Nutritional Immunology

Dietary Zinc Alters Early Inflammatory Responses during Cutaneous Wound

Healing in Weanling CD-1 Mice1,2
Yunsook Lim,* Mark Levy,* and Tammy M. Bray*3
*Department of Human Nutrition, The Ohio State University, Columbus, OH and Linus Pauling Institute,
Oregon State University, Corvallis, OR

ABSTRACT Zinc deficiency is a well-known health problem associated with delayed wound healing, yet the
precise mechanisms that underlie the delay remain unknown. We hypothesized that zinc deficiency delays wound
healing as a result of decreased nuclear factor (NF)B activation, reduced expression of proinflammatory cytokines
[interleukin (IL)-1 and tumor necrosis factor (TNF)-], and a decrease in neutrophil infiltration during the early stage
of cutaneous wound healing. We used a cutaneous, full-thickness excisional wound model in CD-1 mice to
examine the rate of wound closure as well as mRNA levels of inhibitory (I)B, IL-1, and TNF- and infiltration of
neutrophils at the wound site of mice fed a diet containing 1 (deficient), 50 (control), 500, or 1000 g zinc/g diet.
Zinc deficiency reduced the rate of wound closure and mRNA levels of IL-1 and TNF- and attenuated infiltration

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of neutrophils at the wound site compared with controls. Interestingly, zinc supplementation at 1000 g/g delayed
the rate of wound closure and decreased mRNA levels of TNF- and infiltration of neutrophils compared with mice
fed the control diet. These findings demonstrate that zinc deficiency and high-dose zinc supplementation delay
wound healing as a result of altered inflammatory responses and suggest that adequate zinc supplementation may
have beneficial effects on the inflammatory responses to enhance cutaneous wound healing. J. Nutr. 134:
811 816, 2004.

KEY WORDS: wound healing zinc inflammation proinflammatory cytokines

Zinc plays an essential role in growth, immune function,
antioxidant defense, and wound healing (13). Zinc deficiency
was shown to increase the time for wound closure and to
decrease wound strength (4,5). In addition, zinc has been used
as a topical agent to treat diaper rash and as a nutritional
supplement in patients with bedsores, ulcers, and incisional
wounds (6 8). Although the role of zinc in wound healing has
been investigated since the 1950s (9), the mechanisms by
which zinc affects healing processes are not clear.
Wound healing is a complicated network that involves the
interaction and coordination of various cell types, structural
proteins, cytokines, and reactive oxygen species (ROS).4 In
general, there are three major stages of wound healing, i.e.,
inflammation, proliferation, and remodeling (10,11). Al-
though it is a highly dynamic process, inflammation is consid-
ered to be a critical stage for establishing an environment that
facilitates the subsequent stages of the healing process (12).
The initial event during the inflammatory stage is the infil-
1
Presented in part in abstract form at the 9th annual meeting of the Oxygen
Society, November 2002, San Antonio, TX [Lim, Y., Quan, N. & Bray, T.M. (2002)
Zn supplementation affects the early inflammatory phase of cutaneous wound
healing. Free Radic. Biol. Med. 33: S332 (abs.)].
2
Supported by National Institutes of Health Grant DK55847 and NS38315 to
T.M.B.
3
To whom correspondence and reprint requests should be addressed.
E-mail: tammy.bray@oregonstate.edu.
4
Abbreviations used: IB, inhibitory B; IL-1, interleukin-1; NFB, nu-
clear factor B; ROS, reactive oxygen species; TNF-, tumor necrosis factor-;
VEGF, vascular endothelial growth factor.
tration of neutrophils into the wound site to prevent infection
through phagocytic processes and to induce vascular endothe-
lial growth factor (VEGF) production in macrophages by ROS
production (1315). However, excessive ROS production can
cause tissue damage and may impair wound healing (16,17).
The recruitment and function of neutrophils were shown to
be regulated by several chemotactic factors, which are stimu-
lated by interleukin-1 (IL-1) and tumor necrosis factor-
(TNF-) (18,19). Both mR levels of IL-1 and TNF- are
regulated by nuclear factor B (NFB), a critical transcription
factor that regulates the expression of inflammatory mediators
such as cytokines, chemokines, and cell adhesion molecules
(20,21). Hence, NFB plays a pivotal role during the inflam-
matory stage, and NFB activation is thus proposed to be a
critical event of early wound healing.
Despite the considerable importance of inflammation, little
research has focused on the effect of zinc with regard to
pathways that control the early inflammatory responses during
wound healing. Recently, zinc deficiency was shown to reduce
NFB nuclear binding activity in vitro and in vivo (22,23).
However, there is limited evidence that zinc may alter NFB
binding activity and subsequently regulate early wound heal-
ing processes.
We hypothesized that the mechanism by which zinc defi-
ciency delays wound healing involves an alteration in NFB
activation during the inflammatory stage of cutaneous wound
healing. Moreover, we proposed that dietary zinc supplemen-
tation with high levels of zinc may enhance and accelerate

0022-3166/04 $8.00 2004 American Society for Nutritional Sciences.

Manuscript received 2 September 2003. Initial review completed 26 September 2003. Revision accepted 27 January 2004.

811
812 LIM ET AL.

inflammatory responses and promote wound healing. In this
study, we used a cutaneous, full-thickness, excisional wound
model to examine the effects of dietary zinc deficiency and zinc
supplementation on the rate of wound closure, and the tem-
poral kinetics of IL-1, TNF-, and inhibitory (I)B mRNA
expression and neutrophil infiltration during the early inflam-
matory stage of cutaneous wound healing.
MATERIALS AND METHODS
Animals and diets. Female CD-1 mice (3 wk old, 1518 g) were
obtained from Harlan and housed in individual metal wire cages in a
temperature- and humidity-controlled room (12-h light:dark cycle)
with free access to tap water and food. Food intake and body weights
were measured every other day. Mice were acclimated for 2 d before
initiation of dietary treatments. They were fed a modified AIN-93G
rodent powder diet (24) containing 1, 50, 500, or 1000 g zinc/g
diet for 2 wk (Dyets). All mice were used in accordance with animal
protocols approved by the Ohio State University Institutional Lab-
oratory Animal Care and Use Committee.
Zinc concentrations in serum and skin. Serum was separated by
centrifugation at 2000 g for 10 min. Zinc concentration in serum
or skin was assessed according to the method of Luterotti et al. (25).
Dako Company. In situ hybridization was carried out with digoxige-
nin-labeled probes for IB, IL-1, or TNF- mRNA on slide sec-
tions as previously described (26). Diluted antidigoxigenin antibody
was added onto the slides, followed by anti-mouse antibody, primary
streptavidin, biotinyl tyramide solution, and finally CY2-streptavidin.
The slides were then viewed with a microscope through a fluores-
cence filter. The mRNA level was quantified by measuring the
intensity of cells (neutrophils) expressing mRNA of IB, IL-1, and
TNF- using Adobe Photoshop and ImageJ software. mRNA quan-
tified spots were located at the wound edges and within the wound
bed (3 spots per slide). One slide per wound from each of 4 mice/
group was examined.
Statistical analysis. All values are expressed as means SEM.
Data were analyzed by 1-way ANOVA at each time point, and then
differences among means were analyzed using Duncans test. The
paired t test was used to compare differences in zinc concentration
between unwounded and wounded skin in each group. For all tests,
differences were considered significant at P 0.05.
RESULTS
Body weight and food intake. Weight change and food
intake did not differ among the groups throughout the 2-wk
dietary treatment (data not shown).

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Serum or skin tissues were digested in 2 mol/L hydrochloric acid for Zinc concentrations in serum and skin. Serum zinc con-
24 h at room temperature. Samples were then centrifuged at 7000 g
for 25 min, and the supernatant was used for direct measurement of centration was reduced in mice fed the zinc-deficient diet and
metal concentrations using an atomic absorption spectrometer was increased in mice fed zinc-supplemented diets (500 or
(Model AA-5, Varian Techtron). 1000 g/g) compared with mice fed the control diet (P
Wound biopsy. Mice were anesthetized with isoflurane and the 0.05) (Fig. 1A). Zinc concentrations of unwounded and
back of the mouse was shaved and sterilized using an alcohol swab. wounded skin in mice fed the zinc-deficient diet were signifi-
The wound biopsy model used in this experiment was described cantly lower than those of mice fed the control diet and mice
previously (26). The shaven skin was then pinched and folded, and a fed zinc-supplemented diets (Fig. 1B). However, zinc concen-
sterile biopsy punch (3.5-mm diameter, Miltex Instrument Company)
was used to punch through the full-thickness of the folded skin. This
yielded two circular wounds, identical in size, on the dorsum, below
the shoulder blades, of each mouse. A wound placed in this area
cannot be reached by the mice, thereby eliminating self-licking.
Measurement of wound closure. Wounds from individual mice
were photographed digitally every day, beginning on the day of
wounding (d 0) with a standard dot equivalent to the initial wound
area placed beside the wound. Wound closure was quantified using
Canvas 7SE software (Deneba). The rate of wound closure was
expressed as the ratio of wound area (each day after wounding)
compared with the initial wound area. A smaller wound ratio indi-
cates faster wound closure.
Harvesting and histologic analysis. Mice were killed with an
overdose of isoflurane to collect tissue samples at the wound site for
examination of neutrophil infiltration into the wound area. Wounds
at 0, 6, 12, and 24 h after wounding were removed by cutting a square
area that covered the entire wound site from 4 mice from each
treatment group. Harvested tissues were immediately stored in 4%
formaldehyde solution in PBS (pH 7.4) and were then washed in
PBS, dehydrated in a series of alcohols, and embedded in paraffin.
Microtome sections (5-m thick) were cut vertically across the
wound site, adhered to slides, and stained with hematoxylin and
eosin. Photographs of the wound site were obtained; the images were
digitized using Adobe Photoshop (Adobe Systems), and the density of
neutrophil infiltration quantified using ImageJ (NIH). Neutrophil
infiltration into the wound site was quantified at 3 spots on the
wound edge and within the wound bed in each wound by densito-
metric analysis.
In situ hybridization (immunofluorescence). The in situ hybrid-
ization protocols were performed as described previously for ribonu-
cleotide (cDNA) probes (26,27). Antisense probes were transcribed
using the Riboprobe System (Promega Biotech) with T7 RNA poly-
merase. Mouse cDNAs of IB and TNF- were generously provided
by Dr. Rebecca Taub (University of Pennsylvania, Philadelphia, PA)
and Dr. Karl Decker (Albert-Ludwigs-Universitat, Freiburg, Ger- FIGURE 1 Zinc concentrations in serum (A) and in unwounded
many) respectively. A 672-bp DNA sequence of murine IL-1 and wounded skin (B) in mice fed diets containing 1 g zinc/g (ZD),
mRNA was prepared from commercially available insert #963357 in 50 g zinc/g (Control), 500 g zinc/g, or 1000 g zinc/g for 2 wk. Values
vector pT7T3D-pac cloned in host Escherichia coli (American Type are means SEM, n 4. Means for a variable without a common letter
Culture Collection). Immunofluorescence kits were obtained from differ, P 0.05.
 DIETARY ZINC ALTERS EARLY WOUND HEALING 813

tration in wounded skin did not differ from unwounded skin in

the same mouse from all groups. Notably, serum and skin zinc
concentrations did not differ between mice fed the 500 and
the 1000 g/g zinc diet.
Wound closure rate. The rate of wound closure in mice
fed the zinc-deficient diet was significantly slower than that of
mice fed the control diet during the early inflammatory stage
(d 1 4) of wound healing (Fig. 2). The rate of wound closure
in mice fed the 500 g/g zinc diet was faster than that in mice
fed the zinc-deficient diet. Interestingly, the rate of wound
closure of mice fed the 1000 g/g zinc diet was significantly
slower than that of mice fed the control diet or the 500 g/g
zinc diet from d 1 to 4.
mRNA expression of inflammatory mediators and infiltra-
tion of neutrophils. Levels of IB mRNA tended to be
greater (P 0.055 0.085) in mice fed the 500 g/g zinc diet
at each time point compared with mice fed the control diet
(Fig. 3). Moreover, the IB mRNA level in mice fed the 500
g/g zinc diet was significantly higher at 6, 12, and 24 h than
in mice fed the 1000 g/g zinc diet, and at 6 and 24 h
compared with mice fed the zinc-deficient diet (P 0.05).
IL-1 (Fig. 4A) and TNF- (Fig. 4B) mRNAs were de-

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tected in the dermis along the wound edge and clot. The IL-1
mRNA level in mice fed the zinc-deficient diet was lower than
that of control mice at 6 and 12 h after wounding (P 0.05)
(Fig. 4A). On the other hand, TNF- mRNA was differently
expressed in mice fed the various levels of dietary zinc. The
TNF- mRNA level in mice fed the zinc-deficient diet was
lower (43 64%) than in control mice at 6 and 12 h (P 0.05)
(Fig. 4B). The TNF- mRNA level in mice fed the 1000 g/g
zinc diet was lower (4758%) than in control mice at 6 and
24 h after wounding (P 0.05).
Neutrophil infiltration into the wound site of mice fed the
zinc-deficient diet was reduced compared with that of mice fed
the control diet and mice fed the 500 g/g zinc diet at 6, 12,
and 24 h after wounding (P 0.05). In addition, mice fed the
500 g/g zinc diet exhibited greater neutrophil infiltration at
24 h after wounding than mice fed the control diet (P 0.05)
(Fig. 5B). However, neutrophil infiltration in mice fed the
1000 g/g zinc diet was significantly lower than in mice fed the
control diet at all time points.

FIGURE 3 IB mRNA expression in cutaneous wounds of mice

fed diets containing 1 g zinc/g (ZD), 50 g zinc/g (Control), 500 g
zinc/g, or 1000 g zinc/g for 2 wk. (A) Representative photographs of
IB mRNA (bright green color) determined by in situ hybridization
fluorescence with a digoxigenin-labeled antigen probe. i and ii, 12 and
24 h after wounding in control mice. iii and iv, 12 and 24 h after
wounding in zinc-deficient mice. v and vi, 12 and 24 h after wounding
in zinc-supplemented mice at 500 g/g. vii and viii, 12 and 24 h after
wounding in zinc- supplemented mice at 1000 g/g. (B) Quantified IB
mRNA level expression determined by densitometry. Values are mean
SEM, n 4. Means at a time without a common letter differ, P
0.05. ADU: arbitrary density unit.

DISCUSSION
In these experiments, we investigated the effects of dietary
FIGURE 2 The rate of wound closure in mice fed diets containing zinc on cellular and molecular events in the early inflamma-
1 g zinc/g (ZD), 50 g zinc/g (Control), 500 g zinc/g, or 1000 g tory stage of cutaneous wound healing in vivo. Our data
zinc/g for 2 wk. The area of the wound of any time point was relative to demonstrated that dietary zinc deficiency delayed wound clo-
the area of the wound on d 0 (set at 1.0). Values are means SEM, n sure rate and reduced specific markers of the inflammatory
6. Means at a time without a common letter differ, P 0.05. response, including mRNA levels of the proinflammatory cy-
814 LIM ET AL.

supplemented with high levels of zinc (28 30). Hence, both

zinc deficiency and high-dose zinc supplementation may be
deleterious for cutaneous wound healing.
To investigate the molecular mechanisms through which
dietary zinc alters the rate of wound closure, we examined
NFB activation through the expression of IB, a down-
stream gene that is regulated by and mirrors the activity of
NFB (31,32). NFB is a redox-sensitive transcription factor
that regulates inflammatory mediators such as cytokines, che-

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FIGURE 4 IL-1 (A) and TNF- (B) mRNA expression in cutane-
ous wounds of mice fed diets containing 1 g zinc/g (ZD), 50 g
zinc/g (Control), 500 g zinc/g, or 1000 g zinc/g for 2 wk. IL-1 and
TNF- mRNA levels were determined by an in situ hybridization fluo-
rescence method and quantified by densitometry. Values are mean
SEM, n 4. Means at a time without a common letter differ, P
0.05. ADU: arbitrary density unit.

tokines, IL-1 and TNF-, and neutrophil infiltration during

the early inflammatory stage of wound healing. Moreover,
these experiments demonstrated that although zinc supple-
mentation at 500 g/g diet had beneficial effects on the
inflammatory response and wound closure rate, high-dose zinc
supplementation (1000 g/g) negatively affected the inflam-
matory response of wound healing.
As expected, wound and serum zinc levels were significantly
lower, and wound size was significantly greater, in mice fed the
zinc-deficient diet (1 g/g zinc) compared with mice fed the
control diet (50 g/g zinc) or the 500 g/g zinc- supplemented
diet (Figs. 1, 2). However, neither skin nor circulating zinc
level was an unambiguous predictor of the rate of wound
closure. In particular, the rate of wound closure was signifi-
cantly slower in mice fed the high zinc diet (1000 g/g) FIGURE 5 Neutrophil infiltration into cutaneous wounds of mice
compared with mice fed the control diet or the 500 g/g fed diets containing 1 g zinc/g (ZD), 50 g zinc/g (Control), 500 g
zinc-supplemented diet, despite the fact that there was no zinc/g or 1000 g zinc/g for 2 wk. (A) Representative photographs of
significant difference in skin and serum zinc levels between the neutrophil infiltration (arrow heads) into cutaneous wounds determined
two zinc-supplemented groups. Thus, it appears that the del- in hematoxylin and eosin (H&E) stained sections. i and ii, 12 and 24 h
after wounding in control mice. iii and iv, 12 and 24 h after wounding in
eterious effects of dietary zinc supplementation at 1000 g/g
zinc-deficient mice. v and vi, 12 and 24 h after wounding in zinc-
zinc may be mediated by a mechanism that is independent of supplemented mice at 500 g/g. vii and viii, 12 and 24 h after wounding
either serum or wound tissue zinc level. Previous work showed in zinc-supplemented mice at 1000 g/g. (B) Densitometric analysis of
that very high zinc intakes may decrease copper absorption, neutrophil infiltration into cutaneous wounds. Values are expressed as
leading to copper deficiency and anemia and may play an mean SEM, n 4. Means at a time without a common letter differ,
important role in the observed immunodepression of patients P 0.05. ADU: arbitrary density unit.
 DIETARY ZINC ALTERS EARLY WOUND HEALING
mokines, cell adhesion molecules, and extracellular matrix
molecules during inflammation, and is normally maintained in
an inactive form in the cytoplasm bound to the inhibitory
protein, IB (33). During activation by various stimuli such as
UV light, ROS, proinflammatory cytokines, and virus infec-
tion, IB is phosphorylated and degraded, facilitating the
translocation of NFB to the nucleus where it regulates the
expression of immune and inflammatory genes (33). Our data
demonstrated that IB mRNA levels generally were lower in
both the zinc-deficient and the 1000 g/g zinc-supplemented
groups than in mice fed the control or 500 g/g zinc diet (Fig.
3). Furthermore, the delayed rate of wound closure in zinc-
deficient or 1000 g/g zinc-supplemented mice paralleled the
decrease in IB mRNA level. Zinc plays a role as an anti-
oxidant in protecting sulfhydryl groups, essential in protein
stability and activation, from oxidation and prevents superox-
ide and hydroxyl radical production by prooxidant metals,
copper, and iron (2,34,35). Therefore, zinc deficiency may
increase oxidative stressinduced tissue damage by decreasing
antioxidant functions. Previous studies showed that zinc defi-
ciency reduces NFB binding activity after oxidative stress in
vitro and in vivo and impairs NFB translocation in vivo
(22,23,36). Therefore, our data suggest that zinc deficiency
may delay wound healing as a result of impaired NFB binding
activity secondary to increased oxidative stress.
To confirm the effect of dietary zinc on NFB activation
and subsequent events during the early inflammatory stage,
mRNA levels of the proinflammatory cytokines IL-1 and
TNF-, target genes of NFB activation, were investigated at
different time points. Because of their biological roles in the
activation of neutrophils, stimulation of fibroblast and kera-
tinocyte growth, synthesis and breakdown of extracellular ma-
trix proteins, and regulation of immune responses (37), it is
possible that decreased expression of these cytokines may delay
the wound healing process by impairing tissue regeneration,
thus delaying restoration of normal tissue structure and func-
tion. Although the mRNA level of IL-1 was reduced only by
zinc deficiency (Fig. 4A), TNF- mRNA levels were reduced
by zinc deficiency as well as high-dose zinc supplementation
(1000 g/g) (Fig. 4B). Interestingly, mRNA levels of IL-1
and TNF- returned to control levels 24 h after wounding in
zinc-deficient mice, whereas high-dose zinc supplementation
significantly depressed TNF- levels throughout the 24-h pe-
riod. Therefore, these results suggest that zinc deficiency or
high-dose zinc supplementation may exert their deleterious
effects through alterations in the expression of proinflamma-
tory cytokines.
In addition to their putative role in stimulating the expres-
sion of growth factor genes that are important in cutaneous
wound healing (38), IL-1 and TNF- modulate the expres-
sion of chemokines and adhesion molecules necessary for the
recruitment of inflammatory cells to the site of injury (39).
Neutrophils are the predominant cell type at the wound site
within 24 h of wounding where they function not only as
phagocytic cells, but also as cells that synthesize and release
various cytokines and chemokines that are essential to pro-
moting subsequent steps in the wound healing process (40).
Similar to our other results, consumption of either the zinc-
deficient (1 g/g) or the high-zinc (1000 g/g) diet may
impair the wound healing process through their effects on
neutrophil infiltration at the wound site (Fig. 5). However,
neutrophil infiltration at the wound site in mice fed the 500
g/g zinc diet was comparable to that of control mice. Other
studies have shown that not only the recruitment of neutro-
phils but also their chemotactic activity is disturbed by zinc
deficiency or supplementation in vitro (41 43). As noted
815
previously, high-dose zinc supplementation may induce copper
deficiency, which can cause both neutropenia and functional
impairment of neutrophils including the chemotactic response
and phagocytic activity (28 30). Indeed, it was demonstrated
in human studies that a zinc intake 20-fold greater (300 mg/d)
than the current RDA (15 mg/d) decreased several indices of
immune function, including the chemotactic response and
phagocytic activity of neutrophils (44). Hence, these findings
suggest that both zinc deficiency and high-dose zinc supple-
mentation may impair wound healing by adversely affecting
the recruitment and phagocytic activity of neutrophils.
Taken together, the results of this study support the hy-
pothesis that zinc deficiency delays the early inflammatory
response resulting in delayed wound healing. Furthermore,
these data clearly demonstrate that although moderate zinc
supplementation may be beneficial, excessive zinc supplemen-
tation is unequivocally deleterious in terms of its influence on
cutaneous wound healing. Because of the central role of NFB,
which is required for the induction of proinflammatory cyto-
kines such as IL-1 and TNF- during inflammation, de-
creased activation of NFB may lead to a delayed inflamma-
tory cascade, culminating in immunosuppression and delayed
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wound healing in zinc deficiency and high-dose zinc supple-
mentation.
In summary, the current study demonstrated that zinc de-
ficiency exerts negative effects on the early inflammatory re-
sponse of cutaneous wound healing. Understanding the mo-
lecular mechanisms that regulate cutaneous wound healing is
critical not only in zinc deficiency, but also in numerous other
disorders associated with abnormal wound repair. Hence, the
results of this work may provide critical insight into future
nutritional intervention strategies designed to enhance im-
mune function not only in patients suffering from zinc defi-
ciency, but also in patients suffering from malnutrition-related
disorders.
ACKNOWLEDGMENT
We are grateful to Dr. Quan and his laboratory for the technical
support for the histology assay and in situ hybridization.
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