to the basolateral side of the in vitro model, however, was only performed in four of the in vitro transport studies with Caco-2 monocultures or Caco-2/HT-29-MTX co-cultures, respectively, by applying very low (2.5 mg mL −1 ) [238] or physiological albumin concentrations (5% BSA, corresponding to 50 mg mL −1 albumin [71], and 30 mg mL −1 albumin [102,103]). ...
    Article
    Full-text available
    Zinc absorption in the small intestine is one of the main mechanisms regulating the systemic homeostasis of this essential trace element. This review summarizes the key aspects of human zinc homeostasis and distribution. In particular, current knowledge on human intestinal zinc absorption and the influence of diet-derived factors on bioaccessibility and bioavailability as well as intrinsic luminal and basolateral factors with an impact on zinc uptake are discussed. Their investigation is increasingly performed using in vitro cellular intestinal models, which are continually being refined and keep gaining importance for studying zinc uptake and transport via the human intestinal epithelium. The vast majority of these models is based on the human intestinal cell line Caco-2 in combination with other relevant components of the intestinal epithelium, such as mucin-secreting goblet cells and in vitro digestion models, and applying improved compositions of apical and basolateral media to mimic the in vivo situation as closely as possible. Particular emphasis is placed on summarizing previous applications as well as key results of these models, comparing their results to data obtained in humans, and discussing their advantages and limitations.
  • ... Moon et al. (2018) found that Zn could affect the differentiation of neurons and promote the proliferation of mesenchymal stem cells, and its deficiency led to dysplasia of neural progenitor proliferation and differentiation. As a component of proteins necessary to stabilize cell structure, zinc enhances intestinal epithelial barrier function, and Zn deprivation induces a decrease of transepithelial resistance (TEER) and increases the membrane barrier permeability, eventually causes ulcerations of the small intestine (Finamore et al., 2008;Shao et al., 2017;Wang et al., 2013). In addition, Zn has been demonstrated to facilitate the healing of ulcers, inhibit the growth of enteric pathogens and ameliorate oxidative stress (Marreiro et al., 2017). ...
    Article
    Full-text available
    The micronutrient, zinc, plays a vital role in modulating cellular signaling recognition and enhancing intestinal barrier function. However, the precise mechanisms underlying the zinc regulation of intestinal stem cell (ISC) renewal and regeneration ability, which drive intestinal epithelial turnover to maintain the intestinal barrier, under physiological and pathological conditions are unknown. In this study, we used in vivo mouse plus ex vivo enteroid model to investigate thoroughly the protection efficacy of zinc L-aspartate (Zn-Asp) on intestinal mucosal integrity exposed to deoxynivalenol (DON). The results showed that 10 rather than 20 mg/kg body weight (BW) Zn-Asp (calculation in zinc) significantly increased the jejunum mass and ameliorated mucosa injury caused by 2 mg/kg BW DON treatment, including improvement of the intestinal morphology and barrier, as well as enteroid-forming and -budding efficiency, which was expanded from crypt cells isolated from jejunum of mice in each group. The repair process stimulated by Zn-Asp was also accompanied by increased fluorescence signal intensity of KRT20 and Villin; increased numbers of MUC2⁺, CAG⁺, LYZ⁺, BrdU⁺ and Ki67⁺ cells in mouse jejunum; and protein expression of Ki67 and PCNA in the jejunum, crypt and enteroid. Simultaneously, Zn-Asp increased ISC activity to promote intestinal epithelial renewal even under physiological conditions. These results were further verified in ex vivo enteroid culture experiments, which were treated with 100 μmol/L Zn-Asp (calculation in zinc) and 100 ng/mL DON for 72 h. Furthermore, we demonstrated that Zn-Asp improved intestinal integrity or accelerated wound healing along with Wnt/β-catenin signaling upregulation or reactivation. Our findings indicate Zn-Asp, especially Zn, enhances ISC activity to maintain the intestinal integrity by activating the Wnt/β-catenin signaling, which sheds some light upon effective preventive strategies for intestinal injury induced by mycotoxin based on ISCs with exogenous zinc preparations in the proper drugs, health foods or qualified feed.
  • ... 102 No. 12, 2019 ZINC SUPPLEMENTATION DURING IMMUNOACTIVATION 11693 300 enzymes and plays a pivotal role in multiple systems (Prasad, 2012;Kambe et al., 2015). Furthermore, Zn improves epithelial integrity characteristics (mammary and intestinal; Sanz Weng et al., 2018) and immune function (cell adherence, clearance of E. coli, cytokine production; Kidd et al., 1994a;Finamore et al., 2008). Therefore, we hypothesized that supplemental complexed Zn would modify the inflammatory response to an intravenous LPS challenge and in turn alter the leukocyte energetic requirement. ...
    Article
    The objectives of this study were to evaluate the effects of replacing 40 mg/kg of Zn from Zn sulfate (control; CON) with Zn AA complex (AvZn) on metabolism and immunological responses following an intravenous lipopolysaccharide (LPS) challenge in lactating cows. Cows were randomly assigned to 1 of 4 treatments: (1) pair-fed (PF) control (PF-CON; 5 mL of saline; n = 5), (2) PF AvZn (PF-AvZn; 5 mL of saline; n = 5), (3) LPS euglycemic clamp control (LPS-CON; 0.375 μg of LPS/kg of BW; n = 5), and (4) LPS euglycemic clamp AvZn (LPS-AvZn; 0.375 μg of LPS/kg of BW; n = 5). Cows were enrolled in 3 experimental periods (P). During period 1 (3 d), cows received their respective dietary treatments and baseline data were obtained. During period 2 (P2; 2 d), a 12-h LPS euglycemic clamp was conducted or cows were PF to their respective dietary counterparts. During period 3 (P3; 3 d), cows received their dietary treatment and consumed feed ad libitum. Mild hyperthermia (1°C) was observed in LPS cows at 3 h postbolus. Throughout P2, the rectal temperature of LPS-AvZn cows was decreased (0.3°C) relative to LPS-CON cows. Administrating LPS decreased dry matter intake (47%) during P2, and by experimental design the pattern was similar in PF cohorts. During P3, dry matter intake from LPS cows remained decreased (15%) relative to PF cows. Milk yield from LPS cows decreased (54%) during P2 relative to PF cows, but it was similar during P3. During P2, somatic cell count increased 3-fold in LPS cows relative to PF controls. Dietary AvZn tended to decrease somatic cell count (70%) during P3 relative to LPS-CON cows. Insulin increased 7-fold in LPS cows at 12 h postbolus and remained increased (4-fold) for the duration of P2. Circulating glucagon from LPS cows increased (65%) during P2, and supplementing AvZn blunted the increase (30% relative to LPS-CON). During P2, circulating cortisol increased 7-fold post-LPS infusion relative to PF cows, and supplementing AvZn decreased cortisol (58%) from 6 to 48 h postbolus relative to LPS-CON cows. Administrating LPS increased circulating LPS-binding protein and serum amyloid A (3- and 9-fold, respectively) relative to PF cows. Compared with LPS-CON, LPS-AvZn cows had increased circulating serum amyloid A (38%) 24 h postbolus. The 12-h total glucose deficit was 36 and 1,606 g for the PF and LPS treatments, respectively, but was not influenced by Zn source. In summary, replacing a portion of the Zn sulfate with Zn AA complex appeared to reduce the inflammatory response but had no effect on the glucose deficit.
  • Chapter
    Zinc plays a well-documented role in intestinal function, perturbations in zinc homeostasis are associated with impaired barrier function and disease. A large repertoire of zinc transport proteins ( ZnTs, ZIPs), TRP channels, and a distinct zinc receptor, ZnR/GPR39, are expressed along the intestinal epithelial cells. We will discuss the cellular roles of zinc ions, Zn2+, in activating signaling and physiological function of multiple cells along the intestinal epithelium. We will further describe the network of Zn2+-homeostatic proteins that are responsible for maintaining Zn2+ concentrations within the cytosol and their role in maintaining cellular functions. This review will reveal the tip of the iceberg that is currently known regarding the role of intestinal Zn2+ and will elucidate the need for extensive studies that will expound the complete map and physiological roles of Zn2+-homeostatic proteins.
  • Chapter
    Zinc is an essential micronutrient for cell growth, differentiation, and survival and when deficient, is associated with increased susceptibility to infections and inflammatory disorders. Zinc homeostasis is critical for proper immune cell function and is therefore tightly regulated by zinc transporters. Recent evidence has highlighted zinc as an intracellular signaling molecule capable of modulating immune cell signaling. Slight changes in intracellular zinc, either by zinc deficiency or by excess zinc, can alter cellular signaling and immune cell function often resulting in increased inflammation. In this chapter, we discuss zinc signals in inflammation with a focus on zinc dependent modulation of select signaling pathways and the effects on immune cell function in response to potentially damaging challenges.
  • Chapter
    Zinc is an essential trace element that plays an important role in many physiological functions. One of the key functions of zinc is its influence on the immune system. Zinc is required for the development and functioning of immune cells in the innate and the adaptive immune system. Zinc homeostasis is finely controlled within each cell and any deregulation results in impairment of normal functions. Consequences of impaired homeostasis can be observed in many disease models such as infections, allergies, autoimmune diseases, and cancers. Zinc deficiency negatively influences the hematopoiesis and compromises the immune response at multiple molecular, cellular, and systemic levels. This chapter summarizes how zinc is involved in the immune system and how altered zinc levels within cells influence the immune response.
  • Article
    Polarized epithelial cells separate two extremely different cellular milieus. The tight junction (TJ) is the most apical component of the junctional complex and serves as the permeability barrier between these environments. The tight junctional complex appears to be a dynamic and regulated structure. Some of its protein components have been identified and include the transmembrane protein occludin. Nontransmembrane proteins on the cytosolic leaflet including ZO-1, ZO-2, cingulin, 7H6, and several unidentified phosphoproteins are also believed to be part of the TJ. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of TJ structure and may also play a role in the regulation of TJ assembly. Recent progress using the "calcium switch" and the "ATP depletion-repletion" model of TJ formation offers new insight regarding how these structures form. TJ biogenesis appears to be regulated, in part, by classic signal transduction pathways involving heterotrimeric G proteins, release of intracellular Ca2+, and activation of protein kinase C. Although many of the details of the signaling pathways have yet to be defined, these observations may provide insight into how TJs form during tubular development. Furthermore, it may be possible to suggest potential therapeutic targets for intervention in a variety of diseases (e.g., ischemia, toxic injury to the kidney and other epithelial tissue) where TJ integrity has been compromised and reassembly is required.
  • Article
    Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.
  • Article
    Full-text available
    We investigated the potential beneficial effects of Bifidobacterium animalis on intestinal damage using zinc-deficient (ZD) rats as a model for intestinal alterations. The ZD rats were fed diets containing 1 mg Zn/kg for 20 (ZD(20)) or 40 (ZD(40)) d to induce damage that differed in severity. Subgroups of these rats, the ZD(20) + B and ZD(40) + B groups, received a suspension of B. animalis (3.5 x 10(8) colony forming units) daily for the last 10 d. Another subgroup, the ZD(40) + B + 7 d group, was fed the ZD diet for 7 d after the B. animalis treatment period. Zinc deficiency induced ulcerations, edema, inflammatory cell infiltration and dilatation of blood vessels in duodenum, jejunum and ileum, with increasing severity between 20 and 40 d of zinc deficiency. The mucosa of the ZD(20) + B group was well preserved, and most of the morphologic alterations induced by zinc deficiency were normalized in the ZD(40) + B group. The high fecal concentrations of B. animalis in the ZD(40) + B and ZD(40) + B + 7 d groups indicate that these bifidobacteria survived passage through the gastrointestinal tract and proliferated. Electron microscopy confirmed the elevated numbers of bifidobacteria in cecum. Treatment with B. animalis resulted in greater epithelial cell proliferation and disaccharidase activities in the ZD(40) + B group compared with the ZD(40) group. These findings indicate that B. animalis can protect the intestine from alterations induced by zinc deficiency, suggesting that this bacterium may play a role in intestinal mucosal defense.
  • Article
    Zinc is necessary for normal membrane function and stability. We postulated that Zn deficiency may disrupt the integrity of the vascular endothelium by decreasing its barrier function. To test this hypothesis, endothelial cells were cultured on polycarbonate filters and exposed to media enriched with either 1% fetal bovine serum (FBS) (low FBS; total Zn, 1.07 mumols/L medium) or 5% FBS (control; total Zn, 2.29 mumols/L) or low FBS plus two supplemental levels of Zn, 3.36 and 5.66 mumols total zinc/L. Endothelial cell barrier function, expressed as albumin transfer across cultured endothelial monolayers, was significantly lower in cultures exposed to low FBS compared with control medium. Supplementation with 5.66 mumols total Zn/L completely restored endothelial barrier function. A divalent cation chelator, 1,10-orthophenanthroline, was used to induce Zn deficiency in vitro. Compared with control cultures, the presence of 1,10-orthophenanthroline in the culture medium resulted in markedly lower endothelial barrier function that was increased by the addition of Zn but not calcium or magnesium. Activity of the membrane-bound zinc-dependent angiotensin-converting enzyme (ACE) was depressed by low zinc medium, whereas membrane-bound Ca(2+)-ATPase and total ATPase were not depressed. Furthermore, cells cultivated in low zinc medium did not have greater cytosolic release of adenine, indicating no increase in cell injury or death. These data suggest that Zn is vital to endothelial cell integrity and that Zn may play an important role in vascular endothelial barrier function.
  • Article
    A brief review of the clinical and biochemical features of Acrodermatitis enteropathica is given. This condition in now known to be caused by a systemic zinc deficiency secondary to a defect in the intestinal absorption of zinc and it illustrates the metabolic importance of this element in man.
  • Article
    Full-text available
    Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.
  • Article
    Full-text available
    Zinc has been shown to have beneficial effects in vitro on epithelial barrier function, and in vivo to reduce intestinal permeability in malnourished children with diarrhoea. To determine whether malnutrition alters intestinal paracellular permeability, and whether zinc prevents such alterations. Guinea pigs were fed a normal protein diet (NP group), a low protein diet (LP group), or a low protein diet enriched with 1800 ppm zinc (LPZn group) for three weeks. Intestinal permeability was measured on jejunal segments mounted in Ussing chambers by measuring ionic conductance and mucosal to serosal fluxes of 14C-mannitol, 22Na, and horseradish peroxidase. Tight junction morphology was assessed on cryofracture replicas. Mannitol and Na fluxes and ionic conductance increased in the LP group compared with the NP group but remained normal in the LPZn group. Accordingly, jejunal epithelia from the LP group, but not from the LPZn group, showed a small decrease in number of tight junctional strands compared with epithelia from the NP group. Neither malnutrition nor zinc treatment modified horseradish peroxidase fluxes. Malnutrition is associated with increased intestinal paracellular permeability to small molecules, and pharmacological doses of zinc prevent such functional abnormality.
  • Article
    Endothelial cell junctions are complex structures formed by transmembrane adhesive molecules linked to a network of cytoplasmic/cytoskeletal proteins. At least four different types of endothelial junctions have been described (tight junctions, gap junctions, adherence junctions and syndesmos or complexus adhaerentes). Leukocytes adhesion to endothelial cells is frequently followed by their extravasation. The mechanisms which regulate the passage of leukocytes through endothelial clefts remain to be clarified. Many indirect data suggest that leukocytes might transfer signals to endothelial cells both through the release of active agents and adhesion to the endothelial cell surface. These signals could induce the disorganization of interendothelial junctions and facilitate leukocyte transmigration.
  • Article
    The acute phase response is an orchestrated response to tissue injury, infection or inflammation. A prominent feature of this response is the induction of acute phase proteins, which are involved in the restoration of homeostasis. Cytokines are important mediators of the acute phase response. Uncontrolled and prolonged action of cytokines is potentially harmful, therefore mechanisms exist which limit the activity of cytokines; these include soluble cytokine receptors and receptor antagonists. The cytokine signal is transmitted into the cell via membrane-bound receptors. Different intracellular signalling pathways are activated by different cytokine-receptor interactions. Eventually, cytokine-inducible transcription factors interact with their response elements in the promotor region of acute phase genes and transcription is induced. Systemic inflammation results in a systemic acute phase response. However, local inflammatory or injurious processes in the liver may also induce an acute phase response, for example after partial hepatectomy and during hepatic fibrosis. The acute phase proteins induced in these conditions probably act to limit proteolytic and/or fibrogenic activity and tissue damage. The possible function of the acute phase protein alpha 2-macroglobulin in hepatic fibrosis is discussed in some detail.
  • Article
    Polarized epithelial cells separate two extremely different cellular milieus. The tight junction (TJ) is the most apical component of the junctional complex and serves as the permeability barrier between these environments. The tight junctional complex appears to be a dynamic and regulated structure. Some of its protein components have been identified and include the transmembrane protein occludin. Nontransmembrane proteins on the cytosolic leaflet including ZO-1, ZO-2, cingulin, 7H6, and several unidentified phosphoproteins are also believed to be part of the TJ. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of TJ structure and may also play a role in the regulation of TJ assembly. Recent progress using the "calcium switch" and the "ATP depletion-repletion" model of TJ formation offers new insight regarding how these structures form. TJ biogenesis appears to be regulated, in part, by classic signal transduction pathways involving heterotrimeric G proteins, release of intracellular Ca2+, and activation of protein kinase C. Although many of the details of the signaling pathways have yet to be defined, these observations may provide insight into how TJs form during tubular development. Furthermore, it may be possible to suggest potential therapeutic targets for intervention in a variety of diseases (e.g., ischemia, toxic injury to the kidney and other epithelial tissue) where TJ integrity has been compromised and reassembly is required.