Luean
E. Anthony,4
Ph. D., Carole
ABSTRACT
Guinea
fed, and 10
ad libitum
chicken
chicken vitro fed
erythrocytes
SiCr and
rosettes defect
release ad
libitum
with
rabbit
of ascorbic
divided
weeks
cytotoxicity
chicken guinea
a change
pigs. may
The
in
American
workers
ofClinical
Nutrition
groups:
were
comparable cells
marker,
in number
deficient,
intradermally
less of
was
the
an
impairment
or
function
splenic
same
three with
than
with three
cells cells
of
another
cell
cells
from pair-
formed
groups.
function type.
In
from that
dietary
of T lymphocyte
to
groups.
lymphoid spleen
5 x
titers
dietary
lymphoid
in all
pair-
with
antibody
in all incubated
significantly percentage
reflect
ascorbic-acid
immunized
Hemagglutinating
target
was
M.D.
Am.
The in
cell-
I.
Clin.
1979.
vitamin-deficient
Journal
dietary
adjuvant.
The
a T cell
observed thymic atrolaboratory animals and man, it was not until 1958 (1) that the first systematic studies of the role of vitamins in cellular immunity were published by Axeirod et al. (1). Vitamin B6 deficiency has received the greatest attention, and results indicate that lack of pyridoxine decreases delayed skin and systemic hypersensitivity, increased survival time of skin homografts (1, 2) and reduced lymphocyte proliferation in vitro as measured by the mixed lymphocyte reaction (3). Little attention has been given to the effects of other vitamin deficiencies on cellular mechanisms of immunity. Studies of the role of ascorbic acid (AA) on cellular immunity have led to contradictory conclusions. Mud1cr et al. (4) found that scorbutic guinea pigs failed to develop experimental allergic encephalomyelitis or delayed hypersensitivity skin reaction to mycobacterium tuberculosis. However, Trakatellis et al. (5) reported no depression of skin or systemic reactivity after sensitization of AA-deficient guinea pigs to mycobacterium tuberculosis. More recently prolonged survival of skin allografts in scorbutic guinea pigs has been reported (6). The role of AA in the humoral antibody response is also a matter of dispute. Long (7) phy
pigs
B. Taylor,
pigs were
erythrocyte
guinea
deficiency or
three
immunization
erythrocytes, acid
into
later guinea complete
after
labeled control
M. S., and Keith
Freund’s
acid-deficient
Nuir. 32: 1691-1698,
early
in
from
were
Two
2 weeks
of ascorbic
mediated
While
pigs
control.
erythrocytes
spleens
G. Kurahara,
immune
32: AUGUST
observed a deleterious effect on the secondary immune response to diphtheria toxoid by guinea pigs fed an AA-deficient natural diet. Kumar and Axelrod (8) induced severe scurvy in guinea pigs by feeding a purified AA-deficient diet and found no change in secondary antibody response with the same antigen. The primary response in both reports was unaffected. The present work further examines the effect of AA deficiency on cell-mediated immunity and the primary humoral response. Using chicken erythrocytes as target cells, the cytotoxicity of spleen lymphoid cells from immunized AA-deficient guinea pigs was assessed by a 51Cr release technique. The percentage of spleen lymphoid cells binding rabbit erythrocytes (T cell marker), the number ofperipheral white blood cells, the percentage of T cells and the hemagglutinating serum I From partment Hospital,
2
Stanford of Medicine Palo Alto,
Supported
University and the California
by the
School Veterans 94304.
Medical
of Medicine, Administration
Research
Service
of the
Veterans Administration and by United States Health Service Grant 5T32 HL 07034-03. 3 Address reprint requests to: Dr. K. B. Taylor, Hospital, Medical Service, Palo Alto, California 4
1979,
Present
address:
School
at
Texas
77030.
pp.
Houston,
1691-1698.
the
University
Department
Printed
in U.S.A.
of Texas of
Surgery,
Dc-
Public V.A. 94304.
Medical Houston,
1691
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Cell-mediated cytotoxicity and humoral response in ascorbic acid-deficient guinea pigs13
1692
ANTHONY
Materials
in pair-
Ascorbic
also
Clarke
(12), adapted
Diets
the
guinea
Test
Diets,
allowed
Madison,
group of guinea pigs was pair-fed the AA-supplemented diet. Each guinea pig in the pair-fed group received an amount of diet equal to that consumed by its AA-deficient partner during the previous day. Materials
Hanks’
balanced
(Grand
salt solution,
tissue
culture
medium
serum (FCS) and penicillin-streptomycin obtained from Grand Island Biological
Island,
N.Y.),
Ficoll
(Pharmacia
Inc.,
moved, Hanks’ Medium
the remaining balanced salt 199.
cells were solution
washed and then
three times suspended
at 15,000
to develop
Boyum
was
trichloroacetic
The samples were 15 mm at 4 C, and the
for 15 mm at room
temperature
and
cells
(10)
with
were
minor
isolated
modification.
by the
method
The
heparinized
of
blood
was diluted 1:1 with Hanks’ balanced salt solution then layered on a Ficoll and Hypaque gradient and then centrifuged for 35 mm (400 x g). The lymphocyte layer was removed from the interface, suspended in medium 199, and washed twice with the same medium. Cell suspensions were adjusted to 5 x 10’ cells per milliliter. The viability of the lymphocytes was found to be above 95% by trypan blue exclusion. and
rosette
Percentage
ofT
determination
cells was determined
by the formation
of spontaneous nonimmune rosettes with rabbit erythrocytes (14). One hundred microliters of lymphocyte suspension and 100 tl of rabbit red blood cells (80 x l0”/ml) in medium 199 were centrifuged together for 2 mm at 400 x g. The preparations were made in duplicate
and allowed were gently examined lymphocytes
to stand at 4 C for 1 hr or more. resuspended, stained with toluidine microscopically. the percentage
or more cytometer. three
Cytotoxicity
Immunization
x g for
40%
cell preparation
T lymphocyte
in in
et al. (13). Protein
the procedure.
Mononuclear
Pis-
cataway, NJ.) and Hypaque (Winthrop Laboratories, New York, N.Y.) were mixed according to the method of Boyum (10) and used for the separation of lymphocytes. Chicken red cells (CRC) in Alsever’s solution were purchased from Microbiological Media, Concord, Calif. For the T rosette assay blood from the same rabbit (Lit Rabbitry, Aptos, Calif.) was bled from the ear into acidcitrate-dextrose. After plasma and buffy coat were re-
by Zannoni
adding 0.25 ml of of plasma on ice.
read at 525 nm. Standards for each set of experimental samples were determined in duplicate. The tissues from AA-deficient animals were homogenized in 9 volumes and the tissues from control animals in 19 volumes of cold 5% trichloroacetic acid and carried
Blood
Wis.) or the same diet supplemented with AA (4 g/kg). In one experiment to test the effect of inanition and”weight loss on the cytotoxic response an additional
of plasma and tissues was asof the method of Sullivan and
following reagents then added to 0.75 ml of supernatant: 0.05 ml of 85% orthophosphoric acid, 0.05 ml of 8% aa’-dipyridyl in ethanol, and 0.05 ml of 3% aqueous ferric chloride. The ferrous-dipyridyl chromophore was
through the 4-week experimental period fed an AA-deficient diet(9)(Teklad
199, fetal calf mixture were
by ml
centrifuged
Outbred, male, Hartley guinea pigs (Simonsen Laboratories, Gilroy, Calif). weighing 350 to 400 g at the outset were fed a commercial chow diet for 1 week after they were received from the suppliers. Experimental procedures were started immediately after this period of adaptation. The guinea pigs were then maintained on diets and water ad libitum and weighed weekly.
During pigs were
assay
precipitated acid to 2.0
Animals
Co.,
acid
Ascorbic acid content sayed by a modification
methods
and
AL.
erythrocytes
From of
The cells blue and
counts of 200 to 300 lymphocytes binding
was enumerated
in a hema-
assay
guinea pigs from each dietary group were given 5 x 10” washed CRC in 0.4 ml of Freund’s complete adjuvant intradermally in the nuchal region, 0.1 ml per site.
Using chicken erythrocytes as target cells, the cytotoxicity of cells isolated from the spleens of guinea pigs was assessed by the 51Cr release technique described by Loewi and Temple (15). Individually, spleens were aseptically removed, weighed, and minced with scissors in
Blood
Petri dishes containing medium 199. The spleen fragments were passed through a stainless steel tissue sieve and washed twice in medium 199. Final cell dilutions were made in medium 199 containing 10% FCS with 100
After
2 weeks
collection
The animals (Pittman
bled
Moore,
on
and
the
counts
were lightly Inc.,
diet
anaesthetized
Washington
(3 to S ml) by cardiac
puncture.
with Metafane
Crossing,
The
NJ.)
and
concentration
of white blood cells in the samples was enumerated in a hemacytometer utilizing 0.01% crystal violet in 2% acetic acid. Wright-stained blood smears were used for differential white cell counts. Sera were stored at -20 C. Antibody
titration
Two weeks
units of penicillin and liter. All FCS was heat suspension was layered
antibody
titers
elicited
with CRC were measured by the hemagglutination method using doubling dilutions of sera in saline (11).
per milliof the cell
on a Ficoll-Hypaque gradient for preparation of mononuclear cells for the estimation of T rosettes as described above. For labeling with 5tCr, washed CRC were suspended in
after immunization
100 sg of streptomycin inactivated. A portion
medium
199 with
4%
FCS
at a concentration
of 4 x
l0 CRC/0.l ml and incubated at 37 C for I hr in the presence of 100 tCi sodium 5tCr-chromate (specific activity not less than 200 tCi4tg chromium, Amersham/
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/8/1691/4692281 by East Carolina University Health Sciences Library user on 13 January 2019
antibody titers to chicken erythrocytes blood were measured. The effects of feeding on the same parameters were examined.
ET
ASCORBIC
ACID
DEFICIENCY
Arlington Heights, Ill.). After washing four to six in medium 199, the cells were diluted to a concenof iO CRC/ml in medium 199, containing 10% FCS with 100 units of penicillin and 100 g of streptomycin per milliliter. Preliminary experiments using immunized control guinea pigs confirmed the finding of Loewi and Temple ( 15) that cytotoxicity was detectable at an effector:target cell ratio as low as 5: 1 and reached a plateau at a ratio 50: 1. Given the limited number of cells in individual spleens effector:target cell ratios of 10: 1 and 50: 1 were used in subsequent experiments. Cytotoxicity tests were done in 2 ml volumes of medium 199 containing 10% FCS and antibiotics. The cultures contained 0.5 ml spleen effector cells ( 10 x 10 or 50 X iO cells), 0.5 ml guinea pig red blood cells (1 x 10’ cells) and 1 ml CRC (1 X iO cells). Similar cultures omitting the guinea pig red blood cells were also examined. Each tube was set up in duplicate. Incubation was for I 8 hr in a 5% CO-air atmosphere. At the end of the incubation, tubes were centrifuged at 270 x g for 10 mm and 1 ml ofsupernatant was removed. Radioactivity in both pellet and supernatant was counted in a welltype y scintillation counter. Percentage of SiCr release was calculated by the following formula after deduction of
release
spontaneous
obtained
in tubes
similarly
incu-
bated, but containing labeled target cells and guinea pig red blood cells without added effector cells: percentage of 5iCr released = 2 x cpm in supernatant tube/cpm in supernatant tube + cpm in pellet tube x 100. Statistical Group
analysis data
were
compared
using
Student’s
t test
(16).
period. all of during period within The weight mental 12). Liver
GUINEA
1693
PIGS
Other pair-fed guinea their portion of diet the latter part of the were consuming their 1 hr of receipt. ad libitum control continuously throughout period (initial 427 ±
and spleen
pigs consumed each day and experimental diet allotment group the 7; final
gained experi578
±
weight
In guinea pigs consuming the AA-deficient diet for 4 weeks the liver was smaller than the liver of the control guinea pigs but the liver as a percentage of body weight was the same as that of the animals fed the control diet ad libitum or pair fed the control diet (Table 1). The spleens of the guinea pigs fed the AAdeficient diet were larger but the variation within the group was considerable (range 0.46 to 2.44 g) while spleen weight of control guinea pigs ranged between 0.50 to 1. 12 g. The ratio of spleen weight to body weight showed that the spleen was significantly and disproportionately larger in the deficient animals compared to controls. Immunization with CRC did not influence liver or spleen weight.
Results Food
intake
and
body
weight
There were significant changes in food intake and body weight (Fig. 1) of guinea pigs fed the AA-deficient diet. The average food intake (mean ± SEM) during the 1st week and the average body weight at the end of the 1st week were 26.0 ± 0.9 and 464 ± 6 g, respectively. During the 4th week of AA deficiency, the average food intake was reduced to 10.6 ± 1.9 g (P < 0.001) and the average body weight decreased to 3 13 ± 10 g (P < 0.001) at 28 days. However, the reduction in body weight of the AA-deficient guinea pigs was not due entirely to inanition because the average body weight of the pair fed guinea pigs increased over the 28-day period (initial 425 ± 8 g; final 493 ± 9 g). The pair-fed group, however, varied in pattern and amount of food consumed each day. During the initial weeks on the diet some pair-fed animals did not consume all ofthe food given to them and in effect stored diet for use during the latter part of the experimental
I-.
I 0 w
WEEKS
FIG. 1. Weight chart controls were fed a diet kg); I 1 deficient animals
added
ascorbic
acid;
OF
FEEDING
of 30 male guinea pigs. Ten containing ascorbic acid (4 g/ were fed the same diet without
nine
pair-fed
animals
were
control diet in amount consumed by the deficient mals (0) control, (#{149}) deficient, and () pair-fed.
fed ani-
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Searle, times tration
IN
ANTHONY
1694
Liver
AL.
I and
spleen
weight
of guinea
pigs
fed
Spleen body
weight weight
22.04 3.98
5 ± 2.18 ± 0.28
0.66
± 0.07
%
Pair-fed . . immunized
10 ± 1.87 ± 0.21
25.41 4.22
SEM. from
ii
14.16 4.54
0.86 ± 0.10 0. 18 ± 0.02
0.36
Different
pair-fed P < P < 0.02.
AA . deficient
8 ± 1.26 ± 0.19
20.56 4.15
0.81 ± 0.06 0. 14 ± 0.01
0. 12 ± 0.01
(I Results are expressed as mean ± immunized, P < 0.001. d Different 0.02. “ Different from control-immunized, 0.02. h Different from control-immunized,
TABLE
for 4 weeks”
Control . . immunized
Control
No. of animals Liver (g) Liver weight % body weight Spleen (g)
diets
control, g
6 ± 0.70” ± 0.38
(d
16.07 4.41
10 ± 94e.f. ± 0.18
0.93 0.25
± 0.18 ± 0.04”
1.1 1 ± 0.24
from
immunized, 0.05.
AAdeficient . immunized
P < Different
±
0#{149}#{216}9h
P < 0.0 1 . Different from control0.01. e Different from control, P < from pair-fed immunized, P
. 1:32
“Jo
1:16
z
.
#{149}..
..
...#{149}
.
..#{149}..
S..
#{149}#{149}...
.
I.
-
.
1:8
4
Cellular
1695
PIGS
cytotoxicity CONTROL
Spontaneous CRC showed 1% (mean spleen cells
TABLE
release of 51Cr from labeled little variation averaging 8 ± SEM). As shown in Table 4 from nonimmunized animals
±
FIG.
2.
guinea titer
pigs of
an
PAIR-FED
ASCORBIC ACID-DEFICIENT
Scatter diagram of circulating fed diets for 4 weeks. Each individual
antibodies point represents
of
animal.
3
Hematological
data
obtained
from
guinea
pigs
fed
diets
for 4 weeks” 2 wk 0 Control
White blood mm) Lymphocytes Segmented T cells
cell
(WBC)
count
(per
cu
(% WBC) neutrophils
(% lymphoid
(% WBC) cells)
6908
± 645 (24)” 60 ± 4 (3) 40 ± 4 (3) 40 ± 3 (28)
AA
7280
± 967 (10) 62 ± 5 (4) 38 ± 5 (4) 35 ± 3 (16)
deficient
7293
± 760 (15) 58 ± 4
42 ±4 (4) 32 ± 2 (23)
4 wk Control
WBCcount
6444±648
Lymphocytes Segmented T cells
Pair-fed
(% WBC) neutrophils
(% lymphoid
a All values animals were nonimmunized
(% WBC) cells)
expressed immunized guinea
as mean ± SEM. “ Numbers with 5 x 10” CRC 2 weeks pigs fed the control and deficient
(14) 69 ± 3 (12) 31 ± 3 (12) 37 ± 3 (16)
they for
6967
(8) 63 ± 6 (8) 37 ± 6 (8) 33 ± 5 (8)
in parentheses
before diets
AA deficient
4034±635
refer
were
4 weeks
to number
killed. were
of guinea
Since similar
±632
(13) 68 ±4 (8) 32 ± 4 (8) 32 ± 4 (13)
results the
data
pigs.
‘
Pair-fed
in immunized are
not
segregated.
and
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and
GUINEA
showed a negligible 51Cr release. The most active effector cells were from spleens of guinea pigs fed the control diet ad libitum. Spleen cells from pair-fed animals were relatively active. However, the mean value of cell-mediated cytotoxicity was significantly less than that of control guinea pigs (3 1 ± 3% vs 41 ± 3%, P < 0.01) at the 50: 1 ratio. The mean value of cell-mediated toxicity was not significantly different for the pair-fed controls and ad libitum controls when assayed at the 10: 1 ratio. In contrast, the mean value of 5tCr release for the AA-deficient guinea pigs (20 ± 2) was significantly less than that of either pair-fed controls or ad libitum-fed controls.
count was 6908 ± 645 (thean ± SEM) cells per cubic millimeter an the count was not influenced by feeding an AA-deficient diet for 4 weeks. The relative percentage of lymphocytes
IN
ANTHONY
1696
AL.
cells and cells” Cytotoxicity
Control (6)” Control immunized Pair-fed immunized AAdeficient(3) AA deficient (11)
(12) (9)
2 ± 1 41 ± 3 31 ± 3C
immunized
2± 1 20 ± 2d.e
a All values expressed from control immunized, immunized, P < 0.01.
Rosette-forming
Cytotoxicity (% release) 10:1
(% release) 50:1
Group
cells
2 27 32 1 13
as mean ± SEM. ii Numbers in parentheses refer P < 0.01. d Different from control immunized, Different from control immunized, P < 0.05.
in the spleen
To test for changes in effector cell populations isolated from spleen the percentage of ce,lls capable of forming rosettes with rabbit erythrocytes (T cell marker) was estimated. Irrespective of immunization with CRC, approximately 25% of the cells from spleen homogenates isolated on a Ficoll-Hypaque gradient formed rosettes with rabbit erythrocytes (Table 4). In addition, the percentage of rosette-forming cells in spleen was not changed in animals consuming an AA deficient diet. Discussion Guinea pigs are the only common laboratory animal in which AA deficiency can be induced since like primates they can not synthesize the vitamin. That the guinea pigs in this study were rendered AA deficient is reflected in the levels of AA in plasma, liver and spleen (Table 2). We found that the number ofleucocytes in blood is unaffected by deficiency of AA. Further, our studies have shown for the first time that the numbers of lymphocytes, thymus derived lymphocytes (T cells), and segmented neutrophils in peripheral blood are unchanged in AA deficiency. It has been reported that chronic AA deficiency of several months duration depresses leucocyte levels in the blood of guinea pigs (17) although others (19) have found increased number of leucocytes in guinea pigs with scurvy. It is not possible to rule out concommitant infection in the guinea pigs with elevated leucocyte counts and differences in duration and degree of AA deficiency make it difficult to make
± ± ± ± ±
T cells (‘ rosette-forming celLs)
2 3 4 1
27 ± 25 ± 26 ± 19±5 26 ±
3e.f
to numberofguinea P < 0.001 .
e
pigs. Different
2 2 3 2 ‘
from
Different pair fed
direct comparisons with our data. The Mcdical Research Council study (20) of scorbutic subjects has shown there was a modest but insignificant fall in the leukocyte count. The spleen:body weight ratios in the control guinea pigs were identical to those reported by Garcia-Carrillo (21) in a study of a large number ofnormal guinea pigs ranging in weight from 150 to 1000 g. The spleen: body weight ratios of the pair-fed animals although larger were not significantly greater than those ofthe ad libitum controls confirming an earlier report that restricted food intake does not change the spleen:body weight ratio (22). The finding that the spleen:body weight ratio in AA-deficient guinea pigs was significantly increased confirms earlier studies (19, 23) and suggests that AA deficiency causes hypertrophy of this organ. Antibody titer after immunization is one measure ofhumoral immunity. In the present study we failed to observe any inhibition of the primary response in AA-deficient guinea pigs. Long (7) and Kumar and Axelrod (8) have provided similar data in studies on the effects of AA deficiency on circulating antibody production to diphtheria toxoid. Our fmdings that food restriction imposed by pair feeding did not alter antibody response (Fig. 2) agree with those by Kumar and Axelrod (8) and Gershoff et al. (24). Results ofthis study support those of Mud1cr et al. (4) who showed that various cellmediated immune responses are reduced in animals deprived of AA. Using the 51Cr release of labeled chicken erythrocyte target cells, a significant reduction (approximately 50%) occurred in lymphocyte function 4 weeks after the AA-deficient diet was begun and 2 weeks after immunization with CRC.
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TABLE 4 Cytotoxicity assay of splenic lymphoid percentage of T cells in splenic lymphoid
ET
ASCORBIC
ACID
DEFICIENCY
pigs
was
significantly
less
than
that
released
by similar preparations from pair-fed animals (Table 4). These results suggest that although inanition alone had an impact on cell-mediated cytotoxicity, ascorbic acid deficiency itself has an additional negative influence. There may be a synergistic relationship between ascorbic acid deficiency and the resulting inanition that leads to an even greater loss
in immunity.
GUINEA
PIGS
1697
These data provide in vitro evidence that cell-mediated immunity is impaired in AA deficiency. Evidence that AA affects functional cell-mediated immunity and not merely an in vitro response of lymphocytes resides in the fact that AA deficiency prevents the development of experimental allergic encephalomyelitis, which is regarded as a manifestation of hypersensitivity (4) and prolongs survival of skin allografts (6). Pair-feeding is a technique often used in studies of acute nutrient deficiency to compensate for the fall in weight during the terminal stages of avitaminosis. However, such a nutritional regimen may be a source of errors. For example, healthy control animals that are pair-fed consume their food within a short time and fast for the rest of the day. As a result of “meal eating” marked metabolic changes may take place in the animals (e.g., glycogen synthesis:catabolism; lipid synthesis:catabolism) and consequently, factors not connected with the vitamin deficiency come into play. Further, the pattern of food intake in some pair-fed animals also was unlike that of the deficient animals. During the initial stages of feeding some pair-fed animals did not consume their daily ration and in effect “saved” some diet for consumption during the latter stages of the experimental period. The pair-fed animals also showed a decrease in plasma and tissue levels of AA (Table 2) confirming the report by Davies and Hughes (22) that restriction in food intake results in reduction in tissue retention of AA. This may be a further troublesome complication of the pair-fed animal. Clearly AA deficiency had an additional impact on weight loss besides reduced food intake. Whether the deficit was caused by impaired absorption of the diet from the intestinal tract or impaired feed efficiency is not clear. A “weight control,” i.e., feeding a healthy guinea pig a restricted amount of the control diet such that its weight loss is similar to that of a deficient animal may be advantageous. Exogenous vitamin may also be administered by mouth to the weight control. El References 1 . AXELROD, A. E., B. FISHER, E. FISHER, Y. C. P. LEE AND P. WALSH. Effect of pyridoxine deficiency on
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When the animals were immunized with CRC their plasma levels ofAA were deficient but the clinical manifestations of deficiency were undetectable. The exact time of onset of reduced lymphocyte function in AA-deficient animals remains to be determined. Since 5tCr release oflabeled chicken erythrocyte target cells is considered to be a cellmediated immune response involving pnmanly thymus dependent or T cells (25), a reduction in cytotoxicity may reflect a decrease in the number of T cells in the spleen and/or an impairment of the capacity of such cells to effect cytotoxicity. Since the same number of splenic lymphoid cells was used in each cytotoxicity assay and the percentage of T cells in splenic lymphoid cells was about 25% in all dietary groups (Table 4) it appears unlikely that a change in T cell number can account for the differences. Therefore, the defect in AA deficiency could represent a selective dysfunction in a cell type (T lymphocyte) to mediate cytotoxicity. Since peripheral lymphocyte counts and the percentage ofT cells in blood were unchanged (Table 3) AA deficiency seems to disturb T lymphocyte function rather than T cell number. However, more definitive association of cytotoxic activity with T lymphocytes requires selective purification of attacking cells and demonstration that T lymphocytes are indeed mediating cytotoxic activity. The cytotoxicity assay results from the pair-fed animals are anomalous. The reported impact ofinanition and accompanying weight loss on several aspects of cell-mediated immunity in a number of species is inconsistent (4, 26-29). Our data show that 5tCr release by splenic lymphoid cells isolated from pair-fed animals was the same when assayed at the 10: 1 and 50: 1 effector:target cell ratios. It is clear that 51Cr release by splenic lymphocytes from deficient guinea
IN
1698
ANTHONY grafts
FISHER,
in the rat. Science B., A. E. AXELROD,
127: 1388, 1958. E. R. FISHER, S. H. LEE
N. CALVANESE. The favorable effect of pyridoxinc deficiency on skin homograft survival. Surgery 44: 149, 1958. 3. ROBSON, L. C., AND M. R. SCHWARZ. Vitamin B deficiency and the lymphoid system. 1. Effects on cellular immunity and in vitro incorporation of 3Huridine by small lymphocytes. Cell. Immunol. 16: 135, 1975. 4. MUELLER, P. S., M. W. KIES, E. C. ALVORD AND C. SHAW. Prevention of experimental allergic encephalomyelitis (EAE) by vitamin C deprivation. J. Exptl. AND
Med. 5.
1 15: 329,
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The
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fatty
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erides
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Chem. 38: 514, 1955. V., M. LYNCH, S. GOLDSTEIN AND P. SATO. A rapid micromethod for the determination of ascorbic acid in plasma and tissues. Biochem. Med. 11:41, 1974. WILSON, A. B., AND R. R. COOMBS. Rosette-formation between guinea pig lymphoid cells and rabbit erythrocytes-a possible T cell marker. Int. Arch. Allerg. 44: 544, 1973. LOEWI, G., AND A. TEMPLE. Cytotoxicity of immune guinea pig cells I . Investigations ofa correlation with delayed hypersensitivity and a comparison of cyto-
20.
2 1.
22.
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DAVIES,
M. acid
CORBEL,
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S. N.,
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26.
in guinea
J. E. W., AND R. E. HUGHES. A note food restriction on tissue ascorbic Brit. J. Nutr. 38: 299, 1977.
the guinea-pig
25.
liver
2, p. 269. BARTLEY, W., A. A. KREBS AND J. R. P. O’BRIEN. Vitamin C requirement of human adults. Med. Res. Council Spec. Rept. 5cr. no. 280. London: H. M. Stationery Office, 1953. GARCIA-CARRILLO, C. Relationship between body weight and spleen size in guinea-pigs. Lab. Animals 11: 181, 1977.
ascorbic
24.
C deficiency serum,
I, chapt.
effect of guinea-pigs. 23.
vitamin of blood
99: 261. 1969. REID, M. E. Ascorbie acid VIll. Effects of deficiency in animals. In: The Vitamins, edited by W. H. Sebrell
and R. S. Harris.
pu-
reactivity to PPD in pyridoxine-deficient pigs. J. Immunol. 91: 39, 1963. 6. KALDEN, J. R., AND E. A. GUTHY. Prolonged skin allograft survival in vitamin C-deficient guinea-pigs. European Surg. Res. 4: 1 14, 1972. 7. LONG, D. A. Ascorbic acid and the production of antibody in the guinea-pig. Brit. J. Exptl. Pathol. 31: 183, 1950. 8. KUMAR, M., AND A. E. AXELROD. Circulating antibody formation in scorbutic guinea pigs. J. Nutr. 98: 41, 1969. 9. REID, M. E., AND G. M. BRIGGS. Development of a semi-synthetic diet for young guinea pigs. J. Nutr. 51: 341, 1953. 10. BOYUM, A. Isolation of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab. Invest. 2l:(Suppl. 97) 77, 1968. 1 1 . HERBERT, W. J. Passive haemagglutination with special reference to the tanned cell technique. In: Handbook of Experimental Immunology, edited by D. M. Weir. Oxford: Blackwell Scientific Publications, 1973, vol. 1, Chapt. 20, p. 20.12. 12. SULLIVAN, M. X., AND H. C. N. CLARKE. A highly specific procedure for ascorbic acid. J. Assoc. Offic. ZANNONI,
16.
E.
Systemic guinea
13.
toxicity
19. W.
AL.
S. J. SIMONIAN of
amino
in the T.
acid
rat.
J. Nutr.
BRUNNER.
rejection, and 18: 67, 1974.
A.
AND deflcien-
95:
Cell-me-
tumor
D. K., S. J. DUDRICK AND N. I. ABDOU. Immunocompetence of patients with protein-calorie malnutrition. Ann. Internal. Med. 79: 545, 1973. COOPER, W. C., R. A. GOOD AND T. MARIANI. Effects of protein insufficiency on immune responLAW,
siveness.
Am.
28.
NEUMANN,
29.
BISTRIAN,
J. CIin.
Nutr.
27: 647,
1974.
C. 0., C. J. LAWLOR, E. R. STIEHM., M. E. SWENDSIED, C. NEWTON, J. HERBERT, A. J. AMMAN AND M. JACOB. Immunologic responses in malnourished children. Am. J. Clin. Nutr. 28: 89, 1975.
B. R., BLACKBURN, J. P. FLAi-r. Cellular starved states in hospitalized Nutr. 28: 1 148, 1975. SHAW
AND
L., N. S. SCRIMimmunity in semiadults. Am. J. Clin. 0.
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/8/1691/4692281 by East Carolina University Health Sciences Library user on 13 January 2019
skin 2.
ET
E. Anthony,4
Ph. D., Carole
ABSTRACT
Guinea
fed, and 10
ad libitum
chicken
chicken vitro fed
erythrocytes
SiCr and
rosettes defect
release ad
libitum
with
rabbit
of ascorbic
divided
weeks
cytotoxicity
chicken guinea
a change
pigs. may
The
in
American
workers
ofClinical
Nutrition
groups:
were
comparable cells
marker,
in number
deficient,
intradermally
less of
was
the
an
impairment
or
function
splenic
same
three with
than
with three
cells cells
of
another
cell
cells
from pair-
formed
groups.
function type.
In
from that
dietary
of T lymphocyte
to
groups.
lymphoid spleen
5 x
titers
dietary
lymphoid
in all
pair-
with
antibody
in all incubated
significantly percentage
reflect
ascorbic-acid
immunized
Hemagglutinating
target
was
M.D.
Am.
The in
cell-
I.
Clin.
1979.
vitamin-deficient
Journal
dietary
adjuvant.
The
a T cell
observed thymic atrolaboratory animals and man, it was not until 1958 (1) that the first systematic studies of the role of vitamins in cellular immunity were published by Axeirod et al. (1). Vitamin B6 deficiency has received the greatest attention, and results indicate that lack of pyridoxine decreases delayed skin and systemic hypersensitivity, increased survival time of skin homografts (1, 2) and reduced lymphocyte proliferation in vitro as measured by the mixed lymphocyte reaction (3). Little attention has been given to the effects of other vitamin deficiencies on cellular mechanisms of immunity. Studies of the role of ascorbic acid (AA) on cellular immunity have led to contradictory conclusions. Mud1cr et al. (4) found that scorbutic guinea pigs failed to develop experimental allergic encephalomyelitis or delayed hypersensitivity skin reaction to mycobacterium tuberculosis. However, Trakatellis et al. (5) reported no depression of skin or systemic reactivity after sensitization of AA-deficient guinea pigs to mycobacterium tuberculosis. More recently prolonged survival of skin allografts in scorbutic guinea pigs has been reported (6). The role of AA in the humoral antibody response is also a matter of dispute. Long (7) phy
pigs
B. Taylor,
pigs were
erythrocyte
guinea
deficiency or
three
immunization
erythrocytes, acid
into
later guinea complete
after
labeled control
M. S., and Keith
Freund’s
acid-deficient
Nuir. 32: 1691-1698,
early
in
from
were
Two
2 weeks
of ascorbic
mediated
While
pigs
control.
erythrocytes
spleens
G. Kurahara,
immune
32: AUGUST
observed a deleterious effect on the secondary immune response to diphtheria toxoid by guinea pigs fed an AA-deficient natural diet. Kumar and Axelrod (8) induced severe scurvy in guinea pigs by feeding a purified AA-deficient diet and found no change in secondary antibody response with the same antigen. The primary response in both reports was unaffected. The present work further examines the effect of AA deficiency on cell-mediated immunity and the primary humoral response. Using chicken erythrocytes as target cells, the cytotoxicity of spleen lymphoid cells from immunized AA-deficient guinea pigs was assessed by a 51Cr release technique. The percentage of spleen lymphoid cells binding rabbit erythrocytes (T cell marker), the number ofperipheral white blood cells, the percentage of T cells and the hemagglutinating serum I From partment Hospital,
2
Stanford of Medicine Palo Alto,
Supported
University and the California
by the
School Veterans 94304.
Medical
of Medicine, Administration
Research
Service
of the
Veterans Administration and by United States Health Service Grant 5T32 HL 07034-03. 3 Address reprint requests to: Dr. K. B. Taylor, Hospital, Medical Service, Palo Alto, California 4
1979,
Present
address:
School
at
Texas
77030.
pp.
Houston,
1691-1698.
the
University
Department
Printed
in U.S.A.
of Texas of
Surgery,
Dc-
Public V.A. 94304.
Medical Houston,
1691
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Cell-mediated cytotoxicity and humoral response in ascorbic acid-deficient guinea pigs13
1692
ANTHONY
Materials
in pair-
Ascorbic
also
Clarke
(12), adapted
Diets
the
guinea
Test
Diets,
allowed
Madison,
group of guinea pigs was pair-fed the AA-supplemented diet. Each guinea pig in the pair-fed group received an amount of diet equal to that consumed by its AA-deficient partner during the previous day. Materials
Hanks’
balanced
(Grand
salt solution,
tissue
culture
medium
serum (FCS) and penicillin-streptomycin obtained from Grand Island Biological
Island,
N.Y.),
Ficoll
(Pharmacia
Inc.,
moved, Hanks’ Medium
the remaining balanced salt 199.
cells were solution
washed and then
three times suspended
at 15,000
to develop
Boyum
was
trichloroacetic
The samples were 15 mm at 4 C, and the
for 15 mm at room
temperature
and
cells
(10)
with
were
minor
isolated
modification.
by the
method
The
heparinized
of
blood
was diluted 1:1 with Hanks’ balanced salt solution then layered on a Ficoll and Hypaque gradient and then centrifuged for 35 mm (400 x g). The lymphocyte layer was removed from the interface, suspended in medium 199, and washed twice with the same medium. Cell suspensions were adjusted to 5 x 10’ cells per milliliter. The viability of the lymphocytes was found to be above 95% by trypan blue exclusion. and
rosette
Percentage
ofT
determination
cells was determined
by the formation
of spontaneous nonimmune rosettes with rabbit erythrocytes (14). One hundred microliters of lymphocyte suspension and 100 tl of rabbit red blood cells (80 x l0”/ml) in medium 199 were centrifuged together for 2 mm at 400 x g. The preparations were made in duplicate
and allowed were gently examined lymphocytes
to stand at 4 C for 1 hr or more. resuspended, stained with toluidine microscopically. the percentage
or more cytometer. three
Cytotoxicity
Immunization
x g for
40%
cell preparation
T lymphocyte
in in
et al. (13). Protein
the procedure.
Mononuclear
Pis-
cataway, NJ.) and Hypaque (Winthrop Laboratories, New York, N.Y.) were mixed according to the method of Boyum (10) and used for the separation of lymphocytes. Chicken red cells (CRC) in Alsever’s solution were purchased from Microbiological Media, Concord, Calif. For the T rosette assay blood from the same rabbit (Lit Rabbitry, Aptos, Calif.) was bled from the ear into acidcitrate-dextrose. After plasma and buffy coat were re-
by Zannoni
adding 0.25 ml of of plasma on ice.
read at 525 nm. Standards for each set of experimental samples were determined in duplicate. The tissues from AA-deficient animals were homogenized in 9 volumes and the tissues from control animals in 19 volumes of cold 5% trichloroacetic acid and carried
Blood
Wis.) or the same diet supplemented with AA (4 g/kg). In one experiment to test the effect of inanition and”weight loss on the cytotoxic response an additional
of plasma and tissues was asof the method of Sullivan and
following reagents then added to 0.75 ml of supernatant: 0.05 ml of 85% orthophosphoric acid, 0.05 ml of 8% aa’-dipyridyl in ethanol, and 0.05 ml of 3% aqueous ferric chloride. The ferrous-dipyridyl chromophore was
through the 4-week experimental period fed an AA-deficient diet(9)(Teklad
199, fetal calf mixture were
by ml
centrifuged
Outbred, male, Hartley guinea pigs (Simonsen Laboratories, Gilroy, Calif). weighing 350 to 400 g at the outset were fed a commercial chow diet for 1 week after they were received from the suppliers. Experimental procedures were started immediately after this period of adaptation. The guinea pigs were then maintained on diets and water ad libitum and weighed weekly.
During pigs were
assay
precipitated acid to 2.0
Animals
Co.,
acid
Ascorbic acid content sayed by a modification
methods
and
AL.
erythrocytes
From of
The cells blue and
counts of 200 to 300 lymphocytes binding
was enumerated
in a hema-
assay
guinea pigs from each dietary group were given 5 x 10” washed CRC in 0.4 ml of Freund’s complete adjuvant intradermally in the nuchal region, 0.1 ml per site.
Using chicken erythrocytes as target cells, the cytotoxicity of cells isolated from the spleens of guinea pigs was assessed by the 51Cr release technique described by Loewi and Temple (15). Individually, spleens were aseptically removed, weighed, and minced with scissors in
Blood
Petri dishes containing medium 199. The spleen fragments were passed through a stainless steel tissue sieve and washed twice in medium 199. Final cell dilutions were made in medium 199 containing 10% FCS with 100
After
2 weeks
collection
The animals (Pittman
bled
Moore,
on
and
the
counts
were lightly Inc.,
diet
anaesthetized
Washington
(3 to S ml) by cardiac
puncture.
with Metafane
Crossing,
The
NJ.)
and
concentration
of white blood cells in the samples was enumerated in a hemacytometer utilizing 0.01% crystal violet in 2% acetic acid. Wright-stained blood smears were used for differential white cell counts. Sera were stored at -20 C. Antibody
titration
Two weeks
units of penicillin and liter. All FCS was heat suspension was layered
antibody
titers
elicited
with CRC were measured by the hemagglutination method using doubling dilutions of sera in saline (11).
per milliof the cell
on a Ficoll-Hypaque gradient for preparation of mononuclear cells for the estimation of T rosettes as described above. For labeling with 5tCr, washed CRC were suspended in
after immunization
100 sg of streptomycin inactivated. A portion
medium
199 with
4%
FCS
at a concentration
of 4 x
l0 CRC/0.l ml and incubated at 37 C for I hr in the presence of 100 tCi sodium 5tCr-chromate (specific activity not less than 200 tCi4tg chromium, Amersham/
Downloaded from https://academic.oup.com/ajcn/article-abstract/32/8/1691/4692281 by East Carolina University Health Sciences Library user on 13 January 2019
antibody titers to chicken erythrocytes blood were measured. The effects of feeding on the same parameters were examined.
ET
ASCORBIC
ACID
DEFICIENCY
Arlington Heights, Ill.). After washing four to six in medium 199, the cells were diluted to a concenof iO CRC/ml in medium 199, containing 10% FCS with 100 units of penicillin and 100 g of streptomycin per milliliter. Preliminary experiments using immunized control guinea pigs confirmed the finding of Loewi and Temple ( 15) that cytotoxicity was detectable at an effector:target cell ratio as low as 5: 1 and reached a plateau at a ratio 50: 1. Given the limited number of cells in individual spleens effector:target cell ratios of 10: 1 and 50: 1 were used in subsequent experiments. Cytotoxicity tests were done in 2 ml volumes of medium 199 containing 10% FCS and antibiotics. The cultures contained 0.5 ml spleen effector cells ( 10 x 10 or 50 X iO cells), 0.5 ml guinea pig red blood cells (1 x 10’ cells) and 1 ml CRC (1 X iO cells). Similar cultures omitting the guinea pig red blood cells were also examined. Each tube was set up in duplicate. Incubation was for I 8 hr in a 5% CO-air atmosphere. At the end of the incubation, tubes were centrifuged at 270 x g for 10 mm and 1 ml ofsupernatant was removed. Radioactivity in both pellet and supernatant was counted in a welltype y scintillation counter. Percentage of SiCr release was calculated by the following formula after deduction of
release
spontaneous
obtained
in tubes
similarly
incu-
bated, but containing labeled target cells and guinea pig red blood cells without added effector cells: percentage of 5iCr released = 2 x cpm in supernatant tube/cpm in supernatant tube + cpm in pellet tube x 100. Statistical Group
analysis data
were
compared
using
Student’s
t test
(16).
period. all of during period within The weight mental 12). Liver
GUINEA
1693
PIGS
Other pair-fed guinea their portion of diet the latter part of the were consuming their 1 hr of receipt. ad libitum control continuously throughout period (initial 427 ±
and spleen
pigs consumed each day and experimental diet allotment group the 7; final
gained experi578
±
weight
In guinea pigs consuming the AA-deficient diet for 4 weeks the liver was smaller than the liver of the control guinea pigs but the liver as a percentage of body weight was the same as that of the animals fed the control diet ad libitum or pair fed the control diet (Table 1). The spleens of the guinea pigs fed the AAdeficient diet were larger but the variation within the group was considerable (range 0.46 to 2.44 g) while spleen weight of control guinea pigs ranged between 0.50 to 1. 12 g. The ratio of spleen weight to body weight showed that the spleen was significantly and disproportionately larger in the deficient animals compared to controls. Immunization with CRC did not influence liver or spleen weight.
Results Food
intake
and
body
weight
There were significant changes in food intake and body weight (Fig. 1) of guinea pigs fed the AA-deficient diet. The average food intake (mean ± SEM) during the 1st week and the average body weight at the end of the 1st week were 26.0 ± 0.9 and 464 ± 6 g, respectively. During the 4th week of AA deficiency, the average food intake was reduced to 10.6 ± 1.9 g (P < 0.001) and the average body weight decreased to 3 13 ± 10 g (P < 0.001) at 28 days. However, the reduction in body weight of the AA-deficient guinea pigs was not due entirely to inanition because the average body weight of the pair fed guinea pigs increased over the 28-day period (initial 425 ± 8 g; final 493 ± 9 g). The pair-fed group, however, varied in pattern and amount of food consumed each day. During the initial weeks on the diet some pair-fed animals did not consume all ofthe food given to them and in effect stored diet for use during the latter part of the experimental
I-.
I 0 w
WEEKS
FIG. 1. Weight chart controls were fed a diet kg); I 1 deficient animals
added
ascorbic
acid;
OF
FEEDING
of 30 male guinea pigs. Ten containing ascorbic acid (4 g/ were fed the same diet without
nine
pair-fed
animals
were
control diet in amount consumed by the deficient mals (0) control, (#{149}) deficient, and () pair-fed.
fed ani-
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Searle, times tration
IN
ANTHONY
1694
Liver
AL.
I and
spleen
weight
of guinea
pigs
fed
Spleen body
weight weight
22.04 3.98
5 ± 2.18 ± 0.28
0.66
± 0.07
%
Pair-fed . . immunized
10 ± 1.87 ± 0.21
25.41 4.22
SEM. from
ii
14.16 4.54
0.86 ± 0.10 0. 18 ± 0.02
0.36
Different
pair-fed P < P < 0.02.
AA . deficient
8 ± 1.26 ± 0.19
20.56 4.15
0.81 ± 0.06 0. 14 ± 0.01
0. 12 ± 0.01
(I Results are expressed as mean ± immunized, P < 0.001. d Different 0.02. “ Different from control-immunized, 0.02. h Different from control-immunized,
TABLE
for 4 weeks”
Control . . immunized
Control
No. of animals Liver (g) Liver weight % body weight Spleen (g)
diets
control, g
6 ± 0.70” ± 0.38
(d
16.07 4.41
10 ± 94e.f. ± 0.18
0.93 0.25
± 0.18 ± 0.04”
1.1 1 ± 0.24
from
immunized, 0.05.
AAdeficient . immunized
P < Different
±
0#{149}#{216}9h
P < 0.0 1 . Different from control0.01. e Different from control, P < from pair-fed immunized, P
. 1:32
“Jo
1:16
z
.
#{149}..
..
...#{149}
.
..#{149}..
S..
#{149}#{149}...
.
I.
-
.
1:8
4
Cellular
1695
PIGS
cytotoxicity CONTROL
Spontaneous CRC showed 1% (mean spleen cells
TABLE
release of 51Cr from labeled little variation averaging 8 ± SEM). As shown in Table 4 from nonimmunized animals
±
FIG.
2.
guinea titer
pigs of
an
PAIR-FED
ASCORBIC ACID-DEFICIENT
Scatter diagram of circulating fed diets for 4 weeks. Each individual
antibodies point represents
of
animal.
3
Hematological
data
obtained
from
guinea
pigs
fed
diets
for 4 weeks” 2 wk 0 Control
White blood mm) Lymphocytes Segmented T cells
cell
(WBC)
count
(per
cu
(% WBC) neutrophils
(% lymphoid
(% WBC) cells)
6908
± 645 (24)” 60 ± 4 (3) 40 ± 4 (3) 40 ± 3 (28)
AA
7280
± 967 (10) 62 ± 5 (4) 38 ± 5 (4) 35 ± 3 (16)
deficient
7293
± 760 (15) 58 ± 4
42 ±4 (4) 32 ± 2 (23)
4 wk Control
WBCcount
6444±648
Lymphocytes Segmented T cells
Pair-fed
(% WBC) neutrophils
(% lymphoid
a All values animals were nonimmunized
(% WBC) cells)
expressed immunized guinea
as mean ± SEM. “ Numbers with 5 x 10” CRC 2 weeks pigs fed the control and deficient
(14) 69 ± 3 (12) 31 ± 3 (12) 37 ± 3 (16)
they for
6967
(8) 63 ± 6 (8) 37 ± 6 (8) 33 ± 5 (8)
in parentheses
before diets
AA deficient
4034±635
refer
were
4 weeks
to number
killed. were
of guinea
Since similar
±632
(13) 68 ±4 (8) 32 ± 4 (8) 32 ± 4 (13)
results the
data
pigs.
‘
Pair-fed
in immunized are
not
segregated.
and
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and
GUINEA
showed a negligible 51Cr release. The most active effector cells were from spleens of guinea pigs fed the control diet ad libitum. Spleen cells from pair-fed animals were relatively active. However, the mean value of cell-mediated cytotoxicity was significantly less than that of control guinea pigs (3 1 ± 3% vs 41 ± 3%, P < 0.01) at the 50: 1 ratio. The mean value of cell-mediated toxicity was not significantly different for the pair-fed controls and ad libitum controls when assayed at the 10: 1 ratio. In contrast, the mean value of 5tCr release for the AA-deficient guinea pigs (20 ± 2) was significantly less than that of either pair-fed controls or ad libitum-fed controls.
count was 6908 ± 645 (thean ± SEM) cells per cubic millimeter an the count was not influenced by feeding an AA-deficient diet for 4 weeks. The relative percentage of lymphocytes
IN
ANTHONY
1696
AL.
cells and cells” Cytotoxicity
Control (6)” Control immunized Pair-fed immunized AAdeficient(3) AA deficient (11)
(12) (9)
2 ± 1 41 ± 3 31 ± 3C
immunized
2± 1 20 ± 2d.e
a All values expressed from control immunized, immunized, P < 0.01.
Rosette-forming
Cytotoxicity (% release) 10:1
(% release) 50:1
Group
cells
2 27 32 1 13
as mean ± SEM. ii Numbers in parentheses refer P < 0.01. d Different from control immunized, Different from control immunized, P < 0.05.
in the spleen
To test for changes in effector cell populations isolated from spleen the percentage of ce,lls capable of forming rosettes with rabbit erythrocytes (T cell marker) was estimated. Irrespective of immunization with CRC, approximately 25% of the cells from spleen homogenates isolated on a Ficoll-Hypaque gradient formed rosettes with rabbit erythrocytes (Table 4). In addition, the percentage of rosette-forming cells in spleen was not changed in animals consuming an AA deficient diet. Discussion Guinea pigs are the only common laboratory animal in which AA deficiency can be induced since like primates they can not synthesize the vitamin. That the guinea pigs in this study were rendered AA deficient is reflected in the levels of AA in plasma, liver and spleen (Table 2). We found that the number ofleucocytes in blood is unaffected by deficiency of AA. Further, our studies have shown for the first time that the numbers of lymphocytes, thymus derived lymphocytes (T cells), and segmented neutrophils in peripheral blood are unchanged in AA deficiency. It has been reported that chronic AA deficiency of several months duration depresses leucocyte levels in the blood of guinea pigs (17) although others (19) have found increased number of leucocytes in guinea pigs with scurvy. It is not possible to rule out concommitant infection in the guinea pigs with elevated leucocyte counts and differences in duration and degree of AA deficiency make it difficult to make
± ± ± ± ±
T cells (‘ rosette-forming celLs)
2 3 4 1
27 ± 25 ± 26 ± 19±5 26 ±
3e.f
to numberofguinea P < 0.001 .
e
pigs. Different
2 2 3 2 ‘
from
Different pair fed
direct comparisons with our data. The Mcdical Research Council study (20) of scorbutic subjects has shown there was a modest but insignificant fall in the leukocyte count. The spleen:body weight ratios in the control guinea pigs were identical to those reported by Garcia-Carrillo (21) in a study of a large number ofnormal guinea pigs ranging in weight from 150 to 1000 g. The spleen: body weight ratios of the pair-fed animals although larger were not significantly greater than those ofthe ad libitum controls confirming an earlier report that restricted food intake does not change the spleen:body weight ratio (22). The finding that the spleen:body weight ratio in AA-deficient guinea pigs was significantly increased confirms earlier studies (19, 23) and suggests that AA deficiency causes hypertrophy of this organ. Antibody titer after immunization is one measure ofhumoral immunity. In the present study we failed to observe any inhibition of the primary response in AA-deficient guinea pigs. Long (7) and Kumar and Axelrod (8) have provided similar data in studies on the effects of AA deficiency on circulating antibody production to diphtheria toxoid. Our fmdings that food restriction imposed by pair feeding did not alter antibody response (Fig. 2) agree with those by Kumar and Axelrod (8) and Gershoff et al. (24). Results ofthis study support those of Mud1cr et al. (4) who showed that various cellmediated immune responses are reduced in animals deprived of AA. Using the 51Cr release of labeled chicken erythrocyte target cells, a significant reduction (approximately 50%) occurred in lymphocyte function 4 weeks after the AA-deficient diet was begun and 2 weeks after immunization with CRC.
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TABLE 4 Cytotoxicity assay of splenic lymphoid percentage of T cells in splenic lymphoid
ET
ASCORBIC
ACID
DEFICIENCY
pigs
was
significantly
less
than
that
released
by similar preparations from pair-fed animals (Table 4). These results suggest that although inanition alone had an impact on cell-mediated cytotoxicity, ascorbic acid deficiency itself has an additional negative influence. There may be a synergistic relationship between ascorbic acid deficiency and the resulting inanition that leads to an even greater loss
in immunity.
GUINEA
PIGS
1697
These data provide in vitro evidence that cell-mediated immunity is impaired in AA deficiency. Evidence that AA affects functional cell-mediated immunity and not merely an in vitro response of lymphocytes resides in the fact that AA deficiency prevents the development of experimental allergic encephalomyelitis, which is regarded as a manifestation of hypersensitivity (4) and prolongs survival of skin allografts (6). Pair-feeding is a technique often used in studies of acute nutrient deficiency to compensate for the fall in weight during the terminal stages of avitaminosis. However, such a nutritional regimen may be a source of errors. For example, healthy control animals that are pair-fed consume their food within a short time and fast for the rest of the day. As a result of “meal eating” marked metabolic changes may take place in the animals (e.g., glycogen synthesis:catabolism; lipid synthesis:catabolism) and consequently, factors not connected with the vitamin deficiency come into play. Further, the pattern of food intake in some pair-fed animals also was unlike that of the deficient animals. During the initial stages of feeding some pair-fed animals did not consume their daily ration and in effect “saved” some diet for consumption during the latter stages of the experimental period. The pair-fed animals also showed a decrease in plasma and tissue levels of AA (Table 2) confirming the report by Davies and Hughes (22) that restriction in food intake results in reduction in tissue retention of AA. This may be a further troublesome complication of the pair-fed animal. Clearly AA deficiency had an additional impact on weight loss besides reduced food intake. Whether the deficit was caused by impaired absorption of the diet from the intestinal tract or impaired feed efficiency is not clear. A “weight control,” i.e., feeding a healthy guinea pig a restricted amount of the control diet such that its weight loss is similar to that of a deficient animal may be advantageous. Exogenous vitamin may also be administered by mouth to the weight control. El References 1 . AXELROD, A. E., B. FISHER, E. FISHER, Y. C. P. LEE AND P. WALSH. Effect of pyridoxine deficiency on
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When the animals were immunized with CRC their plasma levels ofAA were deficient but the clinical manifestations of deficiency were undetectable. The exact time of onset of reduced lymphocyte function in AA-deficient animals remains to be determined. Since 5tCr release oflabeled chicken erythrocyte target cells is considered to be a cellmediated immune response involving pnmanly thymus dependent or T cells (25), a reduction in cytotoxicity may reflect a decrease in the number of T cells in the spleen and/or an impairment of the capacity of such cells to effect cytotoxicity. Since the same number of splenic lymphoid cells was used in each cytotoxicity assay and the percentage of T cells in splenic lymphoid cells was about 25% in all dietary groups (Table 4) it appears unlikely that a change in T cell number can account for the differences. Therefore, the defect in AA deficiency could represent a selective dysfunction in a cell type (T lymphocyte) to mediate cytotoxicity. Since peripheral lymphocyte counts and the percentage ofT cells in blood were unchanged (Table 3) AA deficiency seems to disturb T lymphocyte function rather than T cell number. However, more definitive association of cytotoxic activity with T lymphocytes requires selective purification of attacking cells and demonstration that T lymphocytes are indeed mediating cytotoxic activity. The cytotoxicity assay results from the pair-fed animals are anomalous. The reported impact ofinanition and accompanying weight loss on several aspects of cell-mediated immunity in a number of species is inconsistent (4, 26-29). Our data show that 5tCr release by splenic lymphoid cells isolated from pair-fed animals was the same when assayed at the 10: 1 and 50: 1 effector:target cell ratios. It is clear that 51Cr release by splenic lymphocytes from deficient guinea
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