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,
*
Laboratory of Experimental Surgery, Surgical Department, and
Department of Pathology, San Raffaele Scientific Institute, Milan, Italy;
University of Milan, Milan, Italy;
§
Department of Immunology and Cell Biology, Mario Negri Institute, Milan, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-dihydroxyvitamin D3
(1,25-(OH)2D3) on differentiation,
maturation, and functions of dendritic cells (DC) differentiated from
human monocytes in vitro in the presence of GM-CSF and IL-4 for 7 days.
Recovery and morphology were not affected by
1,25-(OH)2D3 up to 100 nM. DC differentiated
in the presence of 10 nM 1,25-(OH)2D3
(D3-DC) showed a marked decrease in the expression of CD1a,
while CD14 remained elevated. Mannose receptor and CD32 were
significantly increased, and this correlated with an enhancement of
endocytic activity. Costimulatory molecules such as CD40 and CD86 were
slightly decreased or nonsignificantly affected (CD80 and MHC II).
However, after induction of DC maturation with LPS or incubation with
CD40 ligand-transfected cells, D3-DC showed marginal
increases in MHC I, MHC II, CD80, CD86, CD40, and CD83. The accessory
cell function of D3-DC in classical MLR was also inhibited.
Moreover, allogeneic T cells stimulated with D3-DC were
poor responders in a second MLR to untreated DC from the same or an
unrelated donor, thus indicating the onset of a nonspecific
hyporesponsivity. In conclusion, our data suggest that
1,25-(OH)2D3 may modulate the immune system,
acting at the very first step of the immune response through the
inhibition of DC differentiation and maturation into potent
APC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-Dihydroxyvitamin
D3
(1,25-(OH)2D3)3
is a secosteroid hormone that binds to a nuclear receptor named vitamin
D3 receptor. During the past few years it has
become apparent that
1,25-(OH)2D3, in
addition to its well-known role in mineral and skeletal homeostasis,
regulates the differentiation, growth, and function of a broad range of
cells, including cells of the immune system (1, 2, 3, 4). The
immunological effects of pharmacological levels of
1,25-(OH)2D3 or its
analogues in vivo were demonstrated in studies of autoimmune disease
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) and studies of allograft rejection
(17, 18, 19, 20). The effects of
1,25-(OH)2D3 on the
immune system were ascribed to its action on lymphocytes and
monocytes/macrophages (21, 22, 23). When added to
mitogen-stimulated human peripheral blood lymphocytes in vitro,
1,25-(OH)2D3 inhibits
their proliferation, Ig synthesis, and accumulation of transcripts for
IL-1, IL-2, IL-6, TNF-, and -ß and IFN-
(24, 25, 26).
Of interest is that
1,25-(OH)2D3 induces
promyelocytes to differentiate into monocytes (27); in
addition, 1,25-(OH)2D3
differentiates myeloid leukemia cells to nonproliferating
monocyte/macrophage-like cells in both humans and mice (28, 29) and promotes the differentiation of myeloid stem cells and
normal peripheral blood monocytes toward a macrophage phenotype
(30).
1,25-(OH)2D3 also
affects functional activities of monocytes and macrophages with
contrasting results. Tumor cell cytotoxicity, phagocytosis, and
mycobactericidal activity of monocytes/macrophages are enhanced by
exposure to 1,25-(OH)2D3
(31), but monocyte function as an APC appears be decreased
(32, 33).
Over the past years in vitro methods have been described to
differentiate dendritic cells (DC) from blood monocytes by in vitro
culture with GM-CSF, IL-4 (34, 35, 36), or IL-13
(37). These cultured DC show functional and phenotypic
characteristics typical of the immature stage of differentiation (i.e.,
high capacity of Ag uptake and processing, low capacity to stimulate T
cell proliferation) and can be further differentiated in vitro into
mature DC with TNF-, LPS, IL-1, or CD40L (35, 38). As
they are the most potent APC in vitro and in vivo, DC play a key role
in the initiation of the immune response and are considered promising
tools and targets for immunotherapy (39, 40, 41). It is
therefore important to identify factors that might affect their process
of differentiation and maturation (42, 43).
The aim of our work was to study the effects of
1,25-(OH)2D3 on human
monocyte-derived dendritic cell differentiation, maturation, and
functional activities. Our results demonstrate that
1,25-(OH)2D3 inhibited
DC differentiation and maturation into potent APC. Moreover,
D3-DC promoted the onset of a nonspecific
hyporesponsivity in T cells. These findings may have relevance in the
development of new therapeutic treatments in the field of transplants
and autoimmune diseases
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human recombinant GM-CSF (sp. act., 1.1 x
104 U/mg) was obtained from Novartis (Basel,
Switzerland). Human rIL-4 (sp. act., >2 x
106 U/mg) and human rTNF- (sp. act., >2
x 107 U/mg) were obtained from PeproTech
(London, U.K.). 1,25(OH)2D3
was purchased from Sigma (St. Louis, MO).
DC culture
Highly enriched monocytes (>80% CD14+)
were obtained from buffy coats of 20 blood donors (through the courtesy
of Centro Trasfusionale, Ospedale San Raffaele, Milan, Italy) by Ficoll
and Percoll gradients and were purified by adherence. Monocytes were
cultured for 7 days at 1 x 106/ml in
six-well tissue culture plates (Falcon, Becton Dickinson, Rutherford,
NJ) in RPMI (Biochrom, Berlin, Germany) and 10% FCS (HyClone, Logan,
UT) supplemented with 50 ng/ml GM-CSF and 10 ng/ml IL-4 and with
(D3-DC) or without (ctr-DC) various
concentrations of
1,25-(OH)2D3. In the
control group (GM-CSF plus IL-4) the cell yield was about 80% of input
cells. All cultures were tested for the presence of endotoxin (<0.03
U/ml; Lymulus test, BioWhittaker, Walkersville, MD).
DC maturation
LPS (10 ng/ml) was added to induce maturation of DC for at least 36 h of culture. Alternatively, J558L cells transfected with the ligand for CD40 (J558LmCD40L) were used to induce CD40 triggering on DC. Untransfected J558L cells were used as a control. After irradiation (10,000 rad) J558L cells were seeded together with DC at a 1:1 ratio in 24-well culture plates in culture medium (1 x 106 cells/well). Cells were recovered after 48–72 h of culture.
FACS analysis
Cell staining was performed using mouse mAbs followed by FITC-conjugated, affinity-purified, isotype-specific, goat anti-mouse Abs (Ancell, Bayport, MN). The following mAbs were used: L243 (IgG2a, anti-MHC class II), 32.2 (anti-CD32), and IV.3 (anti-CD64) from American Type Culture Collection (Manassas, VA); UCHM-1 (IgG2a, anti-CD14) and W6/32 (IgG2a, anti-MHC I) from Sigma; SK9 (IgG2b, anti-CD1a) from Becton Dickinson (San Jose, CA); B73.1 (IgG2a, anti-CD16) from Dr. G. Trinchieri (Philadelphia, PA); PAM-1 (IgG1 anti-mannose receptor) (44, 45); BB1 (IgM, anti-CD80), BU63 (IgG1, anti-CD86), and EA-5 (IgG1 anti-CD40) from Ancell; and HB15a (IgG2b, anti-CD83) from Immunotech (Marseilles, France). Results are expressed as the percentage of positive cells or as fluorescence intensity (FI), calculated according to the formula: FI = mean fluorescence (sample) - mean fluorescence (control).
Endocytosis
Mannose receptor (MR)-mediated endocytosis was measured as the cellular uptake of FITC-dextran and was quantified by flow cytometry. Approximately 2 x 105 cells/sample were incubated in medium containing FITC-dextran (1 mg/ml; m.w., 40,000; Sigma) for 0, 60, and 120 min. After incubation cells were washed twice with PBS to remove excess dextran and were fixed in cold 1% formalin. The quantitative uptake of FITC-dextran by the cells was determined by FACS. At least 8,000 cells/sample were analyzed. Fluid phase endocytosis through membrane ruffling was measured as the cellular uptake of 1 mg/ml of Lucifer Yellow (LY) dipotassium salt (Sigma) and was quantified by flow cytometry.
Mixed leukocyte reaction
DC cultured in GM-CSF and IL-4 and with or without
1,25-(OH)2D3 for 7 days
were extensively washed, irradiated (3000 rad from a
137Cs source), and added in graded doses to
1 x 105 responder cells in 96-well
flat-bottom Microtest plates (Costar, Cambridge, MA). Responder cells
were purified allogeneic T cells depleted of autologous APC by passage
with CD14- and CD19-coated Dynabeads (Unypath, Milan, Italy). Each
group was performed in triplicate. Thymidine incorporation was measured
on day 5 by a 16-h pulse with [3H]thymidine (1
µCi/well; spec. act., 5 Ci/mmol; Amersham, Aylesbury, U.K.).
Ag presentation assay
Tetanus toxin (TT)-responsive T cell lines were generated in our laboratory by culturing mononuclear cells with TT (36 µg/ml; Cannaught, Willowdale, Canada) for 1 mo in the presence of IL-2. TT-responsive T cells were tested at least 2 wk after the last PBMC stimulation and 5 days after the last addition of IL-2. DC were obtained from the same donor by culturing monocytes. After 7 days DC were preincubated with TT (6 µg/ml) for 12 h and with LPS for 48 h. Then DC were extensively washed, irradiated (3000 rad), and cocultured with autologous TT-responsive T cell lines for 72 h in 96-well microtiter plates, and [3H]thymidine uptake was measured during the last 12 h of culture (1 µCi/well; sp. act., 5 Ci/mmol; Amersham).
IL-12p70 measurement
After 7 days of culture with GM-CSF (50 ng/ml) and IL-4 (10
ng/ml) in the presence or the absence of
1,25-(OH)2D3, DC were
washed twice and cultured for 3 days at 0.5 x
106/ml in a 24-well flat-bottom plate (Costar).
DC were either nonstimulated or stimulated with TNF- (10 ng/ml), LPS
(50 ng/ml), J558L cells transfected with CD40L (J558LmCD40L), or
untransfected J558m cells. After 3 days medium was collected, and IL-12
p70 was quantified by ELISA (commercial kits from Endogen, Boston,
MA).
Anergy assay
Allogeneic T cells were prepared from human blood using Ficoll and Percoll gradients and subsequent depletion of B cells and monocytes by plastic adherence and by Ab-coated immunomagnetic beads (Unipath, Milan, Italy) according to a standard protocol. T cells were cocultured during the first incubation at a density of 1 x 106/ml with 1 x 104/ml ctr-DC or D3-DC (DC were prepared as described above and matured with LPS). Three days later T cells were separated by Ficoll gradient (Sigma), extensively washed, depleted of DC using Ab-coated immunomagnetic beads (Ab anti CD32 and MR), and rested for 5 days in medium alone. Subsequently, T cells were restimulated with mature ctr-DC generated from the same donor used in the first coculture or from another unrelated donor.
Electron microscopy
DC were processed for electron microscopy. DC were fixed for 2 h in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Then they were postfixed in 1% OsO4 in cacodylate buffer at 4°C for 1 h, dehydrated in graded ethanol up to propylene oxide, and finally embedded in an Epon-Araldite mixture. Well-preserved areas were identified by light microscopy of semithin sections (0.5 mm). Subsequently, serial ultrathin sections (80 nm) were mounted on 200-mesh copper grids, stained with uranyl acetate and lead citrate, and finally examined with a Zeiss CEM 902 electron microscope (New York, NY).
Calculations and statistical analysis
Data were expressed as the mean ± SD. Comparisons were performed using Student’s t test. A p value of <0.05 was considered statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D3 interferes with the
differentiation of DC from human monocytes
To investigate the effect of
1,25-(OH)2D3 on DC
differentiation from monocytes, we cultured monocytes in the presence
of GM-CSF, IL-4 (control group, ctr-DC), and various concentrations
(0.5–100 nM) of
1,25-(OH)2D3
(D3-DC).
1,25(OH)2D3 did not affect
cell recovery at any concentration tested. The standard concentration
of 1,25-(OH)2D3 chosen
for the study was 10 nM, the highest concentration considered
physiological (4); this was also used in previous studies
for leukocyte differentiation (30). Upon culture with
GM-CSF and IL-4 for 7 days cells became nonadherent and clustered, with
abundant cytoplasm and protruding veils typical of DC. Despite a
similar morphology (Fig. 1
), the presence
of 1,25-(OH)2D3 in
culture interfered with the differentiation of monocytes into DC. Fig. 2 shows a representative experiment of
surface phenotype. Control cells expressed high levels of CD1a and were
negative or low positive for CD14 and CD16, while with
1,25-(OH)2D3 the cells
were negative or low positive for CD1a but expressed higher levels of
CD14. Analysis of MHC class I showed an up-regulation of
D3-DC, whereas expression of MHC II, CD40, and
CD86 molecules was decreased. Ag uptake molecules, such as CD32 and MR,
were increased. DC obtained after 7-day culture with GM-CSF and IL-4
could be further differentiated in vitro into fully mature DC by
exposure to LPS or CD40L. D3-DC were not
sensitive to maturation stimuli. In fact, after exposure to LPS (Fig. 3) or CD40L, D3-DC
were unable to up-regulate CD83 as well as the molecules involved in Ag
presentation (MHC I, MHC II, CD80, CD86, and CD40) and to down-regulate
the Ag uptake molecules (CD2 and MR). A summary of six different
experiments is shown in Table I.
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,25-(OH)2D3 showed augmented Ag uptake
capacity and inhibited immunostimulatory capacity
Immature DC, such as cells derived by culturing monocytes with
GM-CSF and IL-4 for 7 days, express a potent ability to uptake external
molecules, essentially via two main mechanisms: a receptor-mediated
endocytosis and a fluid phase endocytosis (macropinocytosis). To study
the endocytic capacity of D3-DC, we used two
fluorescent markers: LY, a nonspecific fluid phase marker, and FITC-DX,
which is mainly taken up via the MR. DC cultured with GM-CSF and IL-4
in the presence of
1,25-(OH)2D3 showed a
vigorous endocytosis of FITC-dextran, higher than control DC (Fig. 4A). The same behavior was
seen when we used LY as marker of fluid phase pinocytosis (Fig. 4B). DC are potent stimulators of allogeneic T cells. We
tested whether D3-DC were able to stimulate
allogeneic T lymphocytes in MLR. D3-DC showed
very little ability to induce allogeneic T lymphocyte proliferation
(Fig. 5A). Moreover, the
immunostimulatory capacity of D3-DC in MLR was
not increased by LPS or was increased to a much lower extent by CD40L,
compared with that of ctr-DC (Fig. 5). In view of the fact that Ag
capture was increased in D3-DC but the
stimulatory capacity was impaired in MLR, we evaluated the ability to
present soluble Ag that need to be taken up and processed. Cells
differentiated in the presence of
1,25-(OH)2D3 showed much
lower efficiency in presenting TT to specific autologous T cell lines
(Fig. 6).
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,25-(OH)2D3 affects IL-12 p70
production by DC
To investigate the capacity of
1,25-(OH)2D3 to
interfere with IL-12 production, after 7 days of culture with GM-CSF
and IL-4 with or without
1,25-(OH)2D3, DC were
washed, seeded in the presence of maturation-inducing stimuli, and
cultured for 3 days. Supernatants were quantified for IL-12 p70. IL-12
p70 production was significantly decreased when
D3-DC were exposed to TNF- (53.5 vs 72.2
pg/0.5 x 106 cells/ml; p =
0.05) or LPS (45.7 vs 79.8 pg/0.5 x 106
cells/ml; p = 0.02), or CD40L (38.6 vs 481.7
pg/0.5 x 106 cells/ml; p =
0.002; Fig. 6
).
Induction of hyporesponsivity in T cells by
1,25-(OH)2D3-treated DC
As T cell stimulation via TCR in the absence of a second signal by
costimulatory molecules and/or secreted cytokines may induce a state of
hyporesponsivity or anergy, we tested whether
D3-treated DC induced an alloantigen-specific
tolerance. In these experiments allogeneic T cells were first
cocultured with ctr-DC or D3-DC and exposed to
LPS for 3 days. Then cells were extensively washed, depleted of
remaining DC by MR or CD2 using Immuno-Dynabeads (Unipath, Milan,
Italy), and rested for 5 days. T cells (viability, >90%) were then
restimulated in a second coculture with mature ctr-DC. T cells first
cocultured with ctr-DC responded vigorously to restimulation with
mature ctr-DC. In contrast, T cells first cocultured with
D3-DC were hyporesponsive to further stimulation
with ctr-DC (Fig. 7A). To
determine whether this hyporesponsivity was alloantigen specific, the
rescued T cells were restimulated with DC generated from an unrelated
donor. In this case also, T cells showed a profound inhibition of
proliferative capacity compared with T cells cocultured with untreated
DC (Fig. 7B). These results indicate that
D3-Dc exposed to LPS induce a state of
hyporesponsivity in T cells, which was not alloantigen restricted. Of
interest, when immature D3-DC were used as
stimulator in the first coculture, T cells were not inhibited (data not
shown).
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,25-(OH)2D3 on already
differentiated immature DC
To evaluate the effects of
1,25-(OH)2D3 on
differentiated immature DC, monocytes were cultured for 7 days in the
presence of GM-CSF and IL-4. Cells were then washed and incubated again
with IL-4, GM-CSF, and
1,25-(OH)2D3 for 3 or 7
additional days. Control cells were incubated with GM-CSF and IL-4 for
the same period of time. Treatment with
1,25-(OH)2D3 partially
reversed DC differentiation, as demonstrated by a down-regulation of
CD1a and an up-regulation of CD14; expression of CD80, CD86, and MHC I
was not affected. In contrast, MHC II and CD40 were significantly
down-regulated (Fig. 8). After exposure
to LPS or CD40L, D3-DC showed a lower expression
of MHC I, MHC II, CD80, CD86, CD83, and CD40. A summary of four
experiments is shown in Table II.
Finally, we evaluated the influence of
1,25-(OH)2D3 on the
endocytic activity of immature DC and on the capacity to stimulate T
lymphocytes in MLR.
1,25-(OH)2D3
significantly increased the uptake of FITC-dextran and Ag uptake
receptor expression, while the capacity to stimulate allogeneic T
lymphocytes was decreased compared with that of untreated DC (data not
shown). Overall, these results demonstrate that
1,25-(OH)2D3 impaired
the maturation of DC even when added tn already differentiated
cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D3, modulates
lymphocyte and macrophage functions (46). We demonstrated
a new target of
1,25-(OH)2D3 action on
the immune system: DC. Because DC have the unique property to activate
naive T cells and are required for the induction of a primary response
(47), the suppression of DC function may very efficiently
control the specific immune response (48).
1,25-(OH)2D3 showed
complex effects on DC.
1,25-(OH)2D3 partially
blocked the GM-CSF- and IL-4-driven differentiation of monocytes to DC.
In fact, in the presence of
1,25-(OH)2D3, despite a
quite similar morphology, the expression of CD1a was inhibited and CD14
expression was increased, a marker of monocytes/macrophages normally
not present on DC (49). In previous studies high
expression of CD14 was found in monocyte-derived DC cultured in the
presence of immunosuppressive factors such as glucocorticoid and IL-10
(50, 51). The intensity of CD14 expression was lower in
vitamin D3-derived DC than in
glucocorticoid-derived or IL-10-derived DC, and CD14 was not seen with
low concentration (5%) of FCS (data not shown) Despite the persistence
of CD14, other markers of monocytes/macrophages, such as CD16 and CD68,
were not present in vitamin D3-treated DC.
Therefore, we conclude that vitamin D3 inhibits a
full differentiation of monocytes into DC, but, unlike IL-10, does not
promotes differentiation toward macrophages. In the past, a clear
effect of D3 has been shown on monocytes, bone
marrow precursors, and monocytic leukemic cell lines on the
differentiation toward macrophages, but under different
culture conditions (28, 30, 52, 53).
1,25-(OH)2D3 is also
important in osteoclast generation, defining the commitment of
monocytes differentiating into osteoclasts as a cooperative associative
mechanism involving osteoblastic cells (54, 55). Monocytes
may be considered relatively immature precursors with multiple
differentiation potentials that depend upon the microenvironment
(56). Our data showed that similar to cytokines such as
M-CSF, GM-CSF, TGF-ß, and IL-4, the hormone
1,25-(OH)2D3 may play an
important role in the final decision determining whether monocytes will
acquire DC, macrophage, or osteoclastic characteristics and functions,
in particular inhibiting DC differentiation.
1,25-(OH)2D3-treated DC
showed other important modifications in phenotype. Normally we can
identify two major phases in the life of DC (40, 41, 57, 58): an immature stage characterized by a high efficiency in
taking up and processing Ags associated with high expression of
molecules involved in Ag uptake as MR, such as CD32; and a mature stage
in which the Ag uptake capacity is lost, the cell migrates toward
regional lymph nodes, and the function shifts to become a potent APC
(59, 60) associated with a high expression of molecules
involved in Ag presentation and T cell stimulation, such as MHC I, MHC
II, CD80, CD86, CD40, and CD83. Exposure of differentiating monocytes
to 1,25-(OH)2D3
increased the expression of molecules involved in Ag capture (CD32,
MR), while some important costimulatory molecules (CD86, CD40) were
inhibited. This phenotype correlates with impaired Ag-presenting
function for T lymphocytes and higher endocytic activity. Moreover,
1,25-(OH)2D3 strongly
inhibited DC maturation, as demonstrated by a low or absent increase in
the expression of MHC I, MHC II, CD80, CD86, CD40, and CD83 and by the
impaired stimulatory capacity for T lymphocytes after exposure to LPS
or CD40L. Finally, as recently reported with already differentiated
DC (61), D3-DC showed impaired IL-12
production after CD40L, LPS, or TNF- exposure. These results extend
the immunosuppressive effects of this hormone and confirm a role for
1,25-(OH)2D3 as a
regulator of immune cell differentiation and function.
The effects of
1,25-(OH)2D3 on immature
DC that have been differentiated for 7 days in the presence of GM-CSF
and IL-4 appear to be similar to, but not identical with, the effects
of 1,25-(OH)2D3 included
at the beginning of the culture. Overall, the effects can be summarized
as follows: a partial conversion to a monocyte/macrophage phenotype, an
impaired capacity to reach maturation, and a decreased ability to
stimulate T cells (the latter not shown). These results confirm the in
vitro instability of immature DC generated with GM-CSF and IL-4
(56, 62). Palucka et al. (56) showed that
upon removal of both GM-CSF and IL-4 and/or reculture with M-CSF,
immature CD1a+/CD14- DC
easily converted to a macrophage phenotype expressing CD14 with a
decreased ability to stimulate allogeneic T cells. Thus
1,25-(OH)2D3 acts at two
different steps of DC life: 1) inhibiting the differentiation from
monocytic precursors and thus impairing the normal turnover of DC in
tissues, and 2) inhibiting the terminal maturation of DC into a
potent APC.
The inhibitory effect of
1,25-(OH)2D3 on DC
maturation and differentiation is very similar to that of IL-10, an
anti-inflammatory cytokine, and to that of glucocorticoids. In
fact, both IL-10 and glucocorticoids were shown to prevent monocyte
differentiation and maturation to DC, to impair IL-12 production, and
to increase Ag uptake (43, 50, 51).
Of interest is the fact that
1,25-(OH)2D3-differentiated
DC matured with LPS or CD40L induced hyporesponsivity of allogeneic T
cells. T cells cocultured with D3-DC showed
impaired proliferation to a second stimulation with control DC from the
same as well as from an unrelated donor. A direct effect of
D3 on T cells is excluded, as
D3-DC were extensively washed before coculture.
Moreover, T cell viability before the second stimulation was >90%,
and T cells were able to proliferate upon addition of exogenous IL-2
(data not shown). The induction of T cell anergy has been reported by
Steinbrink et al. (63) with DC differentiated in the
presence of IL-10, but several differences can be outlined, showing
different mechanisms of action between IL-10 and
D3. In IL-10-treated DC the induction of anergy
is associated only with immature DC and is alloantigen specific. In our
work it was not alloantigen specific and was not observed when we used
immature D3-DC. It appeared at least in part
linked to a soluble factor(s) secreted by D3-DC
exposed to LPS. In fact, supernatants from these cells, when added in a
first coculture to control mature DC and allogeneic T cells, inhibited
T cell proliferation, while supernatants from immature
D3-DC or mature ctr-DC did not. The generation of
DC able to induce T cell hyporesponsivity might be a first step in the
development of treatments for patients at risk of transplant rejection
or with autoimmune or allergic diseases. The therapeutic use of these
cells, however, requires further studies, as the hyporesponsivity was
not specific, and putative factors involved in the induction of T cell
hyporesponsivity remain to be defined.
The effects described in our work were seen in a range of
1,25-(OH)2D3
concentrations from 5 x 10-11 to
10-7. Importantly, calcitriol is effective at
concentrations that are considered physiological
(10-10–10-8 mol/l)
(64) and that correspond to the accepted affinity value
for its receptor (65). The physiological role of vitamin D
in immune responses is not clearly defined. In vivo, both an excess and
a deficiency of vitamin D suppress the delayed hypersensitivity
response (66) or Ig production (67). Vitamin
D-deficient animals and humans have a higher risk of infection,
probably related to impaired macrophage function (68). The
monocyte function as APC seem to be decreased (32). The NK
cell activity is enhanced by
1,25-(OH)2D3. This
enhancing effect of the nonspecific immune defense contrasts with an
inhibition of the Ag-specific immune response, as demonstrated by
decreased T cell proliferation and activity (decreased IL-2, IFN-,
GM-CSF synthesis and secretion). It is very difficult to clarify the
endocrine activity of
1,25-(OH)2D3 in the
immune system. A possible paracrine or autocrine activity may be
postulated. The secretory role of macrophages may be central to the
production of localized concentration of
1,25-(OH)2D3 within
immune microenvironments. Normal macrophages have been shown to
synthesize 1,25-(OH)2D3
when activated by agents such as IFN- and LPS (1, 4).
In granulomatous disorders such as sarcoidosis and tuberculosis,
macrophages are able to produce
1,25-(OH)2D3 and appear
to be insensitive to feedback control by
1,25-(OH)2D3 itself as
well as other regulators, such as calcium and parathyroid hormone. It
is tempting to speculate that
1,25-(OH)2D3 produced by
macrophages, by inhibiting differentiation and function of DC, may
contribute to the peripheral anergy in sarcoidosis and to the
persistence of granulomatous lesion in tuberculosis.
Another site of interest for
1,25-(OH)2D3 action is
the skin. It is likely that the major source of vitamin D for human is
not dietary, but results from its manufacture by a chemical photolysis
reaction in skin. Vitamin D3 itself is a
biologically inert molecule. It must be activated by 25-hydroxylation
in the liver to produce the major circulating form of vitamin D,
25-hydroxyvitamin D3. However, 25-hydroxyvitamin
D3 is also biologically inactive at physiological
concentrations, and it is finally activated in the proximal convoluted
tubule cells of the kidney to produce
1,25-(OH)2D3
(8). Keratinocytes, the most important cells of the skin,
possess both the 24- and 1-hydroxylase enzymes and thus can produce
small amount of
1,25-(OH)2D3. Therefore,
UV exposure induces a systemic and a local increase in
1,25-(OH)2D3 in the
skin. It is known that after UV exposure, Langerhans cells (epidermal
CD1a+ cells) disappear from the healthy skin, and
CD11b+ macrophage-like cells appear in few days
(69). Moreover, UV radiation induces apoptosis and
suppresses the immune function of epidermal Langerhans cells
(70). Although other cytokines, such as IL-10, are known
to play an important role in UV-induced immunosuppression
(71), it is tempting to speculate that
1,25-(OH)2D3 could also
contribute to some of the modifications of Langerhans cells and could
be responsible for the decrease in DC in skin after UV exposition.
In conclusion, our data suggest that
1,25-(OH)2D3 may
modulate the immune system, acting at the very first step of the immune
response through the inhibition of DC differentiation and maturation
into potent APC.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lorenzo Piemonti, Laboratory of Experimental Surgery, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy.
3 Abbreviations used in this paper: 1,25-(OH)2D3, 1,25-dihydroxyvitamin D3; DC, dendritic cells; CD40L, CD40 ligand; MR, mannose receptor; TT, tetanus toxin; 3-DC, DC differentiated in the presence of 10 nM 1,25-(OH)2D3; ctr-DC, control DC; LY, Lucifer Yellow.
Received for publication October 12, 1999. Accepted for publication February 11, 2000.
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