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Original Studies |
,25-Dihydroxyvitamin D3 on the Cytokine Production of Human Peripheral Blood Lymphocytes1
Institute of General and Experimental Pathology (M.W., R.T., K.S., E.B., M.H., R.G., K.B., P.P., O.S., M.P.) and the Department of Gastroenterology and Hepatology (W.R.), Clinic of Internal Medicine IV, University of Vienna, Vienna, Austria
Address all correspondence and requests for reprints to: Martin Willheim, M.D., Institute of General and Experimental Pathology, AKH Wien, Waehringer Guertel 18–20, A-1090 Vienna, Austria. E-mail: martin.willheim{at}akh-wien.ac.at
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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 cytokine
production and differentiation of subsets of CD4+ [T
helper 1 (Th1) and Th2] and CD8+ [T cytotoxic 1 (Tc1) and
Tc2] lymphocytes at the single cell level. PBMC from healthy donors
were cultured with or without 1,25-(OH)2D3
for up to 21 days. On days 0, 7, 14, and 21, the percentage of
cytokine-producing T lymphocytes was analyzed by intracellular cytokine
detection with mAb and flow cytometry. Simultaneous staining for cell
surface markers allowed discrimination of CD4+ and
CD8+ T cell subsets. After culture with
1,25-(OH)2D3 (10-8 mol/L), no
significant effects on the proportion of interferon-
(IFN)- or
interleukin-4 (IL-4)-producing cells were detected, whereas reduced
frequencies of IL-2-producing cells in the CD4+ as well as
in the CD8+ population were found. An increase in the low
percentage of CD4+ and CD8+ T cells producing
the Th2 cytokine IL-13 was noticed. Most interestingly, IL-6-producing
CD4+ and CD8+ T cells could only be detected in
cultures with 1,25-(OH)2D3, reaching a
plateau after 14 days. The percentage of IL-6-producing T cells induced
by 1,25-(OH)2D3 after a given time period
remained stable for at least 7 weeks. Studies of cytokine coexpression
revealed that about 70% of IL-6-producing CD4+ and
CD8+ cells were also positive for IL-2, but more than 90%
were negative for IFN, IL-4, or IL-13, respectively. This suggests
that the IL-6-producing population does not match the Th1/Tc1-like
(IFN+) or Th2/Tc2-like (IL-4+ or
IL-13+) subset. The influence of
1,25-(OH)2D3 on cytokine production by
lymphocytes is probably an important point of intersection between the
endocrine and the immune system. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(IFN) and interleukin-2 (IL-2), but
little or no IL-4 and IL-5, whereas Th2 cells mainly produce IL-4 and
IL-5. Whereas Th1 cells are involved in cell-mediated inflammatory
reactions, Th2 cytokines encourage antibody production, particularly
IgE responses, and enhance eosinophil proliferation and function.
Accordingly, Th2 cells are found in association with strong antibody
and allergic responses. Similar subsets exist in the
CD8+ population [cytotoxic T 1 (Tc1) and Tc2]
(3, 4, 5). There is increasing evidence, that CD4+
as well as CD8+ T lymphocytes are collectively
forming a continuous spectrum in which Th1/Tc1 and Th2/Tc2 cells may be
only two extremes of the possible phenotypes (6, 7). Furthermore, cells
producing high amounts of transforming growth factor-ß have been
described and classified as the Th3 subset (8), indicating that the
original concept is a simplification of the real situation. However,
regardless of whether the variation in T cell cytokine synthesis
represents a continuum or discrete subsets, the Th1-Th2 dichotomy
remains an important functional division in the immune system and has
been implicated in several immune responses concerning infections,
allergy, and autoimmunity (reviewed in Ref. 3).
A number of factors have been held responsible for the differentiation
of Th1/Tc1 and Th2/Tc2 cells, including the dose of antigen, the type
of antigen-presenting cell and/or of the costimulatory pathways
(reviewed in Ref. 9), as well as the local cytokine environment
(reviewed in Refs. 10, 11). Furthermore, the arachidonic acid
metabolite PGE2 (12, 13, 14), and some members of the
steroid hormone family, such as progesterone (15, 16, 17), glucocorticoids
(18, 19), and 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] (20)
have been reported to influence Th and Tc subset development.
1,25-(OH)2D3 is required
for normal calcium and phosphorus homeostasis and regulation of bone
remodeling. In addition,
1,25-(OH)2D3 has
multiple effects on the differentiation and function of hemopoietic,
immunological, epidermal, and cancer cells (21, 22). Synthesis of
1,25-(OH)2D3 from
25-hydroxyvitamin D3 has been shown by various
extrarenal tissues. Activated macrophages can synthesize
1,25-(OH)2D3 in
sarcoidosis, tuberculosis, and other granulomatous diseases, and their
1-hydroxylase activity is increased by IFN (23). Thus, local
production of
1,25-(OH)2D3 has been
discussed to have an autocrine/paracrine function. It has been proposed
that 1,25-(OH)2D3 plays
a role as an immunoregulatory hormone with distinct immunosuppressive
activities (24). Interference with cytokine production of monocytes and
lymphocytes seems to be a key mechanism by which
1,25-(OH)2D3 interacts
with the immune system. Inhibition of secretion of IL-1, IL-2, IL-6,
TNF, and IFN (25, 26, 27, 28, 29) has been reported. Recently, a preferential
inhibition of Th1 functions by the hormone has been suggested (20).
This opened a new point of view on a (patho)-physiological as well
as pharmacological role of
1,25-(OH)2D3. However,
the frequency as well as the precise pattern of cytokine (co)expression
of the T lymphocyte populations that are generated by
1,25-(OH)2D3 have not
yet been defined. Such a clear definition is especially important with
regard to a potential therapeutic application of vitamin
D3 compounds in Th1-mediated clinical situations
such as autoimmunity and transplantation (30, 31, 32).
In the present study we therefore analyzed the regulation of cytokine
production and coexpression in CD4+ and
CD8+ T lymphocytes by
1,25-(OH)2D3 at the
single cell level with a flow cytometric intracellular cytokine
detection method. We demonstrate that
1,25-(OH)2D3 decreases
the frequency of T cells capable of producing IL-2, but not that of
those capable of producing IFN, increases the percentage of
IL-13-positive cells, and induces the appearance of IL-6-producing T
cells. Moreover, we show that T cells producing IL-6 are essentially
negative for IL-4, IL-13, and IFN.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Healthy donors (four women and five men) were members of the
laboratory staff and medical students, with an age range from 25–36
yr. PBMC were isolated from heparinized peripheral blood by density
gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden),
and cultured in Ultra Culture Medium (BioWhittaker, Inc.,
Walkersville, MD). A schematic representation of the cell culture
protocol is shown in Fig. 1
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,25-(OH)2D3 (gift from
Hoffmann La Roche, Basel, Switzerland) at a concentration of
10-8 mol/L was present throughout the culture
period (or absent in control cultures). On day 10, 10 mL fresh medium
were added. Cells were analyzed for their cytokine-producing capability
on day 0 and thereafter every 7 days. Intracellular detection of cytokines
Flow cytometric assessment of T cell cytokine production was
performed essentially by the technique described previously (33). In
brief, PBMC were isolated and cultured as described above. At the
respective time points, cells were stimulated with 10 ng/ml phorbol
12-myristate 13-acetate (PMA) and 1.25 µmol/L ionomycin in the
presence of 2 µmol/L monensin (all from Sigma Chemical Co., St. Louis, MO) for 4 h. Cells were then harvested,
washed, and fixed with 2% formaldehyde. Cells were permeabilized with
0.1% saponin (Sigma Chemical Co.) in phosphate-buffered
saline. Four-color staining was performed, and cytokine production was
analyzed in the CD4+ and
CD8+ lymphocyte populations. Cytokine-specific
rat or mouse anti-human mAb labeled with phycoerythrin [PE;
IL-2(MQ1–17H12), IL-4 (8D4–8), IL-6 (MQ2–13A5), and IL-13
(JES10–5A2)] or fluorescein-isothiocyanate [FITC; IFN (4S.B3)]
as well as the respective isotype controls were obtained from
PharMingen (San Diego, CA). To study the coexpression of
IL-6 with IL-2, IL-4, and IL-13, a FITC-labeled anti-IL-6 mAb
(PharMingen) was used. In this case, a polyclonal
FITC-labeled rabbit anti-rat IgG conjugate (STAR17B,
Serotec, Oxford, UK) had to be used as a second step
reagent to reach sufficient signal intensity for IL-6. Percentages of
IL-6-positive cells after optimal staining were similar with FITC and
PE, and double staining controls confirmed that the population detected
by both techniques was identical. The anti-CD4 mAb was labeled with
allophycocyanin, and the anti-CD8 mAb was labeled with peridinin
chlorophyll (Becton Dickinson and Co., Mountain View, CA).
Figure 2 illustrates the gating strategy
used to analyze cytokine (co-) expression in CD4+
and CD8+ lymphocytes. Cells were gated as
lymphocytes by their light scatter characteristics and subsequently
defined as CD4+ and CD8+.
Cells fulfilling both criteria (lymphocyte and
CD4+ or CD8+) were further
analyzed for their cytokine production pattern.
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At the indicated time points, 1.0-mL aliquots of each culture (n = 5) were transferred to a 24-well plate and stimulated with 10 ng/mL phorbol 12-myristate 13-acetate and 1.25 µmol/L ionomycin for 4 h. IL-6 concentrations in cell culture supernatants were determined with a Medgenix IL-6 EASIA kit (Biosource Technologies Europe S.A., Fleurus, Belgium), according to the protocol of the manufacturer.
Statistics
Data were analyzed using Student’s t test (paired, two-tailed). Significance is expressed by P values (P < 0.05). Enzyme-linked immunosorbent assay (ELISA) data were analyzed using the Mann-Whitney rank sum test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, IL-4, and IL-13 by
1,25-(OH)2D3
Percentages of CD4+ and
CD8+ cells from cultures with or without
1,25-(OH)2D3, producing
the indicated cytokines are presented in Fig. 3, and the relative effects of
1,25-(OH)2D3 on the
frequency of cytokine-producing lymphocytes are shown in Table 1. On day 0, a significantly higher
frequency of IL-2-producing cells was found within the
CD4+ (46.4 ± 9.8%) compared to the
CD8+ (17.8 ± 4.3%) population
(P < 0.005). The rise in the frequency of
IL-2-positive CD4+ and CD8+
cells we observed in control cultures on day 14 was completely blocked
by 1,25-(OH)2D3
(P < 0.005).
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-producing cells were significantly
lower within the CD4+ population on day 0
(13.0 ± 5.1% vs. 24.3 ± 10.9% in
CD8+; P < 0.005). The effects of
1,25-(OH)2D3 on the
percentage of IFN-producing cells were highly variable, leading to
increased, decreased, or unchanged frequencies in experiments from
individual donors. Taken together, no tendency could be detected for
either CD4+ or CD8+
cells.
Few T cells produce IL-4 (3.4 ± 1.7% in
CD4+ vs. 2.0 ± 1.6% in
CD8+; not significant) and IL-13 (3.3 ±
1.2% in CD4+ vs. 1.6 ± 1.0% in
CD8+; P < 0.05) on day 0.
Although both CD4+ and CD8+
T lymphocyte subsets showed a slight tendency to increase their
proportion of IL-4-producing cells after culture with
1,25-(OH)2D3 compared to
controls, no statistical significance was reached. However, the
frequency of cells producing IL-13 significantly increased in cultures
treated with
1,25-(OH)2D3, leading to
8.5 ± 3.2% CD4+ cells staining positively
for IL-13 on day 14. Although significantly higher in
1,25-(OH)2D3 cultures
compared to controls, the percentage of IL-13-producing cells remained
low in CD8+ cells.
Induction of an IL-6-producing T cell population by
1,25-(OH)2D3
On day 0, IL-6-producing T cells were virtually absent, and only
marginal frequencies could be detected in some experiments. Control
cultures without
1,25-(OH)2D3 remained
negative at all time points tested. However, after treatment with
1,25-(OH)2D3, a
significant proportion of CD4+ [6.9 ±
4.5% on day 7 (P < 0.01) and 14.0 ± 9.2% on
day 14 (P < 0.005)] and CD8+
[2.2 ± 1.8% on day 7 (P < 0.05) and 4.3
± 4.8% on day 14 (P < 0.05)] cells produced IL-6.
The percentages of CD4+ and
CD8+ cells producing IL-6, are presented in Fig. 3
(bottom); the relative effects of
1,25-(OH)2D3 on the
frequency of cytokine-producing lymphocytes are shown in Table 1.
Experiments with culture periods of more than 3 weeks revealed that a plateau is reached on day 14 or, in some experiments, on day 21, with a maximum frequency of IL-6-producing CD4+ cells of 20–25% and of IL-6-producing CD8+ cells of 10–13% (data not shown). After having reached a plateau, the percentage of IL-6-positive cells remained stable for at least 62 days, which was the longest period tested (data not shown).
Coexpression of IL-6 with IL-2, IFN, IL-4, and IL-13
Simultaneous staining for the expression of IL-6, on the one hand,
and IL-2, IFN, IL-4, or IL-13, on the other hand, was used to
address the question of cytokine coexpression by
CD4+ and CD8+ cells treated
with 1,25-(OH)2D3 for 14
days. As shown in Figs. 4 and 5, less than 10% of the IL-6-producing
CD4+ cells were also positive for IFN.
Similarly, coexpression of IL-6 with IL-4 or IL-13 was less than 10%
(Fig. 5). In contrast, more than 70% of the IL-6-producing
CD4+ population coexpressed IL-2 (Figs. 4 and 5).
Analogous to CD4+ cells, few IL-6-producing
CD8+ cells coproduced IFN, IL-4, or IL-13. In
the CD8+ population, which exhibited a lower
frequency of IL-2-positive cells, coexpression of IL-6 and IL-2 was
lower than that in CD4+ cells (Fig. 5).
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Culture supernatants were analyzed for secreted IL-6 after
stimulation with phorbol 12-myristate 13-acetate and ionomycin for
4 h without the addition of monensin (n = 5). On day 7, IL-6
could be detected in control cultures (43.2 ± 24.2 pg/mL) as well
as in cultures with
1,25-(OH)2D3 (79.6
± 48.5 pg/mL). On day 14, virtually no production of IL-6 could be
found in controls (4.0 ± 4.7 pg/mL), whereas cultures treated
with 1,25-(OH)2D3
displayed significantly increased levels (113.8 ± 102.2
pg/mL) of secreted IL-6 (P < 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,25-(OH)2D3
recently has attained great interest as an immune modulator with
immunosuppressive activity. This has been attributed to the ability of
the steroid hormone to shift T cell responses from Th1 to Th2 (20).
Here we report for the first time how
1,25-(OH)2D3 affects the
frequency of cytokine-producing human peripheral blood T lymphocytes
and induces a specific pattern of cytokine production in
CD4+ and CD8+ T cells (for
relative effects, see Table 1).
Our results showing that
1,25-(OH)2D3 decreases
the percentages of IL-2-producing CD4+ and
CD8+ cells are in accordance with previous
studies applying ELISA or molecular biology techniques. A concomitant
decrease in absolute cell number was recognizable (data not shown). No
significant effect of
1,25-(OH)2D3 on the
percentage of IFN-producing CD4+ or
CD8+ cells could be detected in our experiments.
Although direct effects of
1,25-(OH)2D3 on IFN
production by T cells (28) have been proposed, Mueller et
al. demonstrated that IL-2, but not IFN, production is
inhibited by
1,25-(OH)2D3 when human
T cell lines are stimulated in the absence of any costimulating cells
(34). This suggests that suppression of IFN production is an
indirect effect of
1,25-(OH)2D3, probably
mediated by inhibition of IL-12 secretion from costimulatory cells,
such as monocytes, dendritic cells, or B cells (20, 35). Endogenous
production of IL-12 may not be an essential requirement for the
appearance of IFN-producing T cells in our differentiation model.
Therefore, no significant effect of
1,25-(OH)2D3 was
detectable. On the other hand, it has been suggested that inhibition of
IL-12 secretion and subsequent suppression of the Th1 response drive
the immune system toward Th2 function (36). It has been reported that
1,25-(OH)2D3 inhibits
the secretion of IL-4 from Th0 cells only at an almost 10-fold higher
concentration than necessary for inhibition of IFN and does not
inhibit IL-4 secretion by Th2 cells (20). Although an increase in the
frequency of IL-4-producing T cells was not significant in our
experiments, there was a highly significant induction of the Th2-type
cytokine IL-13, indicating that
1,25-(OH)2D3, may have
the potency to directly enhance the development of a Th2 response, and
not only via inhibition of Th1 reactions. IL-13 shares many biological
activities with IL-4, although it does not act on T cells, as human T
cells do not express the IL-13 receptor. IL-13 has been shown to
inhibit the production of IL-1, IL-6, and tumor necrosis factor- by
activated monocytes and to enhance the synthesis of IL-1 receptor
antagonist (37). Recently, stimulation of IL-6 production by IL-13 has
been demonstrated in human osteoblasts (38).
IL-6 originally has been assigned the Th2-type cytokine (3). IL-6 is a
pleiotropic cytokine involved in the regulation of immune responses,
the acute phase response, and hemopoiesis (39). Although initially
thought to be a proinflammatory cytokine, recent findings suggest that
IL-6 has many antiinflammatory and immunosuppressive effects (40).
There is substantial evidence that IL-6 and some of the IL-6 type
cytokines (IL-11, leukemia inhibitory factor, and oncostatin M)
have profound effects on bone metabolism by regulating osteoclast and
osteoblast development and function (41). IL-6 is produced by many
different cells after stimulation during infection, trauma, or
immunological challenge. In human peripheral blood, monocytes represent
the primary source of IL-6 (42), although IL-6 production by a fraction
of human peripheral blood T cell has been demonstrated (43, 44).
Inhibition of IL-6 production in human peripheral blood mononuclear
cells (PBMC) as well as in partially purified monocytes, by
1,25-(OH)2D3 has been
reported (29, 24). Others have described variable effects of
1,25-(OH)2D3 on IL-6
secretion by human PBMC depending on the stimulus used (45). However,
data obtained from ELISA and bioassay analysis of bulk culture
supernatants cannot be attributed to a specific cell type, and no
information has been available to date about the regulation of IL-6
production by
1,25-(OH)2D3 in T
lymphocytes. We show that the frequency of IL-6-producing T cells is
very effectively regulated by
1,25-(OH)2D3 (see
relative effects in Table 1).
Monocytes are present in our cultures and are probably responsible for
the detected levels of secreted IL-6 in supernatants from cultures with
or without 1,25-(OH)2D3
on day 7. Absolute and relative number of monocytes decline until day
14 (not shown); therefore, no IL-6 production was detectable in control
cultures on day 14. In our flow cytometric analysis, monocytic cells
were excluded by gating on CD4 and CD8, and T cell lineage of the gated
cells was confirmed by control staining for CD3 (data not shown).
The production of IL-6 by activated CD4+ and
CD8+ T cells in the presence of
1,25-(OH)2D3 is not
transient, and the frequency of positive cells remained stable for
several weeks. As overlap with IFN, IL-4, or IL-13 production is
negligible, IL-6-producing T cells may represent a specific subset
within the continuum of Th and Tc phenotypes, with an as yet unknown
role in the coupling of immune and endocrine system. Thus, the ability
of 1,25-(OH)2D3 to
induce IL-6 production in human T lymphocytes could aid its efficiency
as an anticancer agent, inasmuch as IL-6 released under the influence
of the steroid hormone could stimulate the proliferation of
tumor-infiltrating T lymphocytes (see, among others, Refs. 46, 47)
and thus enhance tumor-specific cytotoxic T cell responses. With
respect to bone remodeling, it should be noted that induction of
osteoclast-like cells by
1,25-(OH)2D3 in naive
mouse bone marrow cultures depends to a sizeable extent on
costimulatory signals via the IL-6/IL-6 receptor pathway (48, 49).
It is remarkable that
1,25-(OH)2D3 regulates
cytokine production in CD8+ and
CD4+ T cells in parallel. This is in line with
reports about the capacity of CD8+ cells to
produce Th1- and Th2-type cytokines and the possibility of triggering
their differentiation into Tc1 and Tc2 with the same agents as
CD4+ cells (33, 50, 51, 52).
Taken together, we show that
1,25-(OH)2D3 induces
differentiation of T cell populations with specific patterns of
cytokine production. Reduced ability of T cells to produce IL-2 and
increased percentages of cells capable of producing IL-13 or IL-6 are
in line with the concept that
1,25-(OH)2D3 acts as an
immunomodulatory agent. Importantly, the fact that
1,25-(OH)2D3 induces a
stable, IL-6-producing T cell population could have far-reaching
consequences for the involvement of both the steroid hormone and the
cytokine in nonimmune systems such as control of tumor cell growth and
bone remodeling.
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Footnotes |
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2 M.W. and R.T. contributed equally to this work.
Received February 22, 1999.
Revised June 9, 1999.
Accepted June 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-2, but not IL-4 and IL-5. J Immunol. 155:4604–4612.[Abstract]
,25-Dihydroxyvitamin D3 inhibits -interferon
synthesis by normal human peripheral blood lymphocytes. Proc Natl Acad
Sci USA. 84:3385–3389.,25-(OH)2D3. J Neuroimmunol. 61:151–160.[CrossRef][Medline]
B downregulation in
transcriptional repression of the p40 gene. J Clin Invest. 101:252–262.[Medline]
production studied at a single-cell
level. Eur J Immunol. 19:1157–1160.[Medline]
,25-dihydroxyvitamin
D3 on interleukin-6 production and osteoclast-like cell
formation in mouse bone marrow primary cultures. Endocrinology. 138:4567–4571.,25-dihydroxyvitamin
D3 on interleukin-6-dependent osteoclast-like cell
formation in mouse bone marrow cell cultures. Bone. 22:341–346.[Medline]
and
IL-4 regulate the growth and differentiation of CD8+ T
cells into subpopulations with distinct cytokine profiles. J
Immunol. 155:2928–2937.
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) or without (
)
10-8 mol/L 1
