• CD4 T cells;
  • Th2 cells;
  • Vitamin D;
  • Asthma;
  • Cytoskeleton rearrangement;
  • Migration


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The fat soluble vitamin D3 metabolite 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its nuclear receptor play an important role in regulating immune responses. While 1,25(OH)2D3 is known to inhibit transcription of cytokine genes that are required for Th1 differentiation or are products of differentiated Th1 cells, its role in regulating differentiation of Th2 cells is less clear. In this study, we show that 1,25(OH)2D3 has anti-inflammatory effects in an in vivo Th2-dependent asthma model. In addition, we demonstrate that 1,25(OH)2D3 down-regulates the cytoskeleton rearrangement required for promoting integrin-mediated adhesion of naive and effector CD4+ T cells. Finally, 1,25(OH)2D3 inhibits chemokine-induced migration of naive cells and their homing to the lymph nodes. Thus, in addition to its regulation of cytokine transcription, 1,25(OH)2D3 regulates migration of cells and thus controls the skewing of various Th subsets in the secondary lymphoid organs and inhibits Th function at sites of inflammation.


1,25-Dihydroxyvitamin D3


VitD3 receptor


Bronchoalveolar lavage

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

1,25-Dihydroxyvitamin D3 [1,25(OH)2D3)], the active metabolite of vitamin D3, is a lypophilic molecule that exerts its actions through a nuclear receptor, the VitD3 receptor (VDR) 1, 2. VDR is a member of the steroid nuclear receptor superfamily. In the presence of its ligand, VDR and its heterodimeric partner, the retinoid X receptor(RXR), act as a transcription factor by binding to VDR-responsive elements present in the promoter regions of target genes, activating or repressing expression of these genes 1, 3, 4.

Although 1,25(OH)2D3 has traditionally been associated with regulation of calcium homeostasis, the discovery of VDR expression in lymphocytes and monocytes 57 suggested an additional role for this hormone in the immune system. A number of recent studies have demonstrated the ability of this receptor-ligand pair to act as a strong immunosuppressor. Activated macrophages express the enzyme 1-αhydroxylase that allows for production of 1,25(OH)2D38, the active form of the vitamin D system, suggesting a role for this steroid hormone in regulation of the immune response. Furthermore, 1,25(OH)2D3 has been demonstrated to inhibit the differentiation and maturation of dendritic cells, modulate the activity of macrophages, reduce the expression of MHC class II and CD40, and inhibit the secretion of IL-11, IL-6, TNF-α and IL-12 913. In T lymphocytes, 1,25(OH)2D3 also diminishes proliferation 9, 14, 15 and regulates skewing towards particular CD4+ T cell subsets.

A key component of the immune defense against pathogens is mediated by CD4+ T lymphocytes, which can differentiate into functionally distinct subsets. Whereas T helper 1 (Th1) cells secrete the cytokines IFN-γ and TNF-β and provide protection against intracellular pathogens, the Th2 subset produces IL-4, IL-5, IL-9 and IL-13, is important in eradicating helminthes and other parasites, and is involved in allergic diseases and the pathogenesis of asthma 9, 14, 15.

Several studies have demonstrated that 1,25(OH)2D3 inhibits cytokines that are either required for Th1 differentiation, such as IL-12, or are products of differentiated Th1 cells (IL-2 and IFN-γ) and augment Th2 cell development 16, 17. The effect of 1,25(OH)2D3 has been studied in Th1 models and was shown to prevent Th1-mediated autoimmune disease in animal models for experimental allergic encephalomyelitis, systemic lupus erythematosus, rheumatoid arthritis and inflammatory bowel disease 1821. These studies indicate that one of the functions of 1,25(OH)2D3 in the immune system is to inhibit Th1 differentiation.

However, recent studies also suggest an inhibitory role of 1,25(OH)2D3 on the production of IL-4 during in vitro polarization of naive T cells, especially when given early during the initial stages of Th differentiation 22. These results point to an inhibitory role for 1,25(OH)2D3 in the proliferation and differentiation of Th2 cells. Furthermore, VDR-deficient mice exhibit enhanced IL-4 production by CD4+ T cells and enhanced proliferation in response to IL-4 23. However, no studies have been conducted to determine the effect of 1,25(OH)2D3 on Th2 disease models in vivo.

Asthma is a chronic inflammatory disease of the airways that is characterized by intermittent episodes of airway obstruction, airway inflammation and wheezing. Although asthma is multifactorial in origin, it has been suggested that T lymphocytes, and in particular CD4+ T cells producing a Th2 pattern of cytokines, have a prominent effect in the pathogenesis of this disease 2427. To determine whether 1,25(OH)2D3 negatively regulates differentiation into Th2 cells, we utilized the asthma ovalbumin model in mice. Our findings demonstrate that 1,25(OH)2D3 dramatically reduces the airway inflammatory response and IL-4 levels in bronchoalveolar lavage (BAL) fluid when given at the initiation of asthma induction or after Th2 cell differentiation has been completed. In addition, we found that 1,25(OH)2D3 inhibits transwell migration and actin polymerization by CD4+ T cells. Thus, we suggest that in addition to its role in cytokine transcription, 1,25(OH)2D3 regulates homing of cells to the lymph nodes, resulting in powerful inhibition of the inflammatory response.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 1,25(OH)2D3 inhibits early Th2 responses in vivo

To determine whether 1,25(OH)2D3 has an in vivo effect in a Th2 model, we analyzed its influence on OVA-sensitized asthmatic mice. The inflammatory response in the asthmatic lung is characterized by infiltration of the airway wall with lymphocytes and eosinophils, and recent advances suggest that T lymphocytes, and in particular CD4+ T cells producing the Th2 pattern of cytokines, have a major effect in the pathogenesis of this disease 2427. 1,25(OH)2D3 was injected i.p. from the 1st day of antigen stimulation, and its effect on the development of asthma was analyzed. As can be seen in Fig. 1A, OVA inhalation induced infiltration of eosinophils (64% OVA/OVA vs. 0.1% control; p=1.25x10–6) and lymphocytes (7.7% OVA/OVA vs. 1.1% control; p=0.005) into the BAL of sensitized mice. However, treatment with 1,25(OH)2D3 dramatically inhibited Ag-induced eosinophil recruitment into the BAL (64% OVA/OVA vs. 9.8% early VitD3; p=8.5x10–5) and reduced lymphocyte migration (7.7% OVA/OVA vs. 3.6% early VitD3; p=0.15). In addition, as shown in Fig. 1B, IL-4 levels in BAL derived from 1,25(OH)2D3-treated mice were reduced as compared to the OVA/OVA PBS-treated mice (33 pg/ml OVA/ OVA vs. 8 pg/ml early 1,25(OH)2D3; p=0.007). Histopathological examination of lung tissue from OVA mice revealed a pleomorphic peribronchial and perivascular infiltrate consisting of eosinophils, lymphocytes, macrophages and neutrophils (Fig. 1C). The peribronchial and perivascular inflammatory infiltrates were given an inflammatory score ranging from 1 to 4 by two different viewers. As can be seen in Fig. 1 C and D, while the inflammatory score in the OVA/OVA-treated mice was significantly increased as compared with the control animals (3.5 OVA/OVA vs. 0 control; p=5.5x10–6), the peribronchial inflammatory score was significantly reduced in the early 1,25(OH)2D3-treated mice (3.5 OVA/OVA vs. 0.33 early VitD3; p=6.3x10–5). These results show that treatment of mice with 1,25(OH)2D3 almost completely blocks the asthmatic response.

While 1,25(OH)2D3 is known to affect calcium homeostasis, the treatment regimen used did not cause hypercalcemia in the treated mice. Similar serum calcium levels were detected in treated and untreated mice (9.67±0.17 mg/100 ml control vs. 9.76±024 mg/100 ml1,25(OH)2D3-treated; p=0.51).

thumbnail image

Figure 1. 1,25(OH)2D3 inhibits the Th2 inflammatory response in an asthma model when administered from the 1st day of antigen stimulation. (A, B) Four untreated mice (control), seven OVA/OVA (asth), and seven OVA-primed mice treated with 1,25(OH)2D3 from the 1st day of antigen stimulation (VitD) were analyzed for (A) BAL cell recovery and (B) IL-4 levels in BAL fluid. (C) Histologic features of OVA-primed mice (OVA/OVA) and OVA-primed mice treated from the 1st day of antigen stimulation (early VitD) or from day 15 after antigen stimulation (late VitD) with 1,25(OH)2D3. (D) Histologic scores in untreated mice (control), OVA-primed mice (asth) and OVA-primed mice treated early with 1,25(OH)2D3 (VitD).

Download figure to PowerPoint

2.2 1,25(OH)2D3 inhibits the asthma-associated inflammatory response mediated by differentiated Th2 cells

1,25(OH)2D3 was previously shown to affect mainly cytokine production, an effect that is most dramatic when this compound is present from the onset of the differentiation process 22. We therefore expected that 1,25(OH)2D3 would show no inhibitory effect on the inflammatory response of mice whose CD4+ population was already skewed towards the Th2 subset. To test this, 1,25(OH)2D3 was injected 2 weeks after the first antigen stimulation, beginning at day 15, 5 min before the first OVA inhalation. Thus 1,25(OH)2D3 treatment began after the differentiation of CD4+ lymphocytes into effector T cells producing Th2 cytokines. This late i.p. injection of 1,25(OH)2D3 still had a significant inhibitory effect on the asthmatic inflammatory response (Fig. 2), inhibiting both Ag-induced eosinophil (OVA/OVA 65% vs. late VitD3 15.3%; p=2.5x10–5) and lymphocyte (OVA/OVA 7.8% vs. late VitD3 3.6%; p=0.02) recruitment into the BAL (Fig. 2A). Moreover, late i.p. injection of 1,25(OH)2D3 significantly reduced the peribronchial and perivascular inflammatory infiltrate in OVA/OVA mice (Fig. 1C). Lung histological changes were given an inflammatory score between 1 and 4 by two different viewers. As shown in Fig. 2B, the inflammatory score in the late 1,25(OH)2D3-treated mice was reduced as compared to untreated OVA/OVA mice (OVA/OVA 3.4 vs. late 1,25(OH)2D3 2; p=0.001). Thus, even with late treatment, where cells were already differentiated to Th2 cells, the response was reduced. Nevertheless, the anti-inflammatory effect of late 1,25(OH)2D3 treatment was reduced as compared to the effect of 1,25(OH)2D3 given before antigen stimulation (early VitD3 0.33 vs. late VitD3 2; p=0.001). Thus, 1,25(OH)2D3 inhibits the inflammatory response in mice whose CD4+ T cells are already differentiated to Th2, but to a lesser extent.

thumbnail image

Figure 2. 1,25(OH)2D3 given 15 days after antigen stimulation inhibits the Th2 inflammatory response in an asthma model. (A) Four untreated mice (control), seven OVA/OVA (asth), and 12 OVA-primed mice treated with 1,25(OH)2D3 from day 15 after antigen stimulation (late VitD) were analyzed for BAL cell recovery. (B) Histologic scores in untreated mice (control), OVA-primed mice (asth) and OVA-primed mice treated late with 1,25(OH)2D3 (VitD).

Download figure to PowerPoint

2.3 1,25(OH)2D3 inhibits T cell migration and actin polymerization

1,25(OH)2D3 showed a significant anti-inflammatory effect, which was most dramatic when given from the onset of the T cell differentiation process. However, 1,25(OH)2D3 had a powerful inhibitory effect even when given after the polarization of cells had ensued. These results could not be explained by the inhibitory function of 1,25(OH)2D3 on IL-4 production, since 1,25(OH)2D3-mediated inhibition is significantly diminished after polarization 22. We therefore proposed that 1,25(OH)2D3 has an additional role on the migratory properties of T cells, preventing them from reaching their site of function. Among the requirements for inducible chemokine-mediated adhesion and migration of cells are an increased rate of actin polymerization and extensive reorganization of the actin-based cytoskeleton. Chemokines such as SDF-1 promote a rapid burst of actin polymerization, which peaks at 30 s to 1 min and subsides to baseline levels within 5 to 10 min post-stimulation. Actin polymerization causes extensive cytoskeleton rearrangement, allowing adhesion of cells to the endothelium and diapedesis, the migration of cells through endothelium and into the lymph nodes or inflamed tissue 28. Because CD4+ T cells producing a Th2 pattern of cytokines have a prominent role in the pathogenesis of asthma, we analyzed the effect of 1,25(OH)2D3 on the ability of Th2 cells to polymerize actin. Total CD4+ T cells were isolated and stimulated with ConA and IL-4, a Th2-inducing cytokine. After 3 days, the cells were washed and analyzed for their response to SDF-1 following pretreatment in the presence or absence 1,25(OH)2D3. Calcitriol pretreatment was used at a dose previously demonstrated to be non-toxic and not inhibitory to T helper cell proliferation 10, 11, 13, 16, 17. While control cells (cells incubated with diluted ethanol) increased their cytoskeleton rearrangement following SDF-1 stimulation, cells treated with 1,25(OH)2D3 were unable to respond (Fig. 3A). To further determine whether 1,25(OH)2D3 inhibition of the migratory properties of cells is restricted to Th2 cells, we analyzed the effect of 1,25(OH)2D3 on naive CD4+ cells. CD4+ T cells were pretreated in the presence or absence of 1,25(OH)2D3 and then stimulated with SDF-1. The cells were immediately fixed and their degree of cytoskeleton rearrangement analyzed. SDF-1 stimulation induced rearrangement of actin in CD4+ T lymphocytes, an elevation that was abolished in cells treated with 1,25(OH)2D3 (Fig. 3B, C).

To determine whether 1,25(OH)2D3 regulates T cell migration, we followed its effect on chemokine-induced transwell migration of purified CD4+ T cells. The transwell migration of these T lymphocytes towards SDF-1 was inhibited by the LDV and RGD peptides, selective blockers of VLA-4 and VLA-5, while the control peptides DVL and RGE did not affect migration (Fig. 4A). Thus, the migration response observed was integrin-mediated. Pretreatment of cells with 1,25(OH)2D3 dramatically inhibited SDF-1-independent and -dependent migration of CD4+ T cells, while the migratory response was barely affected in the presence of reduced levels of 1,25(OH)2D3 (1:100 and 1:1,000 dilutions) (Fig. 4B). A similar inhibitory effect of 1,25(OH)2D3 on SLC-dependent integrin-mediated migration of CD4+ T cells was detected (Fig. 4C). Thus, in addition to its role in cytokine production, 1,25(OH)2D3 inhibits chemokine-induced cytoskeleton rearrangement and migration of naive T cells and Th2 cells. This inhibition might affect the homing of T cells to the lymph nodes, resulting in inhibition of the inflammatory response.To directly demonstrate that treatment with 1,25(OH)2D3 down-regulates in vivo homing of CD4+ T cells to the lymph nodes, purified CD4+ T cells were labeled, and an equal number of live cells were injected i.v. into 1,25(OH)2D3-treated or untreated control mice. The proportion of labeled cells recovered in the spleen and lymph nodes was determined 3.5 h after injection. The accumulation of labeled cells in the spleen was unaffected by the various pretreatments (Fig. 4E). In contrast, migration of cells to the LN of 1,25(OH)2D3-treated mice was significantly decreased. Nevertheless, no change in the microarchitecture of lung lymph nodes was observed in mice treated with 1,25(OH)2D3 (Fig. 4D). Thus, 1,25(OH)2D3, dramatically down-regulates in vivo homing of CD4+ T cells, a process that probably results in regulation of the immune response.

thumbnail image

Figure 3. 1,25(OH)2D3 inhibits cytoskeleton rearrangement in T cells. (A) Th2 cells pretreated with 1,25(OH)2D3 or solvent or (B) CD4+ T cells pretreated with 1,25(OH)2D3 for 30 min were then stimulated with SDF-1. Following fixation, permeabilization, and staining with FITC-phalloidin, the cells were analyzed by FACS. The results obtained for untreated cells were normalized to 100% polymerization of F-actin. The experiments depicted in (A) and (B) are representative of three and five, respectively, performed. (C) CD4+ T cells were incubated in the presence or absence of 1,25(OH)2D3 (VitD) and plated with or without SDF-1. Permeabilized cells were stained with TRITC-phalloidin and mounted on glass coverslips. The polymerized actin was visualized by fluorescence microscopy.

Download figure to PowerPoint

thumbnail image

Figure 4. 1,25(OH)2D3 inhibits chemokine-induced integrin-mediated transwell migration and homing of T cells. (A) CD4+ T cells were placed in the upper well of a 24-well transwell plate in the presence or absence the peptides RGD and LDV or DVL and RGE. The number of cells migrating toward SDF-1 in the lower chamber was evaluated after 3 h by FACS analysis. Percent migration was calculated as the fraction cells found in the lower chamber out of the input cells in the upper chamber. The results presented are representatives of three different experiments. (B, C) CD4+ T cells were pretreated for 15 min in 1 ml medium with or without 1,25(OH)2D3 (VitD) and placed in transwell plates in the presence or absence of SDF-1 (B) or SLC and the inhibitory peptides RGD and LDV (C). Migration after 3 h of incubation was analyzed by FACS. The results presented are representative of three different experiments for each chemokine. (D) Lung lymph node sections from OVA-primed mice (OVA/OVA) and OVA-primed mice treated with 1,25(OH)2D3 from the 1st day of antigen stimulation (VitD). (E) Homing of labeled cells to the LN. CD4+ T cells were labeled with 5 μM CFDA-SE and injected into control or 1,25(OH)2D3-treated mice. After 3.5 h, spleen and LN cells were collected, and the FITC-positive population was analyzed by FACS.

Download figure to PowerPoint

3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The surveillance of the body for foreign antigens is a critical function of the immune system. Lymphocytes migrate from the blood into tissues and secondary lymphoid organs and return to the blood via lymph vessels and the thoracic duct 29. The majority of lymphocytes are capable of tissue-selective trafficking (homing), recognizing organ-specific adhesion molecules on specialized endothelial cells 30. Leukocytes are specifically recruited by a combination of molecular events: blood-borne cells use primary adhesion molecules to tether to and roll on the lumenal wall of postcapillary venules, thereby encountering and rapidly transducing signals to up-regulate secondary adhesion molecules, which then mediate firm arrest 3133.

Naive T lymphocytes traffic through the T cell areas of secondary lymphoid organs in search of antigen presented by dendritic cells 34, 35. These naive cells express CD62 ligand (CD62L) and CC chemokine receptor 7 (CCR7), which are required for cell extravasation at the level of the high endothelial venules 33, 3638. Upon antigen recognition, specific T cells proliferate and, in the presence of polarizing cytokines, differentiate into Th1 or Th2 cells, which produce distinct patterns of cytokines and mediate various types of protective and pathological responses 39, 40. After T cell differentiation into Th1 or Th2 phenotypes, the LN homing receptors are down-regulated, while expression of tissue homing receptors is acquired 4144; Th1 and Th2 cells exhibit distinct migratory capacities in vivo45, 46.

The data presented in this study demonstrate that 1,25(OH)2D3, when given at the onset of antigen stimulation, down-regulates the inflammatory response in an asthma model andreduces IL-4 production in the BAL fluid. These results could be explained solely by the inhibition of IL-4 production by naive T cells. However, we further showed that 1,25(OH)2D3 has a significant anti-inflammatory effect even when given 2 weeks after T cell activation. This observation is consistent with an additional inhibitory role of 1,25(OH)2D3. We suggest that the profound down-regulation of the inflammatory response in the asthma model might result from the combined effect of 1,25(OH)2D3 on the homing of cells to the lymph nodes and on their cytokine production. Our studies show inhibition of the migratory properties of effector CD4+ T cells treated with 1,25(OH)2D3. This down-regulation in cytoskeleton rearrangement is not restricted to Th2 cells, since pretreatment with 1,25(OH)2D3 induced a similar effect in naive CD4+ T cells. Down-regulation of cytoskeleton rearrangement resulted in a dramatic reduction in the ability of the cells to migrate in a transwell assay and inhibited the homing of CD4+ T cells into lymph nodes. Thus, we suggest that in addition to the effect of 1,25(OH)2D3 on cytokine production, it controls homing of naive and effector cells to the LN and sites of inflammation. This inhibition results in reduced exposure of naive T cells to antigen, and as a consequence, their activation is prevented. In addition, 1,25(OH)2D3 inhibits the homing of effector T cells to sites of inflammation where they exert their function. 1,25(OH)2D3 inhibited the inflammatory response in mice whose CD4+ T cells were already skewed to Th2, but to a lesser extent thanwhen administered at the initiation of the asthma response when the cells were still naive. We suggest that since 1,25(OH)2D3 has a dual function, affecting both T cell homing and IL-4 production, the impact of treatment at the onset of the disease is more dramatic; after the cells are already skewed towards the Th2 phenotype, this compound can only regulate cell homing, limiting its effect.

The results presented in this study suggest a potential role for 1,25(OH)2D3 in asthma treatment. Asthma patients are commonly treated with systemic steroids. Most of these patients are also treated with 1,25(OH)2D3 as a preventive measure against steroid-induced osteoporosis, a practice that could have been questioned by previous reports suggesting that 1,25(OH)2D3 directly enhances Th2 differentiation from naive T cells. Our data argue against any aggravating role of 1,25(OH)2D3 on asthma, suggesting instead that the use of 1,25(OH)2D3 in asthma patients may even reduce their inflammatory responses.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Animals

BALB/c and C57BL/6 mice were used at 6–8 weeks of age. All animal procedures were approved by the Animal Research Committee at the Weizmann Institute (Rehovot, Israel).

4.2 Cells

Spleen cells were obtained from mice as previously described 47. CD4+ T cells were enriched using the MACS system (Miltenyi Biotech, Auburn, CA). Th2 cells were obtained as previously described 48. The dose of 1,25(OH)2D3 used in this study was within the range described in other studies; these doses did not induce cell death or inhibition of T helper proliferation 10, 11, 13, 16, 17. 1,25(OH)2D3 was dissolved in 95% ethanol as a stock solution of 10–4 M and diluted with RPMI containing 0.5% FCS to 3.6×10–8 M before use. The control group was incubated with RPMI containing diluted ethanol.

4.3 Transwell migration

Chemotaxis was assayed using transwell chambers (6.5 mm diameter, 5 μm pore size; Corning Inc., Corning, NY) as previously described 49. Migration in the presence or absence of 3.6×10–8 M 1,25(OH)2D3 (Calcitriol, Abbot Labs, Chicago, IL) or the peptides LDV and RGD or DVL and RGE (50 μg/ml) toward the chemokines SDF-1 (100 ng/ml)or SLC (400 ng/ml) placed in the lower part of the apparatus was analyzed after 3 h by FACS.

4.4 Cytoskeleton rearrangement

Cytoskeleton rearrangement in CD4+ T cells was monitored as described previously 49. Cover slides were coated overnight with fibronectin (100 μg/ml) at 4°C. CD4+ T cells (2×105) were incubated in the presence or absence of 1,25(OH)2D3 (Calcitriol, 3.6×10–8 M) and plated with or without SDF-1 (100 ng/ml). After 30 min, the cover slides were washed with PBS and fixed with 3% (V/V) formaldehyde for 30 min at 37°C. The cells were then washed and permeabilized in 1% (V/V) Triton X-100 for 5 min at room temperature. The permeabilized cells were stained with TRITC-phalloidin (10 μg/ml) for 30 min at room temperature, washed and mounted on glass coverslips with Mowiol 4–88 (CalBiochem). The polymerized actin was visualized under a fluorescence microscope.

4.5 OVA sensitization and challenge

BALB/c mice were immunized i.p. on days 0, 7, and 14 as previously described 50. The OVA/OVA group was injected i.p with 300 μl PBS containing 0.9% ethanol on days 0, 7,and 14–19, 5 min before each inhalation. The 1,25(OH)2D3 groups were injected i.p. with 100 ng 1,25(OH)2D3 dissolved in ethanol and further diluted with 300 μl PBS as previously described 21, 51 to a final concentration of 0.9%. The i.p injection was done on days 0, 7, and 14–19 for "early group" treatment, and on days 15–19 for the "late group" treatment.

4.6 Bronchoalveolar lavage

BAL was performed on day 19, 4 h after the last OVA challenge as described previously 50.

4.7 IL-4 ELISA analysis

BAL cells and supernatant were separated by centrifugation. BAL cells were resuspended in 0.5 ml PBS for cytospin slides. ELISA analysis was done on BAL supernatant using anti-IL-4 as the primary Ab and the corresponding biotinylated anti-IL-4 mAb (BD PharMingen, San Diego, CA) following the manufacturer's protocol.

4.8 Lung histology

Lung histology was performed as previously described 50. The tissues were examined blindly by a pathologist and an additional experienced viewer and given an inflammatory score between 1 and 4.

4.9 Tracking of cells in vivo

Mice were injected daily for 3 days with 100 ng 1,25(OH)2D3 or 0.1 ml PBS. On day 3, purified CD4+ cells derived from untreated mice were labeled with 5 μM CFDA-SE (Molecular Probes) for 15 min at room temperature. The cells were injected into treated and untreated mice and analyzed as described previously 52.


  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

This study was supported by The Israel Science Foundation founded by the Academy of Sciences and Humanities and the Minerva foundation. I.S. is the incumbent of the Alvinand Gertrude Levine Career Development Chair of Cancer Research. The authors wish to thank D. Shoseyov for help with the mouse model and the Shachar lab for helpful discussions and ideas and reviewof this manuscript.

  • 1


  • 2


  • 3


  • 4


  • 1
    Lemon, B. D., Fondell, J. D. and Freedman, L. P., Retinoid X receptor:vitamin D3 receptor heterodimers promote stable preinitiation complex formation and direct 1,25-dihydroxyvitamin D3-dependent cell-free transcription. Mol. Cell Biol. 1997. 17: 19231937.
  • 2
    Lowe, K. E., Maiyar, A. C. and Norman, A. W., Vitamin D-mediated gene expression. Crit. Rev. Eukaryot. Gene Expr. 1992. 2: 65109.
  • 3
    Kliewer, S. A., Umesono, K., Mangelsdorf, D. J. and Evans, R. M., Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signaling. Nature 1992. 355: 446449.
  • 4
    Lemon, B. D. and Freedman, L. P., Selective effects of ligands on vitamin D3 receptor- and retinoid X receptor-mediated geneactivation in vivo. Mol. Cell Biol. 1996. 16: 10061016.
  • 5
    Provvedini, D. M., Tsoukas, C. D., Deftos, L. J. and Manolagas, S. C., 1,25-dihydroxyvitamin D3 receptors in human leukocytes. Science 1983. 221: 11811183.
  • 6
    Yu, X. P., Mocharla, H., Hustmyer, F. G. and Manolagas, S. C., Vitamin D receptor expression in human lymphocytes. Signal requirements and characterization by western blots and DNA sequencing. J. Biol. Chem. 1991. 266: 75887895.
  • 7
    Veldman, C. M., Cantorna, M. T. and DeLuca, H. F., Expression of 1,25-dihydroxyvitamin D(3) receptor in the immune system. Arch. Biochem. Biophys. 2000. 374: 334338.
  • 8
    Reichel, H., Koeffler, H. P., Bishop, J. E. and Norman, A. W., 25-Hydroxyvitamin D3 metabolism by lipopolysaccharide-stimulated normal human macrophages. J. Clin. Endocrinol. Metab. 1987. 64: 19.
  • 9
    Tsoukas, C. D., Provvedini, D. M. and Manolagas, S. C., 1,25-dihydroxyvitamin D3: a novel immunoregulatory hormone. Science 1984. 224: 14381440.
  • 10
    Penna, G. and Adorini, L., 1 Alpha,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation, and survival of dendritic cells leading to impaired alloreactive T cell activation. J. Immunol. 2000. 164: 24052411.
  • 11
    Piemonti, L., Monti, P., Sironi, M., Fraticelli, P., Leone, B. E., Dal Cin, E., Allavena, P. and Di Carlo, V., Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells. J. Immunol. 2000. 164: 44434451.
  • 12
    Griffin, M. D., Lutz, W., Phan, V. A., Bachman, L. A., McKean, D. J. and Kumar, R., Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: a vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo. Proc. Natl. Acad. Sci. USA 2001. 98: 68006805.
  • 13
    D'Ambrosio, D., Cippitelli, M., Cocciolo, M. G., Mazzeo, D., Di Lucia, P., Lang, R., Sinigaglia, F. and Panina-Bordignon, P., Inhibition of IL-12 production by 1,25-dihydroxyvitamin D3. Involvement of NF-kappaB down-regulation in transcriptional repression of the p40 gene. J. Clin. Invest. 1998. 101: 252262.
  • 14
    Bhalla, A. K., Amento, E. P., Serog, B. and Glimcher, L. H., 1,25-Dihydroxyvitamin D3 inhibits antigen-induced T cell activation. J. Immunol. 1984. 133: 17481754.
  • 15
    Rigby, W. F., Stacy, T. and Fanger, M. W., Inhibition of T lymphocyte mitogenesis by 1,25-dihydroxyvitamin D3 (calcitriol). J. Clin. Invest. 1984. 74: 14511455.
  • 16
    Lemire, J. M., Archer, D. C., Beck, L. and Spiegelberg, H. L., Immunosuppressive actions of 1,25-dihydroxyvitamin D3: preferential inhibition of Th1 functions. J. Nutr. 1995. 125: 1704S-1708S.
  • 17
    Boonstra, A., Barrat, F. J., Crain, C., Heath, V. L., Savelkoul, H. F. and O'Garra, A., 1alpha,25-Dihydroxyvitamin d3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells. J. Immunol. 2001. 167: 49744980.
  • 18
    Lemire, J. M., Ince, A. and Takashima, M., 1,25-Dihydroxyvitamin D3 attenuates the expression of experimental murine lupus of MRL/l mice. Autoimmunity 1992. 12: 143148.
  • 19
    Lemire, J. M. and Archer, D. C., 1,25-dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J. Clin. Invest. 1991. 87: 11031107.
  • 20
    Cantorna, M. T., Woodward, W. D., Hayes, C. E. and DeLuca, H. F., 1,25-dihydroxyvitamin D3 is a positive regulator for the two anti-encephalitogenic cytokines TGF-beta 1 and IL-4. J. Immunol. 1998. 160: 53145319.
  • 21
    Cantorna, M. T., Hayes, C. E. and DeLuca, H. F., 1,25-Dihydroxycholecalciferol inhibits the progression of arthritis in murine models of human arthritis. J. Nutr. 1998. 128: 6872.
  • 22
    Staeva-Vieira, T. P. and Freedman, L. P., 1,25-dihydroxyvitamin D3 inhibits IFN-gamma and IL-4 levels during in vitro polarization of primary murine CD4+ T cells. J. Immunol. 2002. 168: 11811189.
  • 23
    O'Kelly, J., Hisatake, J., Hisatake, Y., Bishop, J., Norman, A. and Koeffler, H. P., Normal myelopoiesis but abnormal T lymphocyte responses in vitamin D receptor knockout mice. J. Clin. Invest. 2002. 109: 10911099.
  • 24
    Cohn, L., Homer, R. J., Niu, N. and Bottomly, K., T helper 1 cells and interferon gamma regulate allergic airway inflammation and mucus production. J. Exp. Med. 1999. 190: 13091318.
  • 25
    Robinson, D. S., Hamid, Q., Ying, S., Tsicopoulos, A., Barkans, J., Bentley, A. M., Corrigan, C., Durham, S. R. and Kay, A. B., Predominant TH2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 1992. 326: 298304.
  • 26
    Walker, C., Bode, E., Boer, L., Hansel, T. T., Blaser, K. and Virchow, J. C. J., Allergic and nonallergic asthmatics have distinct patterns of T cell activation and cytokine production in peripheral blood and bronchoalveolar lavage. Am. Rev. Respir. Dis. 1992. 146: 109115.
  • 27
    Huang, S. K., Xiao, H. Q., Kleine-Tebbe, J., Paciotti, G., Marsh, D. G., Lichtenstein, L. M. and Liu, M. C., IL-13 expression at the sites of allergen challenge in patients with asthma. J. Immunol. 1995. 155: 26882694.
  • 28
    Franitza, S., Kollet, O., Brill, A., Vaday, G. G., Petit, I., Lapidot, T., Alon, R. and Lider, O., TGF-beta1 enhances SDF-1alpha-induced chemotaxis and homing of naive T cells by up-regulating CXCR4 expression and downstream cytoskeletal effector molecules. Eur. J. Immunol. 2002. 32: 193202.
  • 29
    Gowans, J. L. and Knight, E. J., The route of re-circulation of lymphocytes in the rat. Proc. R. Lond. B. Biol. 1964. 159: 257282.
  • 30
    Butcher, E. C., Scollay, R. G. and Weissman, I. L., Organ specificity of lymphocyte migration: mediation by highly selective lymphocyte interaction with organ-specific determinants on high endothelial venules. Eur. J. Immunol. 1980. 10: 556561.
  • 31
    Butcher, E. C., Leukocyte-endothelial cell recognition: Three (or more) steps to specificity and diversity. Cell 1991. 67: 10331036.
  • 32
    Shimizu, Y., Newman, W., Tanaka, Y. and Shaw, S., Lymphocyte interactions with endothelial cells. Immunol. Today 1992. 13: 106112.
  • 33
    Springer, T. A., Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 1994. 76: 301314.
  • 34
    Butcher, E. C. and Picker, l. J., Lymphocyte homing and homeostasis. Science 1996. 272: 6066.
  • 35
    Banchereau, J. and Steinman, R. M., Dendritic cells and the control of immunity. Nature 1998. 392: 245252.
  • 36
    Cyster, J. G., Chemokines and cell migration in secondary lymphoid organs. Science 1999. 286: 20982102.
  • 37
    Stein, J. V., Rot, A., Luo, Y., Narasimhaswamy, M., Nakano, H., Gunn, M. D., Matsuzawa, A., Quackenbush, E. J., Dorf, M. E. and von Andrian, U. H., The CC chemokine thymus-derived chemotactic agent 4 (TCA-4, secondary lymphoid tissue chemokine, 6Ckine, exodus-2) triggers lymphocyte function-associated antigen 1-mediated arrest of rolling T lymphocytes in peripheral lymph node high endothelial venules. J. Exp. Med. 2000. 191: 6176.
  • 38
    Forster, R., Schubel, A., Breitfeld, D., Kremmer, E., Renner-Muller, I., Wolf, E. and Lipp, M., CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell 1999. 99: 2333.
  • 39
    Abbas, A. K., Murphy, K. M. and Sher, A., Functional diversity of helper T lymphocytes. Nature 1996. 383: 787793.
  • 40
    O'Garra, A., Cytokines induce the development of functionally heterogeneous T helper cell subsets. Immunity 1998. 8: 275283.
  • 41
    Xie, H., Lim, Y. C., Luscinskas, F. W. and Lichtman, A. H., Acquisition of selectin binding and peripheral homing properties by CD4(+) and CD8(+) T cells. J. Exp. Med. 1999. 189: 17651776.
  • 42
    Potsch, C., Vohringer, D. and Pircher, H., Distinct migration patterns of naive and effector CD8 T cells in the spleen: correlation with CCR7 receptor expression and chemokine reactivity. Eur. J. Immunol. 1999. 29: 35623570.
  • 43
    Campbell, J. J. and Butcher, E. C., Chemokines in tissue-specific and microenvironment-specific lymphocyte homing. Curr. Opin. Immunol. 2000. 12: 336341.
  • 44
    Sallusto, F., Mackay, C. R. and Lanzavecchia, A., The role of chemokine receptors in primary, effector, and memory immune responses. Annu. Rev. Immunol. 2000. 18: 593620.
  • 45
    Austrup, F., Vestweber, D., Borges, E., Lohning, M., Brauer,R., Herz, U., Renz, H., Hallmann, R., Scheffold, A., Radbruch, A. and Hamann, A., P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflamed tissues. Nature 1997. 385: 8183.
  • 46
    Randolph, D. A., Huang, G., Carruthers, C. J., Bromley, L. E. and Chaplin, D. D., The role of CCR7 in TH1 and TH2 cell localization and delivery of B cell help in vivo. Science 1999. 286: 21592162.
  • 47
    Shachar, I., Elliot, E. A., Chasnoff, B., Grewal, I. S. and Flavell, R. A., Reconstitution of invariant chain function in transgenic mice in vivo by individual p31 and p41 isoforms. Immunity 1995. 3: 373383.
  • 48
    Flaishon, L., Topilski, I., Shoseyov, D., Hershkoviz, R., Fireman, E., Levo, Y., Marmor, S. and Shachar, I., anti-inflammatory properties of low levels of IFN-γ. J. Immunol.: Cutting edge 2002. 168: 37073711.
  • 49
    Flaishon, L., Lantner, F., Hershkovitz, R., Levo, Y. and Shachar, I., Low levels of IFN-γ down-regulate the integrin-dependent adhesion of B cells by inhibition of cytoskeleton rearrangement. J. Biol. Chem. 2001. 276: 4670146706.
  • 50
    Topilski, I., Harmelin, A., Flavell, R. A., Levo, Y. and Shachar, I., Preferential Th1 immune response in invariant chain-deficient mice. J. Immunol. 2002. 168: 16101617.
  • 51
    Cantorna, M. T., Hayes, C. E. and DeLuca, H. F., 1,25-Dihydroxyvitamin D3 reversibly blocks theprogression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc. Natl. Acad. Sci. USA 1996. 93: 78617864.
  • 52
    Flaishon, L., Hershkovitz, R., Lantner, F., Lider, O., Alon,R., Levo, Y., Flavell, R. A. and Shachar, I., Autocrine secretion of interferon γ negatively regulates homing of immature B cells. J. Exp. Med. 2000. 192: 13811387.