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Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, WI 53706
3To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • calcium • vitamin D • experimental autoimmune encephalomyelitis • multiple sclerosis • mice
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The action of this hormone is via a
steroid hormone-like mechanism through a nuclear receptor, the vitamin D receptor (VDR), which is a member of the steroid hormone
receptor superfamily (Pike 1991
, Ross et al. 1994
). The discovery of VDR in peripheral blood
lymphocytes (Bhalla et al. 1983
, Provvedini et al. 1983
) is the clue that led to the realization that
1,25-(OH)2D3 is a significant regulator of the
immune system. The most striking evidence of a role for
1,25-(OH)2D3 as an immune system regulator
comes from in vivo experiments. 1,25-(OH)2D3
can prevent the development of experimental autoimmune
encephalomyelitis (EAE) (Cantorna et al. 1996
,
Lemire and Archer 1991
) and experimental arthritis
(Cantorna et al. 1998a
), and markedly inhibit transplant
rejection (Bouillon et al. 1995
, Hullett et al. 1998
).
EAE is mediated by CD4+ T cells, which mount an inappropriate
immune-mediated attack on the central nervous system (CNS). Type-1
helper (Th1) cells specific for CNS antigens induce the disease, and
the Th1 cytokines interferon-
(IFN-) and tumor necrosis
factor-
(TNF-) are associated with EAE in mice (Holda and Swanborg 1982
, Powell et al. 1990
). Conversely,
type-2 helper (Th2) cells and other cell types that produce
interleukin-4 (IL-4) and transforming growth factor-ß1 (TGF-ß1) in
response to CNS antigens are known to ameliorate EAE. In vivo,
1,25-(OH)2D3 treatments result in a net loss in
the total number of lymphocytes and a net increase in the expression of
IL-4 and TGF-ß1 (Cantorna et al. 1998b
). Conversely,
the in vivo 1,25-treatments had no effect on IFN- or TNF-
expression (Cantorna et al. 1998b
). The role for calcium
in the regulation of the immune response, if any, remains unclear.
Human multiple sclerosis is most prevalent in females (Grossman et al. 1991
), whereas incidence and severity of EAE in mice
differs in males vs. females, depending on strain (Cantorna et al. 1996
, Cua et al. 1995
). Thus, it is already
known that sex is a major factor in this disease. This study was
designed to determine whether 1,25-(OH)2D3 is
equally effective in both sexes and whether dietary calcium level plays
any role in the development of the disease and the response to
1,25-(OH)2D3. Our results suggest that both
1,25-(OH)2D3 and calcium regulate the immune
response and that 1,25-(OH)2D3 is more
effective against EAE in females fed adequate or high dietary calcium
levels.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The B10.PL mice were produced in our colony using breeding pairs
obtained from Jackson Laboratories (Bar Harbor, ME). During breeding,
the mice were fed Purina diet 5008 Formulab (Richmond, IN), containing
100 IU cholecalciferol/g diet. Mice were used for
experiments at 6–8 wk of age; at that time, the females weighed 18–22
g, and the males 22–26 g. For experiments, all of the mice were fed
synthetic diets (Smith et al. 1987
, Yang et al. 1993
) with the modifications described below. For all
experiments, each mouse was fed 4 g of the experimental diet per
day to ensure that each mouse received its daily dose of
1,25-(OH)2D3 and that controls did not eat more
then 1,25-(OH)2D3-treated mice. Although the
experimental diet was devoid of vitamin D, the mice were exposed to
normal light and therefore were not vitamin D deficient. EAE was
induced in all mice 1 wk after the experimental diets were started. For
mice with severe symptoms of EAE, food was placed in small dishes on
the bottom of the cage. At the end of the experiments, mice were
weighed, killed and exsanguinated.
In the first experimental design, male and female mice were fed diets that contained 1 g calcium/100 g diet, and EAE was induced. In a second series of experiments, groups of 8–12 mice were fed the experimental diet (control treatment) without vitamin D or the experimental diet plus various concentrations of 1,25-(OH)2D3 as indicated. Female mice were fed 1,25-(OH)2D3 ranging from 0 to 200 ng/d and male mice were fed 0–800 ng/d. The experimental diet contained one of three calcium concentrations, i.e., 20 mg (low), 470 mg (medium) or 1 g (high) calcium/100 g diet as indicated. The final experimental design used only male mice; they were fed the control diet or the same diet containing 100 ng 1,25-(OH)2D3/4 g diet. These mice were fed diets that contained low, medium or high calcium as indicated. This feeding protocol resulted in six groups of 6–8 male mice each. The dose of 1,25-(OH)2D3 was chosen because it completely prevented EAE in male mice fed high calcium diets. All of the procedures described were reviewed and approved by the University of Wisconsin-Madison Research Animal Resources Center Committee Review Board on 09/09/94, protocol number A-07–3000-A00755–4-08–94.
EAE disease induction.
Myelin basic protein (MBP) was isolated from guinea pig spinal cords
(Cantorna et al. 1996
), lyophilized and stored at
-20°C. For immunizations, MBP was dissolved in 0.1 mol/L acetic acid
at a concentration of 8 g/L (Cantorna et al. 1996
).
Ether-anesthetized mice were immunized subcutaneously with 0.1 mL
of MBP (400 mg/mouse) emulsified in an equal volume of complete
Freund's adjuvant (Difco Laboratories, Detroit, MI) containing
Mycobacterium tuberculosis H37 Ra. In addition, on the
day of immunization and 2 d later, mice were injected
intraperitoneally with 200 ng of pertussis toxin (LIST Biological
Laboratories, Campbell, CA) suspended in sterile saline. This
immunization protocol resulted in the induction of EAE in 80–100% of
the mice. Male mice were killed on d 21 postimmunization to measure
various immune responses. The EAE severity scoring system was as
follows: 0 = normal; 1 = limp tail; 2 = paraparesis with
a clumsy gait; 3 = hind limb paralysis; 4 = hind- and
forelimb paralysis; 5 = moribund.
Lymphocytes.
Axillary, brachial and inguinal lymph nodes (LN) from six mice were
collected and pooled from control- and
1,25-(OH)2D3-treated mice
on d 21 postimmunization. Each experiment was repeated three times in
its entirety. These LN were chosen because they drained the site of
immunization. Collected LN were disrupted manually using a 23-gauge
needle and a pair of forceps. Total cell numbers in the LN were
determined by counting the number of lymphocytes from control- and
1,25-(OH)2D3-treated mice
and dividing by the number of mice in the group. Flow cytometry of
fluorescent-labeled cell populations (Thy-1, class II, CD4 and CD8)
were done on LN cells from control- and
1,25-(OH)2D3-treated mice
using standard protocols and exactly as described (Smith et al. 1987
). For cytokine polymerase chain reaction (PCR) analysis,
LN cells were saved for total cellular RNA isolation.
Transcript analysis by quantitative competitive PCR.
Cells for mRNA analysis were dissolved in acid guanidinium thiocyanate,
and total RNA was isolated by the phenol chloroform extraction method
(Chomczynski and Sacchi 1987
). Total cellular RNA was
reverse-transcribed using oligo dT primers according to the
manufacturer's protocols (Promega, Madison, WI) and quantitated by
competitive PCR. Primers and mimic DNA specific for
glyceraldehyde-3-phosphate dehydrogenase (G3PDH), IL-4 and TGF-ß1
were obtained from Clontech Laboratories, (Palo Alto, CA)
(Siebert and Larrick 1992
and 1993
). Competitive cDNA
mimics, which included the G3PDH, IL-4 and TGF-ß1 primer sequences
adjoining a neutral DNA segment, were serially diluted and added to
test cDNA aliquots (Siebert and Larrick 1992
and 1993
).
The authentic product/mimic bp sizes were 983/600 for G3PDH, 306/544
for IL-4 and 525/390 for TGF-ß1. The mixture was amplified under
predetermined optimal conditions; the products were resolved by 1.5%
agarose gel electrophoresis and ethidium bromide stained. The cytokine
bands were identified by size with respect to molecular weight
standards. The mimic DNA dilution that yielded a band with a
fluorescence intensity that matched the cytokine band was used to
calculate cytokine cDNA copy number. The G3PDH transcript quantitation
served as a control for reverse transcription efficiency. Values are
reported as cytokine cDNA copies per 1000 copies of G3PDH cDNA.
Serum calcium and 1,25-(OH)2D3 analysis.
After 50 d, the mice were killed; blood was collected by heart
puncture and serum was collected after clot formation. Serum calcium
concentrations were determined using a Perkin Elmer (Norwalk, CT)
atomic absorption spectrometer exactly as described
(Mohamed et al. 1996
).
1,25-(OH)2D3 analysis was done exactly as
described (Arbour et al. 1998
).
Statistics.
Where possible, values reported were averages from multiple mice or
experiments. Because of the variability in EAE induction, peak severity
and cytokine gene expression from one experiment to another, some
values were reported as the values from one representative of three
experiments. A two-sample test for binomial proportions was used
for statistical analysis of all percentage values as described
(Rosner 1986
). Again, where possible, statistical
analyses were done using a statistics program for the Macintosh
(StatView Student, Abacus Concepts, Berkeley, CA). The unpaired
two-group Student's t test (confirmed using the
Mann-Whitney U test) was done and values of P
< 0.05 were considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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When dietary calcium was high, the incidence of EAE was 98% for male
mice and 96% for female mice. Male and female mice that did not
develop EAE by d 50 postimmunization were excluded from the study. For
mice that did develop EAE, the EAE maximum severity was plotted vs. the
day of EAE onset (Fig. 1
). The maximum EAE severity in male mice ranged from 3 to 5, whereas
that in female mice ranged from 2 to 5. An EAE score of 5 was reached
by 47% of male mice, whereas only 17% of females scored a 5
(P 
0.03). The day of EAE onset was also earlier for
males (9–24 d) than females (9–38 d) (P 0.0002).
EAE developed in 34% of female mice 24 d after immunization. Male
mice were more susceptible to EAE then female mice.
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EAE disease severity was unaffected by changes in dietary calcium.
Instead, the incidence of EAE varied with the amount of dietary
calcium. Diets containing high calcium resulted in EAE incidence values
approaching 100% (Fig. 2
). Male and female mice fed low calcium diets had EAE incidence of
~82–83% (Figs. 2
3
4
). The EAE incidence of both male (P 0.07) and female
(P 0.08) mice fed low calcium diets was lower
(although not significantly lower) than that of mice consuming high
dietary calcium. It is well documented that low dietary calcium
stimulates the production of
1,25-(OH)2D3 (DeLuca 1983
). Plasma
1,25-(OH)2D3 values of mice
consuming high calcium diets ranged from 0.06 to 0.18
µmol/L of serum, and values of mice fed low calcium ranged
from 0.10 to 0.36 µmol/L of serum. A median dietary
calcium level resulted in an intermediate EAE incidence of 95–99%
(Fig. 3)
. Mice fed low calcium diets and treated with
1,25-(OH)2D3 showed a
significant (males P 0.007 and females P
0.05) drop in EAE incidence of 40–45% compared with mice that
were not treated with
1,25-(OH)2D3 (Fig. 4)
. The
40–45% decrease in EAE incidence occurred in the absence of an
increase in serum calcium concentrations (2.2 ± 0.2 mmol/L, Fig. 4
).
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When dietary calcium was high, 6 ng/d of
1,25-(OH)2D3 prevented EAE
in female mice, and 100 ng/d of
1,25-(OH)2D3 was needed for
male mice (Fig. 2)
. At these doses of
1,25-(OH)2D3, serum calcium
was elevated. At a medium dietary calcium level, 200 ng/d of
1,25-(OH)2D3 in female mice
and 400 ng/d in male mice were needed to prevent EAE (Fig. 3)
. When
calcium in the diet was low, EAE was not prevented by even high doses
of 1,25-(OH)2D3. These
doses of 1,25-(OH)2D3
raised calcium significantly from 2.0 ± 0.1 to 3.2 ± 0.2
mmol/L calcium (Fig. 4)
. At all levels of dietary calcium, sustained
hypercalcemia was associated with significant weight loss (Table 1
). Finally, at least four times more
1,25-(OH)2D3, and at high
calcium intakes, 17 times more
1,25-(OH)2D3 was required
to prevent EAE in male mice than in female mice. For the
1,25-(OH)2D3-treated mice,
the overall symptoms of EAE (day of onset, paralysis scores) decreased
as the incidence dropped (data not shown).
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1,25-(OH)2D3 administration
to mice consuming high calcium diets resulted in a net loss of
lymphocytes and the increased expression of IL-4 and TGF-ß1
(Cantorna et al. 1998b
). Control mice with EAE had
3.5–4.1 x 107 cells in the LN
regardless of the amount of calcium in their diets (Fig. 5
). The total number of cells in the LN of
1,25-(OH)2D3-treated mice with EAE was the same
as that of controls fed low calcium diets but decreased dramatically
(P 0.001) with the addition of calcium to the
diet (Fig. 5)
. Cell surface analysis showed that regardless of dietary
calcium or 1,25-(OH)2D3 treatment, Thy-1
positive cells made up 52–57% of the LN cells, CD4+ cells were
40–44% of the LN cells, and CD8+ cells were 18–21% of the LN cells.
There was no change in G3PDH mRNA levels by any of the manipulations
used, and because this is considered a "housekeeping" gene, its
level is used as reference. The IL-4 and TGF-ß1 mRNA levels presented
on this basis reflect the IL-4 and TGF-ß1 levels per cell in the LN.
Little IL-4 was detected in the LN of control-fed mice with EAE
regardless of the amount of calcium in the diet (Fig. 6
). IL-4 production in 1,25-(OH)2D3-fed mice was
not different than controls when they were fed low calcium diets. The
amount of IL-4 produced increased as the calcium in the diet increased
but only in cells from mice treated with
1,25-(OH)2D3 (Fig. 6)
. TGF-ß 1 levels in the
LN of control-fed mice were uniformly low compared with those in
mice fed 1,25-(OH)2D3 (Fig. 6)
. Dietary calcium
had no effect on TGF-ß1 mRNA levels (Fig. 6)
. For IL-4 mRNA, mice
with the fewest cells in the LN [high calcium plus
1,25-(OH)2D3-treated] expressed the most IL-4.
For TGF-ß1 mRNA expression, all mice treated with
1,25-(OH)2D3 expressed high levels of TGF-ß1
regardless of the number of cells in the LN.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Therefore, our
results strongly support the involvement of calcium in the action of
1,25-(OH)2D3 for the
regulation of this T-helper cell–dependent autoimmune disease
(Holda and Swanborg 1982
, Powell et al. 1990
). It is of some interest that in mice fed a low calcium diet, doses of 1,25-(OH)2D3 that nevertheless produce frank hypercalcemia produce little or no further reduction in the incidence of EAE. Thus, in the absence of a dietary source of calcium, 1,25-(OH)2D3-mediated increases in serum calcium are ineffective for the further suppression of EAE. On the other hand, there are doses of 1,25-(OH)2D3 that do not cause hypercalcemia but that nevertheless reduce the incidence of EAE. Overall, our results argue that the 1,25-(OH)2D3 may function by both calcium-dependent and calcium-independent mechanisms to suppress EAE.
Manipulating only dietary calcium had no effect on the immune response
to EAE. The total cell number in the LN of mice with EAE was inversely
related to the amount of dietary calcium in mice fed
1,25-(OH)2D3. Similarly,
thymic atrophy resulted as a consequence of
1,25-(OH)2D3-induced
hypercalcemia (Mohamed et al. 1996
). The decreased cell
number in the LN may be due to
1,25-(OH)2D3-induced
decreases in cell expansion or increases in cell death. IL-4 mRNA was
increased, but not that of TGF-ß1, relative to the amount of calcium
fed. In the absence of added dietary calcium,
1,25-(OH)2D3 had no effect
on IL-4 expression.
1,25-(OH)2D3 and calcium
may actually be selectively inhibiting the differentiation and/or
expression of cells that do not make IL-4. The result would seem to be
an increase in IL-4 expression. Therefore, it is possible that IL-4
expression has not changed; rather, the proportion of cells making IL-4
has increased. Conversely, TGF-ß1 increased in response to
1,25-(OH)2D3 regardless of
dietary calcium. This finding is consistent with our previous work,
suggesting that TGF-ß1 gene expression is regulated directly by
1,25-(OH)2D3
(Cantorna et al. 1998b
). Calcium is an important
intracellular messenger, but how dietary calcium might affect
intracellular calcium is unknown. More work is required to understand
the relationship of
1,25-(OH)2D3 and calcium as
immune system regulators.
It is of some interest that low dietary calcium reduces the incidence
of EAE, whereas at the same time it reduces the effectiveness of
1,25-(OH)2D3 in preventing
EAE. The mice used in these experiments were not vitamin D depleted and
thus contained stores of vitamin D and 25-hydroxyvitamin D. Low dietary
calcium markedly stimulates the production of
1,25-(OH)2D3 (DeLuca 1983
). This endogenously produced hormone may well play a role
in reducing the incidence of EAE. On the other hand, it is clear that
even large doses of
1,25-(OH)2D3 cannot
completely prevent EAE in these mice. Low dietary calcium must
therefore play more than one role. These results with low dietary
calcium may well account for some of the divergent results on the
effect of 1,25-(OH)2D3 in
prevention of EAE reported in the literature (Bouillon et al. 1995
, Cantorna et al. 1996
, Lemire and Archer 1991
).
Our results argue that the role of 1,25-(OH)2D3 in preventing EAE and regulating the immune response to EAE involves calcium. Only detailed investigation at the cellular and molecular level will clarify the role of calcium and 1,25-(OH)2D3 in the regulation of the immune system.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Current address: Nutrition Department, Pennsylvania State University, 126S Henderson, University Park, PA 16802.
4 Abbreviations used: CNS, central nervous system; 1,25-(OH)2D3, 1,25-dihydroxycholecalciferol;
EAE, experimental autoimmune encephalomyelitis; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; IFN-, interferon ;
IL-4, interleukin-4; LN, lymph node; MBP, myelin basic protein; MS, multiple sclerosis; PCR, polymerase chain reaction; TGF-ß1,
transforming growth factor ß1; Th1, type-1 helper; Th2, type-2 helper; TNF-, tumor necrosis factor-; VDR, vitamin D receptor.
Manuscript received February 21, 1999. Initial review completed April 8, 1999. Revision accepted July 23, 1999.
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 S. M. Grant, J. A. Wiesinger, J. L. Beard, and M. T. Cantorna Iron-Deficient Mice Fail to Develop Autoimmune Encephalomyelitis J. Nutr., August 1, 2003; 133(8): 2635 - 2638. [Abstract] [Full Text] [PDF]
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T. F. Meehan and H. F. DeLuca CD8+ T cells are not necessary for 1alpha ,25-dihydroxyvitamin D3 to suppress experimental autoimmune encephalomyelitis in mice PNAS, April 16, 2002; 99(8): 5557 - 5560. [Abstract] [Full Text] [PDF]
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 H. F. DELUCA and M. T. CANTORNA Vitamin D: its role and uses in immunology FASEB J, December 1, 2001; 15(14): 2579 - 2585. [Abstract] [Full Text] [PDF]
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M. T. Cantorna, C. Munsick, C. Bemiss, and B. D. Mahon 1,25-Dihydroxycholecalciferol Prevents and Ameliorates Symptoms of Experimental Murine Inflammatory Bowel Disease J. Nutr., November 1, 2000; 130(11): 2648 - 2652. [Abstract] [Full Text] [PDF]
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T. F. Meehan and H. F. DeLuca CD8+ T cells are not necessary for 1alpha ,25-dihydroxyvitamin D3 to suppress experimental autoimmune encephalomyelitis in mice PNAS, April 16, 2002; 99(8): 5557 - 5560. [Abstract] [Full Text] [PDF]
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