Nutrition and Immunology
Effect of Ascorbic Acid Nutriture on Blood Histamine
and Neutrophil Chemotaxis in Guinea Pigs1»2
CAROL S. JOHNSTON AND SHINING HUANG
Foods and Nutrition Laboratory, Department of Family
Resources and Human Development,
Arizona State University, Tempe, AZ 85287
paralleled dietary intake, and these values differed signif
icantly. Blood histamine was significantly depressed in
the high ascorbate group compared to the adequate and
low ascorbate groups, and liver ascorbate was inversely
correlated to blood histamine levels (r - -0.64, P <
0.001). The random migration of neutrophils was not
significantly affected by vitamin dosage. Leukocyte
chemotaxis was significantly impaired in low ascorbate
animals compared to that of animals with adequate as
corbate nutriture. Leukocyte chemotaxis in high as
corbate animals did not differ significantly from that in
the adequate or low ascorbate groups. Furthermore,
chemotaxis was significantly lower when cells extracted
from animals with adequate ascorbate nutriture were
incubated in low ascorbate or high ascorbate serum
rather than in autologous serum. These data suggest
that the histamine-lowering effect of supplemental as
corbate does not appear to enhance leukocyte
chemotaxis and that serum from guinea pigs fed low or
high levels of ascorbate appears to contain factors that
depress chemotaxis. J. Nutr. 121:126-130,
1991.
METHODS
Experimental design. Thirty male Hartley guinea
pigs (-150 g, Charles River Laboratories, Wilmington,
MA) were housed individually in wire-meshed cages
in a temperature-regulated (22"C), light-controlled
(lighted from 0700 to 1900 h) room. Throughout the
study, all animals were fed a convenient scorbutogenie diet, a nonpurified diet designed for rabbits
(Purina rabbit chow, Ralston Purina, St. Louis, MO),
and water ad libitum.
INDEXING KEY WORDS:
•ascorbic acid •histamine
•chemotaxis •guinea pigs
The autooxidation of ascorbic acid to dehydroascorbic acid ruptures the imidazole ring of histamine,
producing the products of histamine degradation (1,
2). Adding ascorbic acid to tissue cultures signifi
cantly reduces both endogenous histamine levels (3,
4) and histidine dccarboxylase activity, a measure of
the histarnine-forming capacity of cells (4). In hu
mans, plasma ascorbate levels > 45.4 umol/L are asso
ciated with low blood histamine levels (5), and the
therapeutic use of the vitamin protects against
0022-3166/91
'Presented at the 1990 Federation of American Societies for
Experimental Biology meeting, Washington D.C. [Johnson, C. S. &
Huang, S. (1990) Effect of ascorbic acid nutrition on neutrophil
chemotaxis. FASEB J. 4:A935. (abs. 3883)].
'Supported by donations to the Arizona State University Foun
dation.
$3.00 ©1991 American Institute of Nutrition. Received 18 December 1989. Accepted 19 June 1990.
126
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histamine-induced anaphylactic shock (6).
Oh and Nakano (4) recently demonstrated that a
parameter of the immune response, lymphocyte blastogenesis, was enhanced by ascorbic acid adminis
tration because the vitamin lowered cell culture
histamine levels that were immunosuppressive. The
present study further examined the possibility that
ascorbic acid promotes immun«function by attenu
ating the effects of immunosuppressive histamine.
Chemotaxis was the immune response chosen for
investigation because it has been established that
chemotaxis is enhanced by ascorbate (7, 8) and
depressed by histamine (9).
Leukocyte chemotaxis in 50% serum was mea
sured using cells and serum extracted from guinea
pigs fed graded doses of ascorbic acid for 4 wk. Our
objective was to determine whether the serum
histamine-lowering effect of supplemental ascorbate
was accompanied by enhanced chemotaxis.
ABSTRACT Histamine suppresses certain immune
responses, including neutrophil chemotaxis. The present
study examined whether the histamine-lowering effect of
ascorbate was accompanied by enhanced chemotaxis in
guinea pigs. Animals were fed low ascorbate, adequate
or high ascorbate diets (0.5, 2.0 or 50 mg ascor
bate-100 g body wt-'-d"1) for 4 wk. Mean liver ascorbate
ASCORBATE
NUTRITURE
AND NEUTROPHIL
CHEMOTAXIS
127
TABLE 1
Effect of ascorbic add nutritore on guinea pig body weight, liver ascorbate, blood histamine and leukocyte motility'
MeasureInitial
body weight, g
Final body weight, g
Liver ascorbate, \imol/g
Blood histamine, ng/ml.
Leukocyte random migration3 mm
Leukocyte chemotactic index3Low
ascorbate1178
ascorbate1173
± 5
± 3
432
± 9
432
±11
0.70 ± 0.05'
1.12 ± 0.05b
151
±15"
142
±11'
3.8 ± 2.5
7.8 ± 3.1
0.08 ± 0.05'Control2176 2.79 ± 0.79bHigh
± 3
437
±17
1.80 ± 0.05C
80
± 4k
3.1 ± 1.5
1.66 ± 0.88'k
'Young male guinea pigs (n - 10/group) were fed a nonpurified, scorbutogenic diet supplemented with a single, daily dose of ascorbic acid
for 4 wk. Values are means ±SEM. Means in a row not sharing a common superscript letter are significantly different, p < 0.05 (least
significant difference test).
^Ascorbic acid dose was 0.5, 2.0 and 50 mg/100 g body wt in the low ascorbate, control and high ascorbate groups, respectively.
3Leukocyte random migration and chcmotaxis in 50% autologous serum.
saline) (16). Leukocytes from the neutrophil-rich
plasma were counted in a hemocytometer and washed
twice in Hanks' balanced salt solution (HBSS),pH 7.2
(17). Cell suspensions (2.5 x IO7 cells/mL) in 50%
autologous plasma, 50% heterologous plasma or
HBSS were used for studies on agarose gel.
The agarose assay is a simple, rapid and inex
pensive method for measuring leukocyte locomotion
(18). Clean microscope slides were dipped in 0.5%
gelatin, rinsed in deionized water and air-dried. A 2%
agarose-0.5% gelatin mixture in deionized water was
dissolved over boiling water, diluted with an equal
volume of HBSS and carefully layered onto micro
scope slides (19, 20). After the gel hardened (4'C for
30 min, two sets of a series of three holes, 2.5mm
apart, were punched in the agarose of each slide using
a metal template. Agarose plugs were removed with a
hypodermic needle. A 10-jiL aliquot of the cell sus
pension was placed in the center well. Ten microliters
of a chemotactic solution (f-Met-Leu-Phe, 1Q-2mol/L
in dimethyl suLfoxide in the presenceof 2 x 10~2mol/L
2-mercaptoethanol; diluted to 10-' mol/L prior to use)
(18) was added to one of the outer wells, and 10 uL of
HBSS with gelatin (0.5%) was placed in the remaining
outer well. Slides were incubated 3 h at 37*C, fixed in
absolute ethanol for 30 min and left overnight in fresh
absolute ethanol (20). The agarose was gently re
moved after immersion (30 min) in water, and the
slide was stained (Wright stain, Diagnostic Systems,
Gibbstown, NJ) and air-dried. Random migration
(mm) and the chemotactic index (movement toward
chemotactic solution divided by movement toward
buffer) were measured microscopically using an eye
piece micrometer disc and a 40 x objective. Each cell
suspension was tested in duplicate.
Statistical analysis. Data are reported as means ±
SEM.One-way analysis of variance and least signif
icant difference tests were used to examine the differ
ences between group means (21). The Pearson
product-moment correlation test was used for corre
lation analysis.
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During the initial 2 wk acclimation period, all
animals received a single, oral dose of L-ascorbic acid
[2.0 mg/(100 g body wt-d], a level adequate for both
growth and incisor development (10). Animals were
then randomly assigned to one of three vitamin
dosage groups: low ascorbate, 0.5 mg ascorbate/(100 g
body wt-d); control, 2.0 mg ascorbate/(100 g body
wt-d); and high ascorbate, 50 mg/(100 g body wt-d).
These dosage levels have been used successfully in
previous work examining ascorbic acid and immune
function (11, 12). All vitamin mixtures were prepared
immediately prior to use in distilled water and ad
ministered orally through rubber tubing (0.125-inch
i.d.) attached to a 1-mL syringe. After a 4-wk experi
mental period, animals were killed by heart puncture
under halothane anesthesia.
Blood histamine and liver ascorbate analysis. All
tissue samples were tested in duplicate. A 5-mL
aliquot of freshly drawn blood anticoagulated with
oxalate (gray top Vacutainer, Becton Dickenson,
Rutherford, NY) was mixed with 4.5 mL deionized
water, to lyse red cells, and 0.5 mL concentrated
perchloric acid, then centrifuged for 10 min at 500 x g
(13). The histamine from the supernatant was ex
tracted by u-butanol and reextracted into an aqueous
phase prior to condensation with o-phthalaldehyde as
originally described by Shore et al. (14). Fluorescence
at 450 nm (filter no. 2A for the Turner fluorometer,
Palo Alto, CA) was measured after activation at 360
nm (filter no. 7-60).
The liver was excised immediately
and
homogenized in 9 mL ice-cold trichloroacetic acid per
gram of tissue. Following centrifugador) (3500 x g,
O'C), the supernatant was stored (-20*C) less than 5 d
and analyzed colorimetrically for vitamin content
using the dinitrophenylhydrazine reagent (15).
Leukocyte chemotaxis. Red cells and mononuclear
leukocytes from freshly heparinized blood (~8 mL,
green top Vacutainer) were sedimented by the ad
dition of 0.75 mL methyl cellulose (2% in normal
128 HUANG0.200
JOHNSTON AND
Buffer^•Low
l
serE3 ascorbate
serumESîHigh
Control
ascorbate
se4-I»'co
•c
.•^"S"
0.150-mfC¡•§
.¿Liaiï1[j1ss
'•
^^^^•^"~\.•
¡ao
0.100-I0
3 2oV1
•0.000
050 •••
0.600
umol/gFIGURE
1.200
1.800
-
2.400
Liver ascorbate,
s
ss
ss
s
sb.riT
0-c15-EV2-I
1 Blood histamine as a function of liver as
acid.Correlation
corbate
in 30 guinea pigs fed three levels of ascorbic
0.001).RESULTSSimilar
was significant (r - -0.64, P <
9.sai•pE
pigsduring weight gains were noted for all guinea
andfinalthe experimental period. The mean initial
notdiffer
body weights for the three dietary groups did
significantly (Table 1). Mean liver ascorbate1
among the three groups (0.70 ±0.05, 1.12 ±0.05 and
1.80 ±0.05 nmol/g for the low ascorbate, control, and
high ascorbate groups, respectively, Table 1). Blood
histamine was inversely related to ascorbate nutritore
(r - -0.64, p < 0.001, Fig. l ); mean blood histamine
values fell from 151 ±15 and 142 ±11ng/mL in the
low ascorbate and control groups, respectively, to 80
±4 ng/mL in the high ascorbate group. The mean
blood histamine levels observed for the low ascorbate
and control groups did not differ significantly, but
both of these values were significantly higher than
that observed for the high ascorbate group (Table 1).
The random migration of leukocytes in 50% autologous serum did not differ significantly among the
dietary treatment groups; however, cells extracted
from the low ascorbate and high ascorbate animals
migrated only 40-50% of the distance traveled by
control cells (Table 1). Leukocyte chemotaxis in 50%
autologous serum was significantly greater for cells
extracted from control sera than for cells extracted
from low ascorbate animals (p < 0.05, Table 1). Al
though the mean chemotactic response in autologous
serum of leukocytes from the high ascorbate animals
was 41 % lower than that noted for control cells, this
difference was not significant.
Neither random migration in autologous serum nor
chemotaxis in autologous serum were related to blood
histamine levels. Furthermore, these parameters were
not related to liver ascorbate.
Figure 2 depicts the chemotaxis and random
migration data for extracted leukocytes incubated in
the various test sera and buffer. Chemotaxis of cells
3-0-°-
hi
$r
STn8
sf|ll111ÕTi1
1•11111i¡^§%
s§§1S1
FIGURE 2 Leukocyte chemotaxis (a) and random
movement (b) in buffer, autologous and heterologous serum.
Leukocytes were extracted from guinea pigs (n - 10/group)
fed
low ascorbate
diet (I),control
diet±
(H)or
ascorbate
diet a (IH)for
4 wk. Values
are means
SEM.high
"Significantly
different from cell movement in low ascorbate and high
ascorbate serum, P < 0.05 (least significant difference test).
from guinea pigs fed a low ascorbate diet was not
altered significantly by the various media, but loco
motion was 57-87% less when these cells were in
cubated in autologous or high ascorbate sera relative
to that in buffer or control sera (Fig. 2a). Chemotaxis
of cells from control guinea pigs was significantly
depressed when cells were incubated in low ascorbate
sera or high ascorbate sera compared to autologous
sera (p < 0.05, Fig. la}. Chemotaxis of cells from
control animal.»was 87% greater in autologous sera
than in buffer, but the difference was not significant.
The directed migration of cells from animals fed the
high levels of ascorbate was about 50% less in low
ascorbate serum than in buffer, control sera or au
tologous sera.
The random migration of leukocytes from guinea
pigs fed low ascorbate diet was 71% greater when
cells were incubated in control sera compared to au
tologous sera, and it was 168% greater when cells
were incubated in high ascorbate sera (Fig. 2b}. A
similar, but less pronounced, trend was noted for cells
from control animals incubated in low ascorbate,
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6'o1
ASCORBATE
NUTRITURE
AND NEUTROPHIL
control and high ascorbate sera. The cells from guinea
pigs fed the high ascorbate diet, however, exhibited
the lowest random migration in autologous sera,- it
was 62% less than the migration in buffer (Fig. 2b).
None of the serum-dependent changes in random
migration were significant.
DISCUSSION
129
present study, leukocyte chemotaxis was impaired
when cells were incubated in low ascorbate serum
relative to control serum, a phenomenon that may be
attributed, in part, to relatively higher levels of
histamine and lower levels of ascorbate. However,
leukotaxis was not further amplified by high
ascorbate-low histamine serum.
High vitamin intake (25 times the control dosage
for 4 wk) did not enhance leukocyte chemotaxis.
Furthermore, serum extracted from animals fed high
levels of ascorbate appeared to contain inhibitors of
chemotaxis since the directed migration of low as
corbate and control leukocytes was lower in high
ascorbate serum than in buffer or control serum. The
inhibitors were specific for chemotaxis and did not
appear to affect the random migration of leukocytes.
Hence, any promotive
effect of the low
histamine-high ascorbate combination was masked
by inhibitory serum factors produced by tissuesaturating levels of the vitamin.
These data conflict with reports from other investi
gators who have demonstrated that supplemental as
corbate enhances leukocyte chemotaxis. Anderson et
al. (32) reported that the chemotaxis of neutrophils
extracted from healthy subjects following 1 wk of
vitamin supplementation (2 or 3 g ascorbate daily)
was elevated two- to four-fold [p < 0.05); however,
chemotaxis was not affected at the l g dosage level. In
addition, numerous investigators demonstrated that
chemotaxis was significantly elevated when normal
leukocytes were incubated in 1 to 5 x 10~3mol/L
ascorbate (7, 8, 24, 29). In these reports, neutrophil
chemotaxis was examined with cells that had been
washed and resuspended in buffer,- hence, serum-de
pendent phenomena were not examined. Our data
suggest that although cellular defects were not ap
parent in leukocytes extracted from high ascorbate
animals, serum from these animals may contain
factors that inhibit chemotaxis. In the present report,
chemotaxis of cells from control animals and
chemotaxis of cells from guinea pigs fed high as
corbate diets were similar when the cells were in
cubated in buffer, but chemotaxis of high ascorbate
cells was only 59% of that noted for control cells
when comparisons were made in autologous serum.
Investigators previously demonstrated that guinea
pigs fed high dietary ascorbate had elevated serum
levels of dehydroascorbic acid, the oxidized form of
the vitamin (1, 33), and that the rise in blood de
hydroascorbic acid was accompanied by a con
comitant increase in blood glucose (1). Blood glucose
levels in animals fed 50 mg ascorbate/100 g body
weight, the dosage used in the present report, rose
from the baseline value of 100 to 284 mg/100 ml.
after 15 d of vitamin treatment (1). The hyper
glycémieeffect of dehydroascorbic acid seems to be
associated with abnormalities of the beta cells of the
islets of Langerhans of the pancreas (34). Since insuffi-
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These data demonstrate that the chemotactic re
sponse of leukocytes was altered by ascorbic acid
nutriture in guinea pigs. Compared to control dosage
levels, suboptimal intake of the vitamin (25% of the
control dosage for 4 wk) significantly depressed cell
chemotaxis, an effect that may involve cellular
defects as well as serum inhibitors. The cellular
defects appeared to be specifically related to the
chemotactic apparatus of the leukocyte and not to
basic locomotion mechanisms because the random
migration of low ascorbate cells in buffer was not less
than that of control cells (Fig. 2b). The chemotactic
index of low ascorbate cells in buffer, however, was
only 28% of that noted for the control cells (Fig. la}.
The presence of chemotaxis inhibitors in serum ex
tracted from animals fed suboptimal ascorbate was
evident since the directed migration of both low as
corbate and control leukocytes tended to be lower
when the cells were incubated in low ascorbate serum
relative to movement in buffer or control serum (Fig.
la}.
Leukocyte chemotaxis, cell movement directed by
external chemical gradients, is probably initiated by
the binding of chemoattractants to membrane recep
tors, followed by second messenger release and subse
quent rearrangement of the cytoskeletal proteins, a
phenomenon known as polymerization (22, 23). Low
leukocyte ascorbate levels may impair the assembly
of these contractile proteins. Boxer et al. (24) demon
strated that ascorbic acid promoted intracellular leu
kocyte microtubule assembly. In a separate report,
these investigators showed that ascorbic acid supple
mentation
significantly
improved
leukocyte
chemotaxis in patients with Chediak-Higashi syn
drome, a disease characterized by impaired neutrophil
microtubule assembly (25). The mechanism of ascorbate-enhanced microtubule assembly is not
known, but the vitamin may alter the redox state of
tubulin sulfhydryl groups (24).
Exogenous agents modulate leukocyte chemotaxis
by altering second messenger release and cell poly
merization. Histamine lowers the chemotactic
responsiveness
of leukocytes,
presumably
by
elevating intracellular cAMP levels via the H-2
receptor (9, 26, 27) and depressing microtubule as
sembly (28). Conversely, exogenous factors that in
crease intracellular cGMP, including ascorbic acid
(29, 30), enhance leukocyte chemotaxis (31). In the
CHEMOTAXIS
130
JOHNSTON AND HUANG
cient insulin impairs leukocyte chemotaxis (35), this
may represent a possible mechanism for chemotaxis
inhibition in the high ascorbate serum.
This study demonstrates that low ascorbate
nutriture impairs leukocyte chemotaxis. Both cellular
defects and serum inhibitors appear to be responsible
for this immunodeficiency. High ascorbate nutriture,
however, does not promote leukotaxis, although
blood histamine, a negative regulator of cell
chemotaxis, is significantly depressed. The data
suggest that tissue-saturating levels of the vitamin
induce serum conditions that depress leukocyte
chemotaxis.
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