1,25DIHYDROXYVITAMIN D, INHIBITS CYTOKINE PRODUCTION BY HUMAN BLOOD MONOCYTES AT THE POST-TRANSCRIPTIONAL LEVEL K. Miiller,l
P.M. Haahr,l
M. Diamant,l K. Bendtzenl
K. Rieneck,’
A. Kharazmi,2
1,25Dihydroxyvitamin D, [1,25-(OH),D,] inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin ((IL)-2 and interferon y. These lymphocyte functions are dependent upon cytokines, including IL-la, IL-lp, IL-6 and tumour necrosis factor cw(TNF-a), produced by the antigen presenting cells. In the present study we examined the effect of 1,25-(OH),D, on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes. The immediate precursor of 1,25(OH),D,, 25OH D,, and the synthetic analogue MC 903 (‘Calcipotriol’) were examined in parallel. 1,25-(OH),D, dose-dependently inhibited the production of IL-q IL-6 and TNF-cu by Escherichiu coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production. MC 903 had comparable effects while 25-OH D, was ineffective. The inhibition caused by 1,25-(OH),D, was not abolished by supraoptimal concentrations of LPS or indomethacin. 1,25-(OH), D, had similar effects on secreted and cell-associated IL-a. Nuclear run-off analysis indicated that inhibition of these cytokines was not caused by impaired production of mRNA. Taken together, the study demonstrates a vitamin D-induced inhibitory effect of LPS-driven monokine production, which is most likely a vitamin D-receptor mediated phenomenon exerted at a post-transcriptional, presecretory level. Impaired monokine production may be of importance in 1,25-(OH),D,-mediated inhibition of lymphocyte functions in vitro.
The steroid hormone 1,25-dihydroxyvitamin D, [1,25-(OH),D,] is best known for its biological role in calcium homoeostasis. Recently, it has become apparent that 1,25-(OH),D, regulates differentiation, growth and function of a broad range of cells. l This has resulted in the successful use of a vitamin D, analogue, MC903 (‘Calcipotriol’), in the treatment of psoriasis.2 Recent evidence indicates that 1,25-(OH),D, may play a role in the regulation of immune functions.3,4 Specific receptors for 1,25-(OH),D, (VDR) are
From the ILaboratory of Medical Immunology and 2Department of Clinical Microbiology, Rigshospitalet University Hospital, DK-2200 Copenhagen, Denmark. Correspondence to: Klaus Miiller, MD, Lab. Med. Immunol., Medical Department ‘M’A 7544, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N. Received 19 February 1992; accepted for publication 4 May 1992 0 1992 Academic Press Limited 1043-4666/92/060506+07 $08.00/O KEY WORDS: 1,2.5-dihydroxyvitamin monocytes/super oxide production 506
D-JMC
903/cytokines/
expressed by activated but not resting lymphocytes.5 This finding has led to studies of the functional significance of their presence. Several reports have shown that 1,25-(OH),D, is a potent inhibitor of lymphocyte proliferation,6,7 immunoglobulin productions10 and release of lymphokines such as interleukin (IL)-2 and interferon (IFN)-y.llJ2 Cells of the monocyte/macrophage lineage also express VDR. However, the effects of 1,25-(OH),D, on these cells are less well defined. It is well documented that 1,25-(OH),D, stimulates maturation of monocyte precursor cell lines such as M1,13J4 HL-6015 and U-937.16 Moreover, 1,25-(OH),D, stimulates monokine production by such cells both at the mRNA14J7-19 and at the protein levels.14J9 Studies of tissue macrophages and peripheral blood monocytes precultured for 3-7 days have also revealed a stimulatory effect of 1,25-(OH),D, leading to enhanced bacterial killing and increased interleukin (IL)-l-like activity. 13~0-22 However, increasing evidence from our and other laboratories suggests that the effect of 1,25-(OH),D, on freshly isolated monocytes is different from the effect on CYTOKINE,
Vol. 4, No. 6 (November),
1992: pp 506512
1,25-(OH),D,
monocyte precursors and mature macrophages. Thus, 1,25-( OH),D, inhibits phytohaemagglutinin-induced production of IL-la by freshly isolated monocytes,u and reduced monokine production may be of importance in 1,25-(OH),D,-mediated inhibition of B cell activation.ia Furthermore, the ability of monocytes to induce antigen-dependent T-cell proliferation is impaired when exposed to 1,25-(OH),D,.24 In the present study we examined the effects of 1,25-(OH),D, and analogues on the production of ILla, IL-Q, IL-6 and tumour necrosis factor CY(TNF-ok) by lipopoly saccharide (LPS)-stimulated freshly isolated human blood monocytes, at the transcriptional level and at the protein level. Another measure of monocyte activation, the superoxide burst response, was examined in parallel. FtESULTS The effect of 1,25-(OH),D, on monokine production by LPS-stimulated, purified monocytes was first examined. LPS, 10-s g/ml, induced production of IL-lo/p, TNF-o and IL-6. The levels of these monokines were below the detection limits in unstimulated cultures with or without 1,25-(OH),D,. 1,25-(OH),D, dose-dependently inhibited the release of IL-la, IL-6 and TNF-a, but not of IL-lp, in 24 h culture supernatants. Significant effects were obtained at 10-s M (Fig. 1). Parallel experiments using cells from different donors suggested that the observed variations in the response to vitamin D were primarily caused by donor differences (not shown). Addition of indomethacin did not abolish the inhibitory effect of 1,25-(OH),D, (not shown).
As shown in Fig. 2, the inhibitory effect of 1,25-(OH),D, in cultures of MNC was of the same magnitude as the effect seen in cultures of partially purified monocytes (see Fig. 1). 1,25-(OH),D, and the analogue MC903 had comparable effects on the release of IL-la and TNF-ok by LPS-stimulated MNC (Fig. 2). The immediate precursor of 1,25-(OH),D,, 25-OH D,, was without effect in both serum-containing and serum-free cultures (Table 1). Two hours of preculture with 1,25-(OH),D,, followed by extensive wash resulted in only moderate reduction of the inhibitory response to 1,25-(OH),D,, measured after 22 h of further incubation (Table 2). In-vitro transcriptional run off assays were performed to determine if the observed inhibition was exerted at the transcriptional level. As shown in Fig. 3, there was no clear change in the levels of the IL-~Ix@--, IL-6- and TNF-a transcripts, as well as in the p-actin transcript, in MNC treated with 10-s M 1,25-(OH),D,. Although IL-la and IL-l@ have similar lymphocyte-activating properties, IL-l@ is largely secreted from the cells, whereas IL-lo is primarily expressed in the plasma membrane.25 As shown in Fig. 4A, 1,25-(OH),D, reduced the contents of immunoreactive IL-lo in both cell lysates and in supernatants. This effect was not overcome by supraoptimal concentrations of LPS (1 l,,tg/ml). The contents of IL-l@ in both compartments were unaffected (Fig 4B). The superoxide burst in response to phorbol myristate acetate (PMA) was examined in similar cultures. 10-s M 1,25-(OH),D, had no effect on the superoxide production after 24 and 72 h of culture, whether or not LPS was present (Table 3). TNF-a
150 150-
IL-la
IL-ID
TNF-a
IL-6
Lt
IL-la
‘i
_
m T
‘c s b s
IOO-
0
I _
/o P I\
50III -12
I -10 -6
I,25 (OH),D,
Figure 1. and IL-6.
Effect
of 1,25-(OH),D,
(log M)
on release
of IL-la,
Purified monocytes were stimulated with LPS or absence of 1,25-(OH),D, at the indicated The contents of the cytokines were measured supernatants. Results are medians and quartiles experiments. *Significantly different from controls
II
T
0* 1
// 1
1.25~(OH),D,
i T PI,, o-~* 7-N Y I1
0*
Ill
I
-12 -10 -*
MC903
III
TNF-(U
in the presence concentrations. in 24 h culture in 6 different (P < 0.05).
Figure 2. and TNF-IX.
Effect
of 1,25-(OH),D,
/
-12 -10 -8
III -I2
I,25 IOH),D,
Concentration
IL-l&
I 507
and
I -J -10 -8
MC903
(log M)
MC903
on release
MNC were stimulated with LPS in the presence vitamin D, analogues at the indicated concentrations. of the cytokines were measured in 24 h culture Results are medians and quartiles in 6 different *Significantly different from controls (P < 0.05).
of IL-b
or absence of The contents supernatants. experiments.
CYTOKINE,
508 i Miiller
et al.
TABLE
Effect of 1,25-(OH),D,
1.
and 25(OH)D, Experiment
on IL-h
Vol. 4, No. 6 (November 1992: 506-512)
release.*
1
Experiment
(%) Serum
control
1,25-(OH),D,
25-(OH)D,
0 2 5 10
304 924 1036 910
144(47) 570(62) 640( 62) 520(57)
316(104) Ego{ 800(88)
control
2.56 1400 1420 1080
1,25-(OH),D,
112(44) ~~:$;~ 644(60)
2 25-(OH)D,
476(186) 1396(100) 1404(99) 1340(124)
*The results are shown as pg/ml IL-k (% of vitamin D-free controls); mean values of duplicate determinations. LPS-stimulated hfNC were incubated with or without vitamin D, analogues. The vitamins were used at a concentration of lo-sM, and the cells were cultured for 24 h before assay for supematant IL-la.
-
LPS
+
Log cont. I,25 (OHU~
Figure cytokine
3. Nuclear run-off gene transcription.
DISCUSSION
+ -8M
analysis.
Effect
of
1,25-(OH),D,
on
10s peripheral blood mononuclear cells at a concentration of 2 x 106iml were pre-incubated with or without l&s M 1,25-(OH),D, for 2 h and then stimulated with 1 pg/ml LPS. Nuclei were isolated 4 h after the initiation of the culture and in vitro elongation of transcription was carried out. Results are from one representative out of three different experiments.
TABLE 2. Effect of pulse exposure to 1,25-(OH),D, TNF-o( release. * Treatment
with 1,25-(OH),D,
Wash after 2 h Exp. 1
+ + +
+ -
1000 675(68) 960 590(61)
on
Exp. 2
1290 790(61) 1400 700(50)
*The results are shown as pgiml TNF-ol (% of vitamin D-free controls); mean values of duplicate determinations. LPS-stimulated MNC were incubated with or without 10-s M 1,25-(OH),D,. LPS was again added to the cells after wash. Similar results were obtained for IL-lol.
The present study shows that 1,25-(OH),D, significantly inhibited the production of IL-la, IL-6 and TNF-CY, but not that of IL-lp, by MNC and partially purified human blood monocytes, without affecting the superoxide burst response. There was no effect of the immediate precursor of 1,25-(OH),D,, 25-OH D,, which has about a lOOO-fold less avidity for VDR.26 Since this difference between 1,25-(OH),D, and 25-OH D, was also found in serum-free cultures, the lack of effect of 25-OH D, was apparently not caused by lower bioavailability of 25-OH D, resulting from a higher degree of protein binding. Therefore, the observed downregulation of cytokine production by 1,25-(OH),D, appears to be a VDR-mediated phenomenon. Although IL-l&p, IL-6 and TNF-a may be produced by both lymphocytes and monocytes, the latter cells are the main source of these cytokines in LPS-stimulated MNC cultures.27 We observed comparable inhibitory effects of 1,25-(OH),D, in MNC cultures and in cultures of partially purified monocytes. This suggests that the reduced monokine production is primarily due to a direct interaction of 1,25-(OH),D, with the monocytes and not an indirect effect, mediated via impaired function of T cells. This was further indicated by the fact that a substantial effect was induced during 2 h of preincubation with 1,25-(OH),D,, at which time T cells do not express significant amounts of VDR.3 The effect of 1,25-(OH),D, was not likely to be mediated via endogenous prostaglandin production, since incorporation of indomethacin in the culture was without effect. It was also not likely to be secondary to interference with secretory processes since cell-associated IL-la was reduced to approximately the same degree as secreted IL-la, whereas IL-ll3 in both compartments was unaffected. It has been reported that the lymphocytederived cytokines, granulocyte-macrophage colonystimulating factor28 and IL-229 are inhibited by 1,25-(OH),D, at a post-transcriptional level. Tran-
1,25-(OH),D,
scriptional run-off analysis in the present study revealed that the monokine-inhibitory activity of the vitamin was also exerted at a post-transcriptional level. This suggests that 1,25-(OH),D, operates by similar mechanisms in monocytes and in lymphocytes, i.e. at a post-transcriptional level of cytokine production. The failure of 1,25-(OH),D, to affect the
LPS (log g/ml)
-II
-10
-9
-8
LPS (log
Figure 4. Effect of l,S(OH),D, IL-lp (B).
-7
-6
g/ml)
on production of IL-la
(A) and
MNC were stimulated with LPS, at the indicated concentrations, in the presence or absence of 10-p M 1,25-(OH),D,. After 24 h, the supernatants were harvested and the cells were lysed by repeated freezing and thawing. The contents of IL-la and IL-lp were measured in supernatants and lysates. Results are from one representative out of 3 different experiments (mean of duplicates). A, Cytokine in cell lysates, 1,25-(OH),D,-free control; A, cytokine in cell lysates, 10-8 M 1,25-(OH),D,; 0, cytokine in supernatants, 1,25-(OH),D,-free control; 0, cytokine in supernatants, 10-8 M 1,25-(OH),D,.
TABLE 3. production. LPS
+ +
Effect of 1,25-(OH),D,
1,25-(OH),D,
+ +
on superoxide
24 h
72 h
6.3(3.5-8.9) 5.8(2.5-9.1) 3.2(2.7-5.4) 3.0(1.7-4.1)
4.3(2.2-5.5) 3.1(1.0-5.9) 3.1(1.9-4.9) 3.1(2.0-6.0)
PMA induced superoxide production was determined in MNC cultured for 24 h (N = 9) and 72 h (N = 6), respectively, with or without 10-s M 1,25-(OH),D3 and/or 10 rig/ml LPS. Superoxide activity is expressed as nanomoles of cytochrome c reduced per min (medians and quartiles) Similar results were obtained with lo-12-10-10 M 1,25-(OH),D, and 0.1 @ml-1 pg/ml LPS.
/ 509
release of immunoreactive IL-10 is in accordance with data reported by Tsoukas et al.23 Using phytohaemagglutinin-activated MNC, they found a pronounced 1,25-(OH),D,-induced reduction in the release of IL-la, but only a slight reduction in the production of IL-lp. These findings suggest differences in the regulation of IL-lp production on the one hand and the production of IL-la, TNF-ok and IL-6 on the other hand. This may also explain the failure to detect a reduction in IL-l-like activity using a thymocyte costimulatory assay.l Previous work has shown that 1,25-(OH),D, induces maturation, cytokine production and cytotoxicity in monocyte precursors and in fully differentiated macrophages. The present study demonstrates that the effect of 1,25-(OH),D, on freshly isolated blood monocytes may be distinct from these effects, showing downregulation of monokine production without reduction of superoxide production. Previously, we have demonstrated that 1,25-(OH),D,-mediated inhibition of poke-weed mitogen-driven immunoglobulin production depends upon the presence of monocytes and is totally reversible by addition of recombinant monokines.10 Taken together, these studies suggest that vitamin D-mediated inhibition of monokine production may be of importance in the control of lymphocyte activation. Results from other laboratories have shown that mononuclear cells isolated from inflammatory sites of patients with sarcoidosis and rheumatoid arthritis are efficient producers of 1,25-(OH),D, in the presence of added 25-OH2D3.30J1 Therefore, 1,25-(OH),D, and its analogues may not only represent potential immunosuppressive drugs, but 1,25-(OH),D, may also play a role as a hormone involved in the control of lymphocyte activation in immunoinflammatory diseases.
MATERIALS
AND METHODS
Vitamin D3 Analogues The vitamin D, analogues 1,25-(OH),D,, 2.5OH D, and MC 903 were kindly donated by Leo Pharmaceutical Products (Ballerup, Denmark). They were dissolved in 98% ethanol and stored at -20°C. Fresh dilutions were made in sterile saline before each experiment. The vitamin D, analogues were tested at final concentrations of lo-12 to 10-s M, and the final concentration of ethanol did not exceed 0.01 % (v/v). This concentration did not affect the monocyte functions.
Cytokine Production Human peripheral blood mononuclear cells (MNC) were obtained by density gradient centrifugation of heparinized blood on Ficoll-Hypaque (Lymphoprep,
510 I Miiller et al.
Nycomed, Oslo, Norway). In some experiments monocytes were partially purified by incubation of 4 x 106 MNC/ml for 90 mitt at 37°C (5% CO,) on plastic Petri dishes in endotoxinfree (Limulus assay) RPM1 with 15% heat-activated normal human serum (NHS). The non-adherent cells were removed by gently washing the plates five times in RPM1 at 37°C with 5% NHS. The adherent cells were loosened by incubation for 20 min with 10-Z M EDTA at 4°C followed by gentle scraping. These cells were > 73% (usually > 90%) leuM3 positive by flow cytometry analysis using a FACStar (Becton Dickinson, Mountain View, CA, USA). The cells were washed and resuspended in RPM1 1640, containing 2 X 10-Z M HEPES (Seromed, Biochrom KG, Berlin, Germany), 8 x 10-4 M glutamine, penicillin, streptomycin and 2% NHS. Escherichiu coli (055:B5) endotoxin (LPS) (Difco, Detroit, MI, USA) and vitamin D, analogues were added at the indicated concentrations at the initiation of the cultures. Some cultures were substituted with 10 &/ml indomethacin (Dumex, Copenhagen, Denmark). The cells were cultured at a concentration of 2 X 106 cells/ml for 24-72 hours. The supernatants were collected and stored at -20°C. 1,25-(OH),D,, in the concentrations used, did not affect cell viability measured by trypan blue exclusion (regularly exceeding 80% after 72 h.)
Assaysfor IL-1 cxand Tumour NecrosisFactor (x (TNF-ol) The contents of IL-la and TNF-a were determined by a previously described ELISA.32 Briefly, specific rabbit antibodies were used for catching and biotinylated rabbit antibodies for detection, followed by incubation with streptavidin-peroxidase. Enzyme activities were quantitated by addition of 1,25-phenylenediamine dihydrochloride and measurements of optical densities at 490 nm. All assayswere performed in duplicate. These ELISAs did not detect IL-l& IL-2, IL-4 or IIN-?/. There was no cross-reaction between IL-la and TNF-o. The detection limits were l&50 pgiml. The intra- and interassay variations were
P.M. Haahr,l
M. Diamant,l K. Bendtzenl
K. Rieneck,’
A. Kharazmi,2
1,25Dihydroxyvitamin D, [1,25-(OH),D,] inhibits lymphocyte proliferation and production of antibodies and lymphokines such as interleukin ((IL)-2 and interferon y. These lymphocyte functions are dependent upon cytokines, including IL-la, IL-lp, IL-6 and tumour necrosis factor cw(TNF-a), produced by the antigen presenting cells. In the present study we examined the effect of 1,25-(OH),D, on the production of these cytokines, as well as superoxide generation by freshly isolated mononuclear cells and partially purified monocytes. The immediate precursor of 1,25(OH),D,, 25OH D,, and the synthetic analogue MC 903 (‘Calcipotriol’) were examined in parallel. 1,25-(OH),D, dose-dependently inhibited the production of IL-q IL-6 and TNF-cu by Escherichiu coli lipopolysaccharide (LPS)-stimulated monocytes, without affecting superoxide production. MC 903 had comparable effects while 25-OH D, was ineffective. The inhibition caused by 1,25-(OH),D, was not abolished by supraoptimal concentrations of LPS or indomethacin. 1,25-(OH), D, had similar effects on secreted and cell-associated IL-a. Nuclear run-off analysis indicated that inhibition of these cytokines was not caused by impaired production of mRNA. Taken together, the study demonstrates a vitamin D-induced inhibitory effect of LPS-driven monokine production, which is most likely a vitamin D-receptor mediated phenomenon exerted at a post-transcriptional, presecretory level. Impaired monokine production may be of importance in 1,25-(OH),D,-mediated inhibition of lymphocyte functions in vitro.
The steroid hormone 1,25-dihydroxyvitamin D, [1,25-(OH),D,] is best known for its biological role in calcium homoeostasis. Recently, it has become apparent that 1,25-(OH),D, regulates differentiation, growth and function of a broad range of cells. l This has resulted in the successful use of a vitamin D, analogue, MC903 (‘Calcipotriol’), in the treatment of psoriasis.2 Recent evidence indicates that 1,25-(OH),D, may play a role in the regulation of immune functions.3,4 Specific receptors for 1,25-(OH),D, (VDR) are
From the ILaboratory of Medical Immunology and 2Department of Clinical Microbiology, Rigshospitalet University Hospital, DK-2200 Copenhagen, Denmark. Correspondence to: Klaus Miiller, MD, Lab. Med. Immunol., Medical Department ‘M’A 7544, Rigshospitalet, Tagensvej 20, DK-2200 Copenhagen N. Received 19 February 1992; accepted for publication 4 May 1992 0 1992 Academic Press Limited 1043-4666/92/060506+07 $08.00/O KEY WORDS: 1,2.5-dihydroxyvitamin monocytes/super oxide production 506
D-JMC
903/cytokines/
expressed by activated but not resting lymphocytes.5 This finding has led to studies of the functional significance of their presence. Several reports have shown that 1,25-(OH),D, is a potent inhibitor of lymphocyte proliferation,6,7 immunoglobulin productions10 and release of lymphokines such as interleukin (IL)-2 and interferon (IFN)-y.llJ2 Cells of the monocyte/macrophage lineage also express VDR. However, the effects of 1,25-(OH),D, on these cells are less well defined. It is well documented that 1,25-(OH),D, stimulates maturation of monocyte precursor cell lines such as M1,13J4 HL-6015 and U-937.16 Moreover, 1,25-(OH),D, stimulates monokine production by such cells both at the mRNA14J7-19 and at the protein levels.14J9 Studies of tissue macrophages and peripheral blood monocytes precultured for 3-7 days have also revealed a stimulatory effect of 1,25-(OH),D, leading to enhanced bacterial killing and increased interleukin (IL)-l-like activity. 13~0-22 However, increasing evidence from our and other laboratories suggests that the effect of 1,25-(OH),D, on freshly isolated monocytes is different from the effect on CYTOKINE,
Vol. 4, No. 6 (November),
1992: pp 506512
1,25-(OH),D,
monocyte precursors and mature macrophages. Thus, 1,25-( OH),D, inhibits phytohaemagglutinin-induced production of IL-la by freshly isolated monocytes,u and reduced monokine production may be of importance in 1,25-(OH),D,-mediated inhibition of B cell activation.ia Furthermore, the ability of monocytes to induce antigen-dependent T-cell proliferation is impaired when exposed to 1,25-(OH),D,.24 In the present study we examined the effects of 1,25-(OH),D, and analogues on the production of ILla, IL-Q, IL-6 and tumour necrosis factor CY(TNF-ok) by lipopoly saccharide (LPS)-stimulated freshly isolated human blood monocytes, at the transcriptional level and at the protein level. Another measure of monocyte activation, the superoxide burst response, was examined in parallel. FtESULTS The effect of 1,25-(OH),D, on monokine production by LPS-stimulated, purified monocytes was first examined. LPS, 10-s g/ml, induced production of IL-lo/p, TNF-o and IL-6. The levels of these monokines were below the detection limits in unstimulated cultures with or without 1,25-(OH),D,. 1,25-(OH),D, dose-dependently inhibited the release of IL-la, IL-6 and TNF-a, but not of IL-lp, in 24 h culture supernatants. Significant effects were obtained at 10-s M (Fig. 1). Parallel experiments using cells from different donors suggested that the observed variations in the response to vitamin D were primarily caused by donor differences (not shown). Addition of indomethacin did not abolish the inhibitory effect of 1,25-(OH),D, (not shown).
As shown in Fig. 2, the inhibitory effect of 1,25-(OH),D, in cultures of MNC was of the same magnitude as the effect seen in cultures of partially purified monocytes (see Fig. 1). 1,25-(OH),D, and the analogue MC903 had comparable effects on the release of IL-la and TNF-ok by LPS-stimulated MNC (Fig. 2). The immediate precursor of 1,25-(OH),D,, 25-OH D,, was without effect in both serum-containing and serum-free cultures (Table 1). Two hours of preculture with 1,25-(OH),D,, followed by extensive wash resulted in only moderate reduction of the inhibitory response to 1,25-(OH),D,, measured after 22 h of further incubation (Table 2). In-vitro transcriptional run off assays were performed to determine if the observed inhibition was exerted at the transcriptional level. As shown in Fig. 3, there was no clear change in the levels of the IL-~Ix@--, IL-6- and TNF-a transcripts, as well as in the p-actin transcript, in MNC treated with 10-s M 1,25-(OH),D,. Although IL-la and IL-l@ have similar lymphocyte-activating properties, IL-l@ is largely secreted from the cells, whereas IL-lo is primarily expressed in the plasma membrane.25 As shown in Fig. 4A, 1,25-(OH),D, reduced the contents of immunoreactive IL-lo in both cell lysates and in supernatants. This effect was not overcome by supraoptimal concentrations of LPS (1 l,,tg/ml). The contents of IL-l@ in both compartments were unaffected (Fig 4B). The superoxide burst in response to phorbol myristate acetate (PMA) was examined in similar cultures. 10-s M 1,25-(OH),D, had no effect on the superoxide production after 24 and 72 h of culture, whether or not LPS was present (Table 3). TNF-a
150 150-
IL-la
IL-ID
TNF-a
IL-6
Lt
IL-la
‘i
_
m T
‘c s b s
IOO-
0
I _
/o P I\
50III -12
I -10 -6
I,25 (OH),D,
Figure 1. and IL-6.
Effect
of 1,25-(OH),D,
(log M)
on release
of IL-la,
Purified monocytes were stimulated with LPS or absence of 1,25-(OH),D, at the indicated The contents of the cytokines were measured supernatants. Results are medians and quartiles experiments. *Significantly different from controls
II
T
0* 1
// 1
1.25~(OH),D,
i T PI,, o-~* 7-N Y I1
0*
Ill
I
-12 -10 -*
MC903
III
TNF-(U
in the presence concentrations. in 24 h culture in 6 different (P < 0.05).
Figure 2. and TNF-IX.
Effect
of 1,25-(OH),D,
/
-12 -10 -8
III -I2
I,25 IOH),D,
Concentration
IL-l&
I 507
and
I -J -10 -8
MC903
(log M)
MC903
on release
MNC were stimulated with LPS in the presence vitamin D, analogues at the indicated concentrations. of the cytokines were measured in 24 h culture Results are medians and quartiles in 6 different *Significantly different from controls (P < 0.05).
of IL-b
or absence of The contents supernatants. experiments.
CYTOKINE,
508 i Miiller
et al.
TABLE
Effect of 1,25-(OH),D,
1.
and 25(OH)D, Experiment
on IL-h
Vol. 4, No. 6 (November 1992: 506-512)
release.*
1
Experiment
(%) Serum
control
1,25-(OH),D,
25-(OH)D,
0 2 5 10
304 924 1036 910
144(47) 570(62) 640( 62) 520(57)
316(104) Ego{ 800(88)
control
2.56 1400 1420 1080
1,25-(OH),D,
112(44) ~~:$;~ 644(60)
2 25-(OH)D,
476(186) 1396(100) 1404(99) 1340(124)
*The results are shown as pg/ml IL-k (% of vitamin D-free controls); mean values of duplicate determinations. LPS-stimulated hfNC were incubated with or without vitamin D, analogues. The vitamins were used at a concentration of lo-sM, and the cells were cultured for 24 h before assay for supematant IL-la.
-
LPS
+
Log cont. I,25 (OHU~
Figure cytokine
3. Nuclear run-off gene transcription.
DISCUSSION
+ -8M
analysis.
Effect
of
1,25-(OH),D,
on
10s peripheral blood mononuclear cells at a concentration of 2 x 106iml were pre-incubated with or without l&s M 1,25-(OH),D, for 2 h and then stimulated with 1 pg/ml LPS. Nuclei were isolated 4 h after the initiation of the culture and in vitro elongation of transcription was carried out. Results are from one representative out of three different experiments.
TABLE 2. Effect of pulse exposure to 1,25-(OH),D, TNF-o( release. * Treatment
with 1,25-(OH),D,
Wash after 2 h Exp. 1
+ + +
+ -
1000 675(68) 960 590(61)
on
Exp. 2
1290 790(61) 1400 700(50)
*The results are shown as pgiml TNF-ol (% of vitamin D-free controls); mean values of duplicate determinations. LPS-stimulated MNC were incubated with or without 10-s M 1,25-(OH),D,. LPS was again added to the cells after wash. Similar results were obtained for IL-lol.
The present study shows that 1,25-(OH),D, significantly inhibited the production of IL-la, IL-6 and TNF-CY, but not that of IL-lp, by MNC and partially purified human blood monocytes, without affecting the superoxide burst response. There was no effect of the immediate precursor of 1,25-(OH),D,, 25-OH D,, which has about a lOOO-fold less avidity for VDR.26 Since this difference between 1,25-(OH),D, and 25-OH D, was also found in serum-free cultures, the lack of effect of 25-OH D, was apparently not caused by lower bioavailability of 25-OH D, resulting from a higher degree of protein binding. Therefore, the observed downregulation of cytokine production by 1,25-(OH),D, appears to be a VDR-mediated phenomenon. Although IL-l&p, IL-6 and TNF-a may be produced by both lymphocytes and monocytes, the latter cells are the main source of these cytokines in LPS-stimulated MNC cultures.27 We observed comparable inhibitory effects of 1,25-(OH),D, in MNC cultures and in cultures of partially purified monocytes. This suggests that the reduced monokine production is primarily due to a direct interaction of 1,25-(OH),D, with the monocytes and not an indirect effect, mediated via impaired function of T cells. This was further indicated by the fact that a substantial effect was induced during 2 h of preincubation with 1,25-(OH),D,, at which time T cells do not express significant amounts of VDR.3 The effect of 1,25-(OH),D, was not likely to be mediated via endogenous prostaglandin production, since incorporation of indomethacin in the culture was without effect. It was also not likely to be secondary to interference with secretory processes since cell-associated IL-la was reduced to approximately the same degree as secreted IL-la, whereas IL-ll3 in both compartments was unaffected. It has been reported that the lymphocytederived cytokines, granulocyte-macrophage colonystimulating factor28 and IL-229 are inhibited by 1,25-(OH),D, at a post-transcriptional level. Tran-
1,25-(OH),D,
scriptional run-off analysis in the present study revealed that the monokine-inhibitory activity of the vitamin was also exerted at a post-transcriptional level. This suggests that 1,25-(OH),D, operates by similar mechanisms in monocytes and in lymphocytes, i.e. at a post-transcriptional level of cytokine production. The failure of 1,25-(OH),D, to affect the
LPS (log g/ml)
-II
-10
-9
-8
LPS (log
Figure 4. Effect of l,S(OH),D, IL-lp (B).
-7
-6
g/ml)
on production of IL-la
(A) and
MNC were stimulated with LPS, at the indicated concentrations, in the presence or absence of 10-p M 1,25-(OH),D,. After 24 h, the supernatants were harvested and the cells were lysed by repeated freezing and thawing. The contents of IL-la and IL-lp were measured in supernatants and lysates. Results are from one representative out of 3 different experiments (mean of duplicates). A, Cytokine in cell lysates, 1,25-(OH),D,-free control; A, cytokine in cell lysates, 10-8 M 1,25-(OH),D,; 0, cytokine in supernatants, 1,25-(OH),D,-free control; 0, cytokine in supernatants, 10-8 M 1,25-(OH),D,.
TABLE 3. production. LPS
+ +
Effect of 1,25-(OH),D,
1,25-(OH),D,
+ +
on superoxide
24 h
72 h
6.3(3.5-8.9) 5.8(2.5-9.1) 3.2(2.7-5.4) 3.0(1.7-4.1)
4.3(2.2-5.5) 3.1(1.0-5.9) 3.1(1.9-4.9) 3.1(2.0-6.0)
PMA induced superoxide production was determined in MNC cultured for 24 h (N = 9) and 72 h (N = 6), respectively, with or without 10-s M 1,25-(OH),D3 and/or 10 rig/ml LPS. Superoxide activity is expressed as nanomoles of cytochrome c reduced per min (medians and quartiles) Similar results were obtained with lo-12-10-10 M 1,25-(OH),D, and 0.1 @ml-1 pg/ml LPS.
/ 509
release of immunoreactive IL-10 is in accordance with data reported by Tsoukas et al.23 Using phytohaemagglutinin-activated MNC, they found a pronounced 1,25-(OH),D,-induced reduction in the release of IL-la, but only a slight reduction in the production of IL-lp. These findings suggest differences in the regulation of IL-lp production on the one hand and the production of IL-la, TNF-ok and IL-6 on the other hand. This may also explain the failure to detect a reduction in IL-l-like activity using a thymocyte costimulatory assay.l Previous work has shown that 1,25-(OH),D, induces maturation, cytokine production and cytotoxicity in monocyte precursors and in fully differentiated macrophages. The present study demonstrates that the effect of 1,25-(OH),D, on freshly isolated blood monocytes may be distinct from these effects, showing downregulation of monokine production without reduction of superoxide production. Previously, we have demonstrated that 1,25-(OH),D,-mediated inhibition of poke-weed mitogen-driven immunoglobulin production depends upon the presence of monocytes and is totally reversible by addition of recombinant monokines.10 Taken together, these studies suggest that vitamin D-mediated inhibition of monokine production may be of importance in the control of lymphocyte activation. Results from other laboratories have shown that mononuclear cells isolated from inflammatory sites of patients with sarcoidosis and rheumatoid arthritis are efficient producers of 1,25-(OH),D, in the presence of added 25-OH2D3.30J1 Therefore, 1,25-(OH),D, and its analogues may not only represent potential immunosuppressive drugs, but 1,25-(OH),D, may also play a role as a hormone involved in the control of lymphocyte activation in immunoinflammatory diseases.
MATERIALS
AND METHODS
Vitamin D3 Analogues The vitamin D, analogues 1,25-(OH),D,, 2.5OH D, and MC 903 were kindly donated by Leo Pharmaceutical Products (Ballerup, Denmark). They were dissolved in 98% ethanol and stored at -20°C. Fresh dilutions were made in sterile saline before each experiment. The vitamin D, analogues were tested at final concentrations of lo-12 to 10-s M, and the final concentration of ethanol did not exceed 0.01 % (v/v). This concentration did not affect the monocyte functions.
Cytokine Production Human peripheral blood mononuclear cells (MNC) were obtained by density gradient centrifugation of heparinized blood on Ficoll-Hypaque (Lymphoprep,
510 I Miiller et al.
Nycomed, Oslo, Norway). In some experiments monocytes were partially purified by incubation of 4 x 106 MNC/ml for 90 mitt at 37°C (5% CO,) on plastic Petri dishes in endotoxinfree (Limulus assay) RPM1 with 15% heat-activated normal human serum (NHS). The non-adherent cells were removed by gently washing the plates five times in RPM1 at 37°C with 5% NHS. The adherent cells were loosened by incubation for 20 min with 10-Z M EDTA at 4°C followed by gentle scraping. These cells were > 73% (usually > 90%) leuM3 positive by flow cytometry analysis using a FACStar (Becton Dickinson, Mountain View, CA, USA). The cells were washed and resuspended in RPM1 1640, containing 2 X 10-Z M HEPES (Seromed, Biochrom KG, Berlin, Germany), 8 x 10-4 M glutamine, penicillin, streptomycin and 2% NHS. Escherichiu coli (055:B5) endotoxin (LPS) (Difco, Detroit, MI, USA) and vitamin D, analogues were added at the indicated concentrations at the initiation of the cultures. Some cultures were substituted with 10 &/ml indomethacin (Dumex, Copenhagen, Denmark). The cells were cultured at a concentration of 2 X 106 cells/ml for 24-72 hours. The supernatants were collected and stored at -20°C. 1,25-(OH),D,, in the concentrations used, did not affect cell viability measured by trypan blue exclusion (regularly exceeding 80% after 72 h.)
Assaysfor IL-1 cxand Tumour NecrosisFactor (x (TNF-ol) The contents of IL-la and TNF-a were determined by a previously described ELISA.32 Briefly, specific rabbit antibodies were used for catching and biotinylated rabbit antibodies for detection, followed by incubation with streptavidin-peroxidase. Enzyme activities were quantitated by addition of 1,25-phenylenediamine dihydrochloride and measurements of optical densities at 490 nm. All assayswere performed in duplicate. These ELISAs did not detect IL-l& IL-2, IL-4 or IIN-?/. There was no cross-reaction between IL-la and TNF-o. The detection limits were l&50 pgiml. The intra- and interassay variations were
