Physiol. Genomics Track the topics, authors and articles important to you
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Physiol. Genomics 18: 141-151, 2004. First published May 11, 2004; doi:10.1152/physiolgenomics.00003.2004
1094-8341/04 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
18/2/141    most recent
00003.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spach, K. M.
Right arrow Articles by Hayes, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spach, K. M.
Right arrow Articles by Hayes, C. E.
Received 8 January 2004; accepted in final form 29 April 2004.
Physiological Genomics 18:141-151 (2004)
1094-8341/04 $5.00 © 2004 American Physiological Society

Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis

Karen M. Spach 1, Laura B. Pedersen 2, Faye E. Nashold 2, Tsuyoshi Kayo 4, Brian S. Yandell 3, Tomas A. Prolla 4 and Colleen E. Hayes 2

1 Department of Nutritional Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706
2 Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin 53706
3 Department of Horticulture and Statistics, University of Wisconsin-Madison, Madison, Wisconsin 53706
4 Department of Genetics and Medical Genetics, University of Wisconsin-Madison, Madison, Wisconsin 53706


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
Multiple sclerosis (MS) is a debilitating autoimmune disease of the central nervous system (CNS) that develops in genetically susceptible individuals who are exposed to undefined environmental risk factors. Epidemiological, genetic, and biological evidence suggests that insufficient vitamin D may be an MS risk factor. However, little is known about how vitamin D might be protective in MS. We hypothesized that 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] might regulate gene expression patterns in a manner that would resolve inflammation. To test this hypothesis, experimental autoimmune encephalomyelitis (EAE) was induced in mice, 1,25-(OH)2D3 or a placebo was administered, and 6 h later, DNA microarray hybridization was performed with spinal cord RNA to analyze the gene expression patterns. At this time, clinical, histopathological, and biological studies showed that the two groups did not differ in EAE disease, but changes in several 1,25-(OH)2D3-responsive genes indicated that the 1,25-(OH)2D3 had reached the CNS. Compared with normal mice, placebo-treated mice with EAE showed increased expression of many immune system genes, confirming the acute inflammation. When 1,25-(OH)2D3 was administered, several genes like glial fibrillary acidic protein and eukaryotic initiation factor 2{alpha} kinase 4, whose expression increased or decreased with EAE, returned to homeostatic levels. Also, two genes with pro-apoptotic functions, calpain-2 and caspase-8-associated protein, increased significantly. A terminal deoxynucleotidyl transferase-mediated dUTP nicked end labeling study detected increased nuclear fragmentation in the 1,25-(OH)2D3-treated samples, confirming increased apoptosis. Together, these results suggest that sensitization of inflammatory cells to apoptotic signals may be one mechanism by which the 1,25-(OH)2D3 resolved EAE.

vitamin D; multiple sclerosis; autoimmune disease; DNA microarray


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 
MULTIPLE SCLEROSIS (MS) is a debilitating autoimmune disease of the central nervous system (CNS). In MS, an abnormal immune response to myelin antigens causes mononuclear cell infiltration, demyelination, oligodendrocyte loss, and axonal degeneration (4). The etiology of MS is uncertain, but the available immunologic, genetic, and epidemiological data suggest that MS develops in genetically susceptible individuals who are exposed to as-yet undefined causal environmental risk factors (53). Genetic heterogeneity exists with respect to MS susceptibility, likely reflecting the action of several genes, each with moderate effects. The MS susceptibility alleles are incompletely penetrant, indicating that environmental risk factors determine whether the disease develops. Identifying these environmental risk factors and how they influence disease development are important goals of MS research.

Several studies have shown that a lack of ultraviolet B (UVB) sunlight exposure is an environmental risk factor for MS (1, 24, 27, 75, 76). Because MS prevalence decreases with increasing UVB exposure, and UVB light is essential for vitamin D3 synthesis, we have hypothesized that the vitamin D3 hormone, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], may be a natural inhibitor of MS (29). Consistent with this hypothesis, we and others found that 1,25-(OH)2D3 profoundly inhibited the induction of experimental autoimmune encephalomyelitis (EAE) (11, 13, 46, 49, 51) and rapidly reversed established EAE disease (50). EAE, an induced disease, is a widely used MS model (67). Like MS, EAE occurs in relapsing-remitting or chronic progressive forms, shows strong genetic susceptibility associated with the major histocompatibility complex, reflects environmental triggers related to prior infection, and exhibits white matter lesions associated with T helper type 1 (Th1) cells and gray matter pathology with axonal degeneration.

Although UVB light catalyzes vitamin D3 synthesis, two further metabolic steps are required to convert this biologically inactive compound into a hormone (54). A constitutive liver enzyme, vitamin D3-25-hydroxylase (25-OHase), catalyzes a C-25 hydroxylation. Thereafter, an inducible kidney enzyme, 25-hydroxyvitamin D3-1{alpha}-hydroxylase (1{alpha}-OHase), catalyzes a C-1 hydroxylation. It is highly significant that microglia (25, 81), neurons (25, 81), and interferon-{gamma} (IFN-{gamma})-activated macrophages (2, 21, 31, 56) express the 1{alpha}-OHase, so the CNS could be a site of active 1,25-(OH)2D3 synthesis. Furthermore, the myeloid and lymphoid lineage cells, astrocytes, glial cells, neurons, and oligodendrocytes all express the nuclear vitamin D receptor (VDR) (30) that binds 1,25-(OH)2D3 and forms an activated transcription regulatory complex (28, 39). The presence of the 1{alpha}-OHase and the VDR in the CNS led us to hypothesize that given sufficient sunlight exposure and vitamin D3, the neurons, microglia, and IFN-{gamma}-activated macrophages could produce high levels of 1,25-(OH)2D3 in the CNS, thereby altering gene expression patterns in a manner that would attenuate inflammation (30).

The purpose of the experiments reported here was to identify the rapid 1,25-(OH)2D3-mediated gene expression changes in the CNS that preceded the resolution of EAE lesions and the reversal of clinical EAE disease (50). We expected that these gene expression changes might provide new insight into how sunlight and vitamin D3 could inhibit MS. We previously developed an experimental system to study the molecular basis for rapid 1,25-(OH)2D3-mediated reversal of established EAE (50). EAE was induced, and animals with severe EAE were treated with 1,25-(OH)2D3 or placebo to begin disease reversal in a synchronous manner. Kinetic studies using this EAE treatment model showed that within 24 h of treatment, the hormone dramatically reduced the inflammatory cells in the CNS lesions (50). These data suggested that the 1,25-(OH)2D3 may have sensitized the inflammatory cells to apoptosis. Here, we have used the EAE treatment model to analyze how 1,25-(OH)2D3-mediated activation or repression of vitamin D-responsive genes might reverse EAE. A DNA microarray study examined gene expression patterns in the CNS of hormone-treated and placebo-treated mice with EAE, and a terminal deoxynucleotidyl nicked end labeling (TUNEL) study identified cells undergoing apoptosis. The results strongly suggest that the 1,25-(OH)2D3 altered gene expression patterns in a manner that would sensitize the inflammatory cells to apoptosis, thereby contributing to lesion resolution in this autoimmune disease model.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 References
 
Mice.
The B10.PL(73NS)/Sn breeding pairs were originally obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were bred in the Biochemistry Department’s pathogen-free mouse colony. The mice were fed laboratory diet 5008 (PMI Nutrition International, Brentwood, MO), containing 3.3 IU vitamin D3 per gram diet and 1% calcium, and maintained at 23°C with 40–60% humidity and 12:12-h light-dark cycles. The drinking water was provided ad libitum. All animal experimentation was conducted in accord with accepted standards of humane animal care, as outlined in the Ethical Guidelines. The experimental protocols were approved by the Institutional Animal Care and Use Committee.

EAE induction and 1,25-(OH)2D3 treatment.
EAE was induced in adult male mice (age 6–8 wk) with myelin basic protein and pertussis toxin (List Biological Laboratories, Campbell, CA) as described (50), and EAE disease severity was scored daily (11). Mice with an EAE clinical score of 2.5 to 3 were randomized into two groups, and injected intraperitoneally with 200 ng of 1,25-(OH)2D3 in 0.1 ml of soybean oil (Hunt-Wesson Foods) or soybean oil only as the placebo. The 1,25-(OH)2D3 stock solution (1 mg/ml absolute ethanol; Tetrionix, Madison, WI) was stored in the dark at –20°C prior to dilution in oil. The placebo- and hormone-treated mice with EAE were euthanized, then perfused with saline, and the spinal cords were removed. Spinal cord samples were obtained from placebo- and hormone-treated mice at 6 h posttreatment. Spinal cords were also obtained from normal male mice. The spinal cords were fixed in 4% paraformaldehyde or flash frozen in liquid nitrogen and stored at –80°C for histopathology or RNA extraction, respectively.

Histopathology.
The paraformaldehyde-fixed spinal cords were embedded in paraffin, sectioned longitudinally at 10 µm, stained with Gill’s no. 3 hematoxylin and eosin Y (Sigma Diagnostics, St. Louis, MO), and examined using a Zeiss Axioskop microscope equipped with a Plan-Neofluar 20x/0.5 objective. Bright-field images were acquired with AxioVision 3.0 software controlling an Axiocam digital camera. Each of 2 spinal cord sections per mouse was divided into 10 segments, and each segment was further subdivided into 2 parts. Each of the 40 subdivided parts was scored in a blinded fashion as 0 or 1, based on the absence or presence, respectively, of infiltrating inflammatory cells. The histopathology score was recorded as the percentage of the 40 subdivided spinal cord parts that showed a readily identifiable inflammatory cell infiltrate.

Polymerase chain reaction.
Total RNA was extracted from frozen spinal cord samples using TRI Reagent (Molecular Research Center, Cincinnati, OH). To assess whether the RNA was intact, a sample was reverse transcribed from an oligo(dT) primer using the Reverse Transcription System (Promega, Madison, WI), and the GAPDH transcripts were PCR amplified using published primers (49). Only RNA samples that yielded a GAPDH amplicon were employed for further studies.

Real-time PCR was performed as described with minor modifications (10). The PCR reactions (25 µl) contained 0.5–1 µg cDNA, 50 nM of each primer, 12.5 µl of 2x SYBR Green PCR Master Mix (PE Applied Biosystems, Foster City, CA), and 2.25 µl H2O. The amplification was accomplished with a GeneAmp 5700 Sequence Detection Systems instrument (PE Applied Biosystems) programmed for incubations of 2 min at 50°C and 10 min at 95°C, followed by 40 cycles of denaturation for 15 s at 95°C and annealing/extension for 1 min at 60°C.

The GAPDH primers have been published previously (57). The glial fibrillary acidic protein (GFAP) PCR primers were designed using Primer Express software (PE Applied Biosystems) and purchased from Integrated DNA Technologies (Coralville, IA). The GFAP primers were forward 5'-GAA GGT CCG CTT CCT GGA A-3' and reverse 5'-GGC TCG AAG CTG GTT CAG TT-3'. The CD45 primers were forward 5'-GAA CCC CGG AGC CTA CCA-3' and reverse 5'-CAG AAC CAT TGG CAG CAT GT-3'. The calpain-2 primers were forward 5'-TAA CCA ACT GAG CCA GGT GTG-3' and reverse 5'-GCC AGC TTG GTT GTT TCA AG-3'. The caspase-8-associated protein 2 primers were forward 5'-TCA ATG GAG AAA ATC CCA CA-3' and reverse 5'-AGC TTT TGG TCT CAG TGG CT-3'. To generate a standard curve, cDNA representing each specific amplicon was gel purified and quantified by absorbance at 260 nm. Each real-time PCR included reactions with serially diluted standard cDNA. The standard cDNA copy number, calculated from the absorbance and the dilution was plotted vs. the threshold cycle, CT. The transcript copy number in each unknown sample was determined from CT by reference to the appropriate standard curve. The data were calculated as transcript abundance relative to GAPDH as the internal standard.

DNA microarray analysis.
Each intact RNA sample was used to synthesize biotin-labeled cRNA exactly as described (45). Each biotin-labeled cRNA sample was hybridized to one microarray (murine U74Av2 GeneChips; Affymetrix) exactly as described (44). The cRNA from Escherichia coli, B. subtilis, and the P1 bacteriophage served as hybridization controls. The GeneChips were washed, stained first with streptavidin phycoerythrin, then with biotinylated goat antibodies to streptavidin, and finally with streptavidin-phycoerythrin again to amplify the signal. The stained GeneChips were subsequently washed in an Affymetrix fluidics station (model 800101) and scanned at 6-µm resolution on a Hewlett-Packard GeneArray scanner (model 900154).

The gene expression data were analyzed with DNA-Chip Analyzer (dChip) software (47), leading to estimates of log base 2 expression. The log-transformed gene expression data for the 1,25-(OH)2D3-treated mice (n = 3) were compared with the data for the placebo-treated mice (n = 3). For genes whose expression increased with 1,25-(OH)2D3 treatment, the fold change was calculated as (signalhormone/signalplacebo). For genes whose expression decreased with 1,25-(OH)2D3 treatment, the fold change was calculated as (signalplacebo/signalhormone). The comparison between the placebo-treated mice (n = 3) and the normal mice (n = 3) was done in an analogous manner. The data have been submitted to the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) and can be found under GEO accession numbers GSM13048 through GSM13056 and GSE842.

Gene expression differences were considered to be statistically suggestive when P ≤ 0.05 for the Student’s t-test and P ≤ 0.058 for the Mann-Whitney rank sum test (5). Overall ANOVAs comparing all three treatments were conducted but are not reported here. No multiple comparison correction was made for the two tests described above. Instead, we limited our analysis to genes with a ≥|2.0|-fold change. As a further guard against false discoveries amongst the 12,488 statistical tests that were conducted on the data set, we used q values to estimate the gene-specific false recovery rate based on the P value derived from the Student’s t-test (68). The q value estimates the minimum false discovery rate of one particular gene that has been deemed significant based on the Student’s t-test P value (68). Hierarchical cluster analysis was performed using robust multi-array analysis (RMA) with the R software package (http://bioconductor.org/CRAN) (36). The genes determined to be significant by the criteria described above were separated into clusters using the average distance method.

In situ apoptosis detection.
Spinal cord samples for apoptosis studies were obtained from placebo- and hormone-treated mice at 6 h posttreatment. They were fixed for 24 h in 4.0% paraformaldehyde followed by 70% ethanol in water. The fixed tissue was embedded in paraffin, sectioned longitudinally at 6 µm, and deparaffinized by heating 5 min at 58°C. After a 10-min wash in xylene, the sections were rehydrated by washing sequentially in 100%, 95%, and 70% ethanol in water. Apoptotic cells were detected by TUNEL using a kit (NeuroTACS II in situ apoptosis detection kit; R & D systems, Minneapolis, MN) according to the manufacturer’s directions. Apoptosis was evaluated in blinded fashion by determining the percentage of lesions with at least 5% apoptotic cells (as evidenced by intense nuclear TUNEL staining). Bright-field images were acquired as above with a Plan-Apochromat 63x/1.4 oil objective.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 References
 
Clinical and biological analysis of mice with EAE.
Knowledge of rapid gene expression changes in the CNS following hormone administration to mice with EAE could provide important insights into how 1,25-(OH)2D3 produced locally in the CNS might inhibit MS. To acquire such knowledge on the broadest possible scale, we used DNA microarrays to compare patterns of gene expression in spinal cord cells obtained from hormone-treated and placebo-treated mice with EAE. Severe EAE disease was induced, and mice with a mean clinical score of 2.7 ± 0.2 were randomized to receive an injection of 200 ng 1,25-(OH)2D3 in oil or oil only as the placebo. This dose was selected for this and prior studies because it effectively inhibited EAE in male mice without causing hypercalcemia (12, 49, 50). It is not clear whether this dose achieved a physiological level of 1,25-(OH)2D3 in the inflamed CNS, because the 1,25-(OH)2D3 level in the inflamed CNS is unknown. It could be significantly higher than the 1,25-(OH)2D3 level in plasma, because the IFN-{gamma}-activated macrophages have a very high 1,25-(OH)2D3 biosynthetic capacity (22).

Measurements of mouse weight, EAE clinical disease score, and CNS histopathology (Table 1 and Fig. 1), and also flow cytometry of spinal cord cell samples (data not shown), showed that the hormone-treated and placebo-treated mice did not differ with respect to EAE disease parameters or cell populations present in the inflamed CNS at 6 h posttreatment. A histopathological examination of the hematoxylin and eosin-stained spinal cord sections showed no observable differences between the placebo- and hormone-treated samples. Both samples had characteristic hyperacute EAE lesions with an inflammatory cell infiltrate composed primarily of neutrophils and also macrophages and T lymphocytes. Changes in gene expression were evident at this time point, indicating that the hormone had reached the CNS within 6 h. Accordingly, spinal cord RNA samples were collected at 6 h posttreatment for gene expression analysis.


View this table:
[in this window]
[in a new window]
  		Table 1. The 1,25-(OH)2D3- and placebo-treated groups did not differ with regard to EAE disease severity or spinal cord inflammatory cell infiltrate

 


View larger version (58K):
[in this window]
[in a new window]
  		Fig. 1. The spinal cords from placebo-treated and 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-treated animals showed equivalent lesion severity and inflammatory cell infiltration at 6 h posttreatment. Mice with an experimental autoimmune encephalomyelitis (EAE) clinical score of 2.5–3 were randomized into two groups. Mice in one group received an intraperitoneal injection of 200 ng 1,25-(OH)2D3 in 0.1 ml oil, whereas mice in the other group received oil only as the placebo. The spinal cord samples were collected 6 h later. The spinal cords were fixed in paraformaldehyde, embedded in paraffin, sectioned longitudinally and stained with hematoxylin and eosin. Slides were examined using a Zeiss Axioskop microscope equipped with a Plan-Neofluar 20x/0.5 objective. Bright-field images were acquired with AxioVision 3.0 software controlling an Axiocam digital camera. The nuclei of infiltrating, inflammatory cells appear as dark blue spots. Images are representative of 3 mice per group.

 
Prior to gene expression analysis, an RT-PCR for GAPDH was performed to ascertain the RNA sample quality. Three individual RNA samples per group were selected on the basis of a robust GAPDH signal and hybridized to individual GeneChips. To determine the completeness of the hybridizations, the signal intensity ratios of the 5' and 3' probes specific for GAPDH, ß-actin, and 18S RNA were analyzed. These ratios ranged from 0.8–1.2 for all nine samples, indicating complete hybridization. Neither EAE disease nor hormone treatment affected these genes, as judged by comparisons with normal controls. Thereafter, the remaining gene expression data were log-transformed and analyzed.

EAE disease-induced gene expression changes in the CNS.
We first compared the gene expression patterns in the CNS of the placebo-treated mice with EAE to the normal control mice. This acute inflammatory disease appeared to alter the expression patterns of 2.6% of the genes analyzed (327 of 12,488), as determined by applying statistical (Student’s t-test P ≤ 0.05 and Mann-Whitney test P ≤ 0.058) and fold change (≥|2.0|) criteria for the significance of differences. However, the estimated false discovery rate (q value) was >30% for these P values (68). Therefore, a more stringent criterion of P < 0.005 (Student’s t-test) was applied to hold the false discovery rate below 30%. By this criterion, 0.8% (95 of 12,488) of the genes had altered expression in EAE disease. Table 2 presents the results for a selected subset of these genes; Supplemental Table S1 presents all of them. (Supplementary Material is available at the Physiological Genomics web site.)1


View this table:
[in this window]
[in a new window]
  		Table 2. Selected genes whose expression in the CNS was altered by EAE induction

 
The EAE disease increased the expression of many genes associated with antigen processing, presentation, and the immune response. Two proteosome subunits and cathepsins B, C, D, L, and S increased with EAE disease. The proteosome and cathepsins participate in antigen processing (59) and presentation (32), respectively. Several major histocompatibility (MHC) class I genes associated with antigen presentation, and genes involved with MHC class II molecule assembly, increased significantly in EAE. There were also gene expression increases consistent with leukocyte infiltration and activation. The CD45 (72), CD52 (19), CD72, and lymphocyte antigen 6 (78) genes expressed on activated T and B cells, and the CD86 (15), CD9 (55), S100A8 (33, 79), and toll-like receptor 6 (71) genes expressed on monocytes and dendritic cells, all increased in EAE disease compared with normal mice. A real-time PCR analysis confirmed the trend toward increased CD45 gene expression in EAE disease, but this trend did not reach significance (P = 0.07; data not shown). These gene expression changes were consistent with the clinical and histopathological data indicating that EAE disease induction led to a strong inflammatory autoimmune response in the CNS.

A hallmark of MS pathology is degradation of the myelin sheath due to oligodendrocyte injury or loss (4). Since the complement system may inflict damage on oligodendrocytes in the presence of antibodies to myelin basic protein (74), it is noteworthy that several genes encoding complement-associated molecules increased. In addition, two genes encoding myelin sheath proteins decreased (Supplemental Table S1), suggesting that the oligodendrocytes were producing less proteolipid protein (–5.3-fold, P = 0.009) and myelin oligodendrocyte glycoprotein (–2.2-fold, P = 0.009) due to cell injury or loss, consistent with the EAE histopathology analysis (Fig. 1).

1,25-(OH)2D3-induced changes in gene expression.
We next compared the gene expression patterns in the CNS of the 1,25-(OH)2D3-treated and placebo-treated mice with EAE (Table 3). In this acute EAE disease model, primarily CD4+ T cells and CD11b+ macrophages infiltrate the CNS (50). The CD4 and CD11b gene expression did not differ between the hormone-treated and placebo-treated mice with EAE, indicating that the two groups did not differ with respect to CD4+ T cell and CD11b+ macrophage abundance in the spinal cords, consistent with the clinical and histopathological data (Table 1) and the flow cytometric data (not shown). In addition, no significant differences were observed for the inflammatory leukocyte surface markers discussed above. Therefore, the gene expression differences discussed below are attributable to hormone-induced changes and not to differences in the inflammatory cell populations in the spinal cord samples.


View this table:
[in this window]
[in a new window]
  		Table 3. Selected genes whose expression in the inflamed CNS was altered by 1,25-(OH)2D3 treatment

 
Three known vitamin D-responsive genes were among the genes whose expression changed in the 1,25-(OH)2D3-treated mice compared with the placebo-treated mice, confirming that the 1,25-(OH)2D3 reached the CNS within 6 h. The genes encoding fatty acid binding protein 3 (16) and calcium-activated neutral protease calpain-2 (62) increased (Table 3), whereas the tubulin-{alpha}3 gene (52) decreased (Supplemental Table S2).

Comparing the gene expression patterns of the 1,25-(OH)2D3-treated and placebo-treated mice with EAE, we found that the 1,25-(OH)2D3 treatment appeared to alter the expression patterns of 0.7% of the genes analyzed (84 of 12,488; Student’s t-test P ≤ 0.05; Mann-Whitney test P ≤ 0.058; fold change ≥|2.0|). The q values for this comparison did not allow a meaningful interpretation, because they led to an estimated false discovery rate near 100%. However, a real-time PCR analysis of 5 genes from this list of 84 genes confirmed 4 of them as true positive discoveries for a false discovery rate of about 20% (Fig. 2). Table 3 presents the results for a selected subset of the vitamin D-responsive genes; Supplemental Table S2 presents all of them.



View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Real-time PCR analysis confirmed the glial fibrillary acidic protein (GFAP), calpain-2, and caspase-8-associated protein gene expression results. The experimental design is given in the legend to Fig. 1. At 6 h posttreatment, the spinal cord RNA was isolated and reverse transcribed, and quantitative PCR was performed. The results are means and SD; n = 4 naive mice/group; n = 6–9 sick, placebo- or 1,25-(OH)2D3-treated mice/group. **P < 0.02 and ***P < 0.01 (Mann-Whitney test).

 
Several genes whose expression decreased with EAE disease increased to near normal gene expression levels in the 1,25-(OH)2D3-treated mice. These genes were eukaryotic translation initiation factor 2{alpha} kinase 4 (eIF-2{alpha} kinase-4), uromodulin, bone morphogenetic protein 15, procollagen type IV, and cadherin-related neuronal receptor I. Conversely, some genes whose expression increased with EAE disease decreased to near normal gene expression levels in the 1,25-(OH)2D3-treated mice. Examples were GFAP, neurotrophic tyrosine kinase receptor type 2 (TrkB), and the retinoic acid receptor-{gamma} (RAR-{gamma}). A real-time PCR analysis confirmed the GFAP gene expression result (Fig. 2). In summary, these data suggest the return of several genes to homeostatic expression levels in the hormone-treated mice.

1,25-(OH)2D3-induced changes in astrocyte gene expression.
It is not known which cells are targeted by the 1,25-(OH)2D3 during EAE resolution, since immune cells (7, 60), neurons (9, 61, 77), astrocytes (43), oligodendrocytes (6), and microglia (69) express the VDR and are involved in the complex interactions of EAE disease. To gain insight into the target cells for 1,25-(OH)2D3 and VDR-mediated action in the inflamed CNS, we looked for cell type-specific genes whose expression changed in response to hormone treatment. The astrocyte-specific gene GFAP (8) increased with EAE disease, signaling the formation of reactive astrocytes, and decreased with 1,25-(OH)2D3 treatment (Table 3), indicating a return of the reactive astrocytes to their homeostatic state. The astrocyte-specific gene calcium/calmodulin-dependent protein kinase II-{delta} increased with 1,25-(OH)2D3 treatment (8). These results indicated that astrocytes are early target cells for 1,25-(OH)2D3-mediated action.

1,25-(OH)2D3-induced changes in apoptosis-related gene expression.
The 1,25-(OH)2D3 treatment also altered the expression of several apoptosis-related genes. The DNA microarray data (Table 3) and real-time PCR data (Fig. 2) showed significant increases in the transcripts encoding the pro-apoptotic proteins calpain-2 and caspase-8-associated protein 2 in the CNS of the 1,25-(OH)2D3-treated mice with EAE compared with the placebo-treated mice. Also, the transcripts for two anti-apoptotic proteins, mouse inhibitor of apoptosis (MIAP-2) and CXCL12 (also termed stromal-derived differentiation factor-1) (70), decreased. These changes in calpain-2, caspase-8-associated protein 2, MIAP-2, and CXCL12 gene expression suggested the hypothesis that the 1,25-(OH)2D3 might increase the inflammatory cell sensitivity to apoptosis as one contribution to EAE resolution.

To investigate the apoptosis hypothesis, TUNEL staining was performed on spinal cord sections. The DNA fragmentation that is a hallmark of cells undergoing apoptosis was visualized by detecting the labeled DNA ends immunohistochemically. An increased frequency of apoptotic cells was evident in the spinal cord sections from the hormone-treated mice compared with the placebo-treated mice (Fig. 3). The TUNEL-stained cells were associated with the inflammatory lesions in the meningeal region and did not occur in the white matter or the gray matter of the spinal cord section. This location is consistent with the cells being inflammatory cells responsible for lesional damage, such as neutrophils, macrophages, or CD4+ T cells. Thus the TUNEL results are consistent with the altered gene expression patterns and highlight possible hormone-mediated sensitization of macrophages or CD4+ T cells to apoptotic signals.



View larger version (135K):
[in this window]
[in a new window]
  		Fig. 3. The 1,25-(OH)2D3 treatment rapidly increased inflammatory cell apoptosis in the central nervous system (CNS) of mice with severe EAE. The experimental design is given in the legend to Fig. 1. Six hours posttreatment, spinal cords were isolated, fixed, embedded, and sectioned, and in situ TUNEL staining for apoptotic cells was performed. The slides were examined and the images were acquired as described in the legend to Fig. 1. The fragmented DNA of the apoptotic cells appears as darkly stained material. Images are representative of 3 mice per group.

 
A hierarchical RMA cluster analysis was performed (36). The cluster analysis on the EAE-associated genes showed that inflammatory genes in clusters 1, 3, 4, and 5 increased in expression for the placebo-treated mice with EAE compared with the normal mice (Supplemental Fig. S1 and Supplemental Table S3). The cluster dendrogram for the vitamin D-responsive genes was markedly asymmetrical, reflecting the nonnormal distribution of the data, as was also evident in the q value calculation (Supplemental Fig. S2 and Supplemental Table S4). The cluster data showed that there were very few large gene expression changes when the 1,25-(OH)2D3-treated and placebo-treated mice with EAE were compared. This result indicated that the 6 h time point probably represented initial hormone-induced gene expression changes in the disease resolution pathway.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 References
 
The experiments reported here have provided new insights into how 1,25-(OH)2D3-mediated activation or repression of vitamin D-responsive genes inhibited EAE, an MS model. It is our hypothesis that 1,25-(OH)2D3 produced in the CNS performs essential immunoregulatory functions that inhibit the development of MS (30). Administering 1,25-(OH)2D3 to mice with severe EAE rapidly decreased the inflammatory CD11b+ cell burden in the CNS and reversed the clinical disease signs (50). Here, the cellular targets and mechanisms underlying these effects were investigated through gene expression analysis. The studies were carried out at a time when the injected hormone had reached the CNS but the CNS cell populations had not yet been altered. The results suggested that 2.6% of the transcripts analyzed changed with EAE disease; many of these changes were associated with antigen processing and presentation, leukocyte infiltration and activation, and a pathogenic autoimmune response. In comparison, 0.7% of the transcripts analyzed changed with 1,25-(OH)2D3 treatment of mice with EAE. Some of the alterations reflected a return to homeostatic levels of transcripts that changed with EAE. Modulations in cell type-specific transcripts indicated that astrocytes were probably direct targets of protective 1,25-(OH)2D3 actions. In addition, changes in apoptosis-associated genes, together with the TUNEL analysis, indicated that the 1,25-(OH)2D3 had probably sensitized inflammatory cells to apoptotic signals. The return of particular EAE-associated transcripts to homeostatic levels, the protection of astrocytes, and the sensitization of inflammatory cells to apoptosis would all be consistent with rapid resolution of clinical and histopathological disease (50).

DNA microarrays provide a powerful approach to study hormone-responsive genes and disease mechanisms, but some considerations affect the design and interpretation of microarray studies. Because these studies are costly, the number of treatment groups, time points, and replicates per group is often limited. Our study did not examine the effect of 1,25-(OH)2D3 on gene expression in the normal spinal cord, because 1,25-(OH)2D3 does not appear to have major functional roles in the normal CNS; mice lacking the hormone (17) or the receptor (80) have no neurological phenotype. Our study focused instead on the effect of 1,25-(OH)2D3 on gene expression in the inflamed spinal cord, where high level 1,25-(OH)2D3 synthesis and inflammatory cell responsiveness would be expected. Accordingly, it is possible that a fraction of the gene expression changes reported here could be part of the normal CNS response to 1,25-(OH)2D3, rather than being characteristic of the inflamed CNS response to the hormone. The study was done at one time point, so some very early gene expression changes may have been missed and some of the observed gene expression changes may represent indirect hormone effects. Also, although stringent statistical and fold change criteria were applied, false positives and negatives may be represented in the data. This could be addressed in part by a larger microarray experiment; we had only three replicates per treatment. Finally, the microarrays detect transcript changes that may not be reflected at the protein level. Despite these limitations, the microarray approach is valuable, because gene expression changes may suggest disease resolution mechanisms worthy of further investigation.

The present DNA microarray study examined spinal cord mRNA derived from a heterogeneous mixture of neurons, oligodendrocytes, astrocytes, microglia, and immune cells interacting in a complex micro-environment. Spinal cord mRNA was analyzed to preserve the effects of cell-cell interactions during the hormone treatment. However, gene expression changes in rare cell populations may have been missed. Microarray hybridization analysis can detect gene expression changes in cells that comprise ≥5% of the total cell population (3). The CD4+ T cells comprised <2% of spinal cord cells from mice with EAE (50). This low frequency may explain why IFN-{gamma}, the signature cytokine of pathogenic CD4+ T cells, was not detected in our experiments, or prior EAE (14, 34) or MS (48) studies.

Our results were consistent with the two previous gene expression profiling studies done on spinal cords from mice with acute EAE, yet differences in experimental design and analysis led to some distinctive differentially expressed genes. (14, 34). We confirmed results from Ibrahim et al. (34) for CD53, CD52, complement component 1q ß-chain, ferritin light chain 1, complement component 3, and Ia-associated invariant chain. We also confirmed results from Carmody et al. (14) for MHC class I molecules (e.g., H-2 D region locus 1 and others), chemokine receptors (e.g., chemokine C-C receptor 1), cytokines (e.g., small inducible cytokine A9), IFN-inducible transcripts (e.g., IFN-inducible GTPase), cathepsins (e.g., cathepsin C), proteosome subunits, leukocyte surface markers (e.g., CD53 and toll-like receptor 6), S100 calcium binding protein A11 (also known as calgizzarin), and others. Importantly, our microarray study also suggested an increase in CD45 transcripts, consistent with inflammatory cell infiltration. CD45 is a plasma membrane tyrosine phosphatase that regulates T and B lymphocyte receptor signaling (72). A CD45 point mutation has been implicated in the development of MS in some families (37). The three EAE studies are consistent with a gene expression analysis of MS lesions compared with normal control samples (48). Noteworthy in the MS study were increases in MHC class I and II, complement 1 and 3, calgizzarin, and markers of T cells, B cells, and macrophages. In summary, there was a remarkable consistency in the three studies of EAE and the study of MS in that all of the studies detected genes involved in antigen processing and presentation, leukocyte infiltration and activation, and other immune-related molecules.

When 1,25-(OH)2D3 was administered to mice with severe acute EAE, the leukocyte marker genes did not change, consistent with the clinical, histopathological, and biological data indicating that the hormone had not altered the leukocyte populations. Several genes that changed with EAE returned to near normal expression levels. Examples are GFAP, eIF-2{alpha} kinase 4, MIAP-2, TrkB, cadherin-related neuronal receptor I, and RAR-{gamma}. It was surprising that genes associated with antigen processing and presentation, leukocyte infiltration and activation, and immune-mediated pathology were not among the vitamin D-responsive genes. In particular, the placebo and 1,25-(OH)2D3-treated groups did not differ significantly in their IL-1, IL-2, IL-4, IL-12, TNF-{alpha}, IFN-{gamma}, or iNOS gene expression, despite prior in vitro studies reporting that 1,25-(OH)2D3 regulated these genes (reviewed in Ref. 30). It is possible that these genes are indirectly regulated by 1,25-(OH)2D3 in vivo, or that the 1,25-(OH)2D3 level achieved in the CNS in the present experiments was lower than the level used in the in vitro studies, or that these were false-negative data points in our study due to the small sample size or large within group variance.

Our data indicate that the 1,25-(OH)2D3 rapidly affected reactive astrocytes. Astrocytes perform both pro-inflammatory (20) and anti-inflammatory (26, 58) functions, contributing to homeostasis in the CNS (20). The 1,25-(OH)2D3 decreased GFAP transcripts. GFAP is an astrocyte-specific intermediate filament protein that increases significantly in the reactive astrocytosis of acute MS (23, 65). On the other hand, the 1,25-(OH)2D3 increased calcium/calmodulin-dependent protein kinase II-{delta} transcripts. This kinase functions as a Ca2+ spike frequency detector (73), phosphorylating PEA-15 (phosphoprotein enriched in astrocytes) and preventing astrocyte apoptosis (42). Together, these data suggest that the 1,25-(OH)2D3 likely decreased the astrocyte immune activation state and enhanced astrocyte survival.

Our microarray and PCR data showed a significant increase in transcripts encoding calpain-2 and caspase-8-associated protein 2 in the 1,25-(OH)2D3-treated animals. The experimental design does not allow us to determine which cells are undergoing apoptosis, although a vast literature exists linking calpain-2 and caspase-8-associated protein 2 to T cell apoptosis. Calpain-2 initiates an apoptosis program in response to an increase in cytosolic calcium (38), and T cell receptor (TCR) engagement causes an increase in cytosolic calcium (40, 66). Moreover, mature, activated T lymphocytes are known to have a calcium- and calpain-2-dependent apoptosis program that is triggered through the TCR (64). Thus, if the 1,25-(OH)2D3 increased the calpain-2 in the T cells, then TCR engagement might induce cell death rather than stimulation. Similarly, caspase-8-associated protein 2 (also known as FLASH, for "FLICE-associated huge protein") is essential for caspase-8 activation (35), and caspase-8 activation is essential for CD95 (FAS) death receptor-mediated apoptosis in T lymphocytes (63). Thus, if the 1,25-(OH)2D3 increased the caspase-8-associated protein 2 in the T cells, then these cells might have greater sensitivity to CD95 (FAS)-mediated apoptosis triggered by astrocytes expressing CD95L (18, 41, 58). Since there are few links between calpain-2, caspase-8-associated protein 2, apoptosis, and CD11b+ cells, we hypothesize the 1,25-(OH)2D3 sensitizes T cells to both TCR-mediated and CD95 (FAS)-mediated apoptosis through upregulation of calpain-2 and caspase-8-associated protein 2. Current experiments are examining this possibility. Apoptotic elimination of encephalitogenic CD4+ T cells is a well-documented early step in recovery from EAE and is essential for preventing MS (26, 58).

In summary, this study of 1,25-(OH)2D3-induced gene expression changes in the EAE treatment model has provided important new insights into how 1,25-(OH)2D3 may regulate CNS inflammation (30). If there is sufficient sunlight and vitamin D3, then the microglia (25, 81), neurons (25, 81), and IFN-{gamma}-activated macrophages could produce 1,25-(OH)2D3 locally in the CNS. The locally produced 1,25-(OH)2D3 could then act via a paracrine pathway to regulate nearby cells. Our results indicated that the hormone probably exerted protective effects on astrocytes and, at the same time, may have increased the sensitivity of encephalitogenic CD4+ T cells to TCR- and death receptor-mediated apoptotic signals. It is our hope that increasing our understanding of the complex interactions between the vitamin D endocrine system, the immune system, and the CNS will bring us closer to a time when vitamin D-based strategies can be devised to prevent and treat MS.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
References
 
The National Multiple Sclerosis Society provided research grant RG-3107 to support this study. K. M. Spach was supported by Predoctoral Training Grant DK-07665-08 awarded to the Department of Nutritional Sciences by the National Institutes of Health.


   ACKNOWLEDGMENTS
 
We thank Dr. Cheol-Koo Lee (Univ. of Wisconsin Department of Genetics and Medical Genetics), Dr. Denise Ney (Univ. of Wisconsin Department of Nutritional Sciences), and Dr. Ian Imanna (Univ. of Wisconsin Department of Biochemistry) for thoughtful comments on the research and on this manuscript. We thank Dr. Michael Hart (Univ. of Wisconsin Medical School, Department of Pathology) for comments and consultation on CNS pathology. We also thank Wayne Nehls (Univ. of Wisconsin Department of Biochemistry) for careful animal husbandry.


   FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: C. E. Hayes, Dept. of Biochemistry, Univ. of Wisconsin-Madison, 433 Babcock Drive, Madison, WI 53706 (E-mail: hayes{at}biochem.wisc.edu).

10.1152/physiolgenomics.00003.2004.

1 The Supplementary Material for this article (Supplemental Tables S1–S4 and Supplemental Figs. S1 and S2) is available online at http://physiolgenomics.physiology.org/cgi/content/full/00003.2004/DC1. Back


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 References
 

  1. Acheson ED, Bachrach CA, and Wright FM. Some comments on the relationship of the distribution of multiple sclerosis to latitude, solar radiation and other variables. Acta Psychiat Scand 35, Suppl 147: 132–147, 1960.
  2. Adams JS and Gacad MA. Characterization of 1{alpha}-hydroxylation of vitamin D3 sterols by cultured alveolar macrophages from patients with sarcoidosis. J Exp Med 161: 755–765, 1985.[Abstract/Free Full Text]
  3. Afshari CA. Perspective: microarray technology, seeing more than spots. Endocrinology 143: 1983–1989, 2002.[Abstract/Free Full Text]
  4. Al-Omaishi J, Bashir R, and Gendelman HE. The cellular immunology of multiple sclerosis. J Leukoc Biol 65: 444–452, 1999.[Abstract]
  5. Altman DG. Practical Statistics for Medical Research. London: Chapman and Hall, 1991, p. 532–533.
  6. Baas D, Prufer K, Ittel ME, Kuchler-Bopp S, Labourdette G, Sarlieve LL, and Brachet P. Rat oligodendrocytes express the vitamin D(3) receptor and respond to 1,25-dihydroxyvitamin D(3). Glia 31: 59–68, 2000.[CrossRef][Web of Science][Medline]
  7. Bhalla AK, Amento EP, Clemens TL, Holick MF, and Krane SM. Specific high-affinity receptors for 1,25-dihydroxyvitamin D3 in human peripheral blood mononuclear cells: presence in monocytes and induction in T lymphocytes following activation. J Clin Endocrinol Metab 57: 1308–1310, 1983.[Abstract/Free Full Text]
  8. Bignami A, Eng LF, Dahl D, and Uyeda CT. Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res 43: 429–435, 1972.[CrossRef][Web of Science][Medline]
  9. Brewer LD, Thibault V, Chen KC, Langub MC, Landfield PW, and Porter NM. Vitamin D hormone confers neuroprotection in parallel with downregulation of L-type calcium channel expression in hippocampal neurons. J Neurosci 21: 98–108, 2001.[Abstract/Free Full Text]
  10. Bustin SA. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25: 169–193, 2000.[Abstract]
  11. Cantorna MT, Hayes CE, and Deluca HF. 1,25-Dihydroxyvitamin D3 reversibly blocks the progression of relapsing encephalomyelitis, a model of multiple sclerosis. Proc Natl Acad Sci USA 93: 7861–7864, 1996.[Abstract/Free Full Text]
  12. Cantorna MT, Humpal-Winter J, and Deluca HF. Dietary calcium is a major factor in 1,25-dihydroxycholecalciferol suppression of experimental autoimmune encephalomyelitis in mice. J Nutr 129: 1966–1971, 1999.[Abstract/Free Full Text]
  13. Cantorna MT, Woodward WD, Hayes CE, and Deluca HF. 1,25-Dihydroxyvitamin D3 is a positive regulator for the two anti-encephalitogenic cytokines TGF-beta 1 and IL-4. J Immunol 160: 5314–5319, 1998.[Abstract/Free Full Text]
  14. Carmody RJ, Hilliard B, Maguschak K, Chodosh LA, and Chen YH. Genomic scale profiling of autoimmune inflammation in the central nervous system: the nervous response to inflammation. J Neuroimmunol 133: 95–107, 2002.[CrossRef][Web of Science][Medline]
  15. Carreno BM and Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol 20: 29–53, 2002.[CrossRef][Web of Science][Medline]
  16. Chou SY, Hannah SS, Lowe KE, Norman AW, and Henry HL. Tissue-specific regulation by vitamin D status of nuclear and mitochondrial gene expression in kidney and intestine. Endocrinology 136: 5520–5526, 1995.[Abstract]
  17. Dardenne O, Prud’homme J, Arabian A, Glorieux FH, and St-Arnaud, R. Targeted inactivation of the 25-hydroxyvitamin D(3)-1(alpha)-hydroxylase gene (CYP27B1) creates an animal model of pseudovitamin D-deficiency rickets. Endocrinology 142: 3135–3141, 2001.[Abstract/Free Full Text]
  18. Dittel BN, Merchant RM, and Janeway CA Jr. Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis. J Immunol 162: 6392–6400, 1999.[Abstract/Free Full Text]
  19. Domagala A and Kurpisz M. CD52 antigen: a review. Med Sci Monit 7: 325–331, 2001.[Medline]
  20. Dong Y and Benveniste EN. Immune function of astrocytes. Glia 36: 180–190, 2001.[CrossRef][Web of Science][Medline]
  21. Dusso A, Brown A, and Slatopolsky E. Extrarenal production of calcitriol. Semin Nephrol 14: 144–155, 1994.[Web of Science][Medline]
  22. Dusso AS, Kamimura S, Gallieni M, Zhong M, Negrea L, Shapiro S, and Slatopolsky E. {gamma}-Interferon-induced resistance to 1,25-(OH)2 D3 in human monocytes and macrophages: a mechanism for the hypercalcemia of various granulomatoses. J Clin Endocrinol Metab 82: 2222–2232, 1997.[Abstract/Free Full Text]
  23. Eddleston M and Mucke L. Molecular profile of reactive astrocytes: implications for their role in neurologic disease. Neuroscience 54: 15–36, 1993.[CrossRef][Web of Science][Medline]
  24. Freedman DM, Dosemeci M, and Alavanja MC. Mortality from multiple sclerosis and exposure to residential and occupational solar radiation: a case-control study based on death certificates. Occup Environ Med 57: 418–421, 2000.[Abstract/Free Full Text]
  25. Garcion E, Wion-Barbot N, Montero-Menei CN, Berger F, and Wion D. New clues about vitamin D functions in the nervous system. Trends Endocrinol Metab 13: 100–105, 2002.[CrossRef][Web of Science][Medline]
  26. Gold R, Hartung HP, and Lassmann H. T-cell apoptosis in autoimmune diseases: termination of inflammation in the nervous system and other sites with specialized immune-defense mechanisms. Trends Neurosci 20: 399–404, 1997.[CrossRef][Web of Science][Medline]
  27. Hammond SR, English DR, and McLeod JG. The age-range of risk of developing multiple sclerosis: evidence from a migrant population in Australia. Brain 123: 968–974, 2000.[Abstract/Free Full Text]
  28. Haussler MR, Whitfield GK, Haussler CA, Hsieh JC, Thompson PD, Selznick SH, Dominguez CE, and Jurutka PW. The nuclear vitamin D receptor: biological and molecular regulatory properties revealed. J Bone Miner Res 13: 325–349, 1998.[CrossRef][Web of Science][Medline]
  29. Hayes CE, Cantorna MT, and Deluca HF. Vitamin D and multiple sclerosis. Proc Soc Exp Biol Med 216: 21–27, 1997.[CrossRef][Medline]
  30. Hayes CE, Nashold FE, Spach KM, and Pedersen LB. The immunological functions of the vitamin D endocrine system. Cell Mol Biol (Noisy-le-grand) 49: 277–300, 2003.[Web of Science][Medline]
  31. Hewison M, Zehnder D, Bland R, and Stewart PM. 1{alpha}-Hydroxylase and the action of vitamin D. J Mol Endocrinol 25: 141–148, 2000.[Abstract]
  32. Honey K and Rudensky AY. Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol 3: 472–482, 2003.[CrossRef][Web of Science][Medline]
  33. Hu SP, Harrison C, Xu K, Cornish CJ, and Geczy CL. Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide. Blood 87: 3919–3928, 1996.[Abstract/Free Full Text]
  34. Ibrahim SM, Mix E, Bottcher T, Koczan D, Gold R, Rolfs A, and Thiesen HJ. Gene expression profiling of the nervous system in murine experimental autoimmune encephalomyelitis. Brain 124: 1927–1938, 2001.[Abstract/Free Full Text]
  35. Imai Y, Kimura T, Murakami A, Yajima N, Sakamaki K, and Yonehara S. The CED-4-homologous protein FLASH is involved in Fas-mediated activation of caspase-8 during apoptosis. Nature 398: 777–785, 1999.[CrossRef][Medline]
  36. Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, and Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249–264, 2003.[Abstract]
  37. Jacobsen M, Schweer D, Ziegler A, Gaber R, Schock S, Schwinzer R, Wonigeit K, Lindert RB, Kantarci O, Schaefer-Klein J, Schipper HI, Oertel WH, Heidenreich F, Weinshenker BG, Sommer N, and Hemmer B. A point mutation in PTPRC is associated with the development of multiple sclerosis. Nat Genet 26: 495–499, 2000.[CrossRef][Web of Science][Medline]
  38. Johnson DE. Noncaspase proteases in apoptosis. Leukemia 14: 1695–1703, 2000.[CrossRef][Web of Science][Medline]
  39. Jones G, Strugnell SA, and Deluca HF. Current understanding of the molecular actions of vitamin D. Physiol Rev 78: 1193–1231, 1998.[Abstract/Free Full Text]
  40. Kim MJ, Jo DG, Hong GS, Kim BJ, Lai M, Cho DH, Kim KW, Bandyopadhyay A, Hong YM, Kim Do H, Cho C, Liu JO, Snyder SH, and Jung YK. Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death. Proc Natl Acad Sci USA 99: 9870–9875, 2002.[Abstract/Free Full Text]
  41. Kohji T, Tanuma N, Aikawa Y, Kawazoe Y, Suzuki Y, Kohyama K, and Matsumoto Y. Interaction between apoptotic cells and reactive brain cells in the central nervous system of rats with autoimmune encephalomyelitis. J Neuroimmunol 82: 168–174, 1998.[CrossRef][Web of Science][Medline]
  42. Kubes M, Cordier J, Glowinski J, Girault JA, and Chneiweiss H. Endothelin induces a calcium-dependent phosphorylation of PEA-15 in intact astrocytes: identification of Ser104 and Ser116 phosphorylated, respectively, by protein kinase C and calcium/calmodulin kinase II in vitro. J Neurochem 71: 1307–1314, 1998.[Web of Science][Medline]
  43. Langub MC, Herman JP, Malluche HH, and Koszewski NJ. Evidence of functional vitamin D receptors in rat hippocampus. Neuroscience 104: 49–56, 2001.[CrossRef][Web of Science][Medline]
  44. Lee CK, Allison DB, Brand J, Weindruch R, and Prolla TA. Transcriptional profiles associated with aging and middle age-onset caloric restriction in mouse hearts. Proc Natl Acad Sci USA 99: 14988–14993, 2002.[Abstract/Free Full Text]
  45. Lee CK, Klopp RG, Weindruch R, and Prolla TA. Gene expression profile of aging and its retardation by caloric restriction. Science 285: 1390–1393, 1999.[Abstract/Free Full Text]
  46. Lemire JM and Archer DC. 1,25-Dihydroxyvitamin D3 prevents the in vivo induction of murine experimental autoimmune encephalomyelitis. J Clin Invest 87: 1103–1107, 1991.[Web of Science][Medline]
  47. Li C and Wong WH. Model-based analysis of oligonucleotide arrays: expression index computation and outlier detection. Proc Natl Acad Sci USA 98: 31–36, 2001.[Abstract/Free Full Text]
  48. Lock C, Hermans G, Pedotti R, Brendolan A, Schadt E, Garren H, Langer-Gould A, Strober S, Cannella B, Allard J, Klonowski P, Austin A, Lad N, Kaminski N, Galli SJ, Oksenberg JR, Raine CS, Heller R, and Steinman L. Gene-microarray analysis of multiple sclerosis lesions yields new targets validated in autoimmune encephalomyelitis. Nat Med 8: 500–508, 2002.[CrossRef][Web of Science][Medline]
  49. Nashold FE, Hoag KA, Goverman J, and Hayes CE. Rag-1-dependent cells are necessary for 1,25-dihydroxyvitamin D(3) prevention of experimental autoimmune encephalomyelitis. J Neuroimmunol 119: 16–29, 2001.[CrossRef][Web of Science][Medline]
  50. Nashold FE, Miller DJ, and Hayes CE. 1,25-Dihydroxyvitamin D3 treatment decreases macrophage accumulation in the CNS of mice with experimental autoimmune encephalomyelitis. J Neuroimmunol 103: 171–179, 2000.[CrossRef][Web of Science][Medline]
  51. Nataf S, Garcion E, Darcy F, Chabannes D, Muller JY, and Brachet P. 1,25 Dihydroxyvitamin D3 exerts regional effects in the central nervous system during experimental allergic encephalomyelitis. J Neuropathol Exp Neurol 55: 904–914, 1996.[Web of Science][Medline]
  52. Nemere I, Theofan G, and Norman AW. 1,25-Dihydroxyvitamin D3 regulates tubulin expression in chick intestine. Biochem Biophys Res Commun 148: 1270–1276, 1987.[CrossRef][Web of Science][Medline]
  53. Oksenberg JR, Baranzini SE, Barcellos LF, and Hauser SL. Multiple sclerosis: genomic rewards. J Neuroimmunol 113: 171–184, 2001.[CrossRef][Web of Science][Medline]
  54. Omdahl JL, Morris HA, and May BK. Hydroxylase enzymes of the vitamin D pathway: expression, function, and regulation. Annu Rev Nutr 22: 139–166, 2002.[CrossRef][Web of Science][Medline]
  55. Oritani K, Aoyama K, Tomiyama Y, Kincade PW, and Matsuzawa Y. Stromal cell CD9 and the differentiation of hematopoietic stem/progenitor cells. Leuk Lymphoma 38: 147–152, 2000.[Web of Science][Medline]
  56. Overbergh L, Decallonne B, Valckx D, Verstuyf A, Depovere J, Laureys J, Rutgeerts O, Saint-Arnaud R, Bouillon R, and Mathieu C. Identification and immune regulation of 25-hydroxyvitamin D-1-alpha-hydroxylase in murine macrophages. Clin Exp Immunol 120: 139–146, 2000.[CrossRef][Web of Science][Medline]
  57. Overbergh L, Valckx D, Waer M, and Mathieu C. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 11: 305–312, 1999.[CrossRef][Web of Science][Medline]
  58. Pender MP and Rist MJ. Apoptosis of inflammatory cells in immune control of the nervous system: role of glia. Glia 36: 137–144, 2001.[CrossRef][Web of Science][Medline]
  59. Princiotta MF, Finzi D, Qian SB, Gibbs J, Schuchmann S, Buttgereit F, Bennink JR, and Yewdell JW. Quantitating protein synthesis, degradation, and endogenous antigen processing. Immunity 18: 343–354, 2003.[CrossRef][Web of Science][Medline]
  60. Provvedini DM, Tsoukas CD, Deftos LJ, and Manolagas SC. 1,25-Dihydroxyvitamin D3 receptors in human leukocytes. Science 221: 1181–1183, 1983.[Abstract/Free Full Text]
  61. Prufer K and Jirikowski GF. 1,25-Dihydroxyvitamin D3 receptor is partly colocalized with oxytocin immunoreactivity in neurons of the male rat hypothalamus. Cell Mol Biol (Noisy-le-grand) 43: 543–548, 1997.[Medline]
  62. Ravid A, Koren R, Rotem C, Garach-Jehoshua O, Glaser T, and Liberman UA. 1,25-Dihydroxyvitamin D3 increases the cellular content of the calcium-activated neutral protease mu-calpain in renal cell carcinoma. Endocrinology 135: 2822–2825, 1994.[Abstract]
  63. Salmena L, Lemmers B, Hakem A, Matysiak-Zablocki E, Murakami K, Au PY, Berry DM, Tamblyn L, Shehabeldin A, Migon E, Wakeham A, Bouchard D, Yeh WC, McGlade JC, Ohashi PS, and Hakem R. Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. Genes Dev 17: 883–895, 2003.[Abstract/Free Full Text]
  64. Sarin A, Nakajima H, and Henkart PA. A protease-dependent TCR-induced death pathway in mature lymphocytes. J Immunol 154: 5806–5812, 1995.[Abstract]
  65. Smith ME and Eng LF. Glial fibrillary acidic protein in chronic relapsing experimental allergic encephalomyelitis in SJL/J mice. J Neurosci Res 18: 203–208, 1987.[CrossRef][Web of Science][Medline]
  66. Squier MK and Cohen JJ. Calpain, an upstream regulator of thymocyte apoptosis. J Immunol 158: 3690–3697, 1997.[Abstract]
  67. Steinman L. Optic neuritis, a new variant of experimental encephalomyelitis, a durable model for all seasons, now in its seventieth year. J Exp Med 197: 1065–1071, 2003.[Free Full Text]
  68. Storey JD and Tibshirani R. Statistical significance for genomewide studies. Proc Natl Acad Sci USA 100: 9440–9445, 2003.[Abstract/Free Full Text]
  69. Stumpf WE, Bidmon HJ, Li L, Pilgrim C, Bartke A, Mayerhofer A, and Heiss C. Nuclear receptor sites for vitamin D-soltriol in midbrain and hindbrain of Siberian hamster (Phodopus sungorus) assessed by autoradiography. Histochemistry 98: 155–164, 1992.[CrossRef][Web of Science][Medline]
  70. Suzuki Y, Rahman M, and Mitsuya H. Diverse transcriptional response of CD4(+) T cells to stromal cell-derived factor (SDF)-1: cell survival promotion and priming effects of SDF-1 on CD4(+) T cells. J Immunol 167: 3064–3073, 2001.[Abstract/Free Full Text]
  71. Takeda K, Kaisho T, and Akira S. Toll-like receptors. Annu Rev Immunol 21: 335–376, 2003.[CrossRef][Web of Science][Medline]
  72. Tchilian EZ and Beverley PC. CD45 in memory and disease. Arch Immunol Ther Exp (Warsz) 50: 85–93, 2002.[Medline]
  73. Vallano ML, Beaman-Hall CM, Mathur A, and Chen Q. Astrocytes express specific variants of CaM KII delta and gamma, but not alpha and beta, that determine their cellular localizations. Glia 30: 154–164, 2000.[CrossRef][Web of Science][Medline]
  74. Van Beek J, Elward K, and Gasque P. Activation of complement in the central nervous system: roles in neurodegeneration and neuroprotection. Ann NY Acad Sci 992: 56–71, 2003.[Web of Science][Medline]
  75. Van Der Mei IA, Ponsonby AL, Blizzard L, and Dwyer T. Regional variation in multiple sclerosis prevalence in Australia and its association with ambient ultraviolet radiation. Neuroepidemiology 20: 168–174, 2001.[CrossRef][Web of Science][Medline]
  76. Van Der Mei IA, Ponsonby AL, Dwyer T, Blizzard L, Simmons R, Taylor BV, Butzkueven H, and Kilpatrick T. Past exposure to sun, skin phenotype, and risk of multiple sclerosis: case-control study. BMJ 327: 316, 2003.[Abstract/Free Full Text]
  77. Walbert T, Jirikowski GF, and Prufer K. Distribution of 1,25-dihydroxyvitamin D3 receptor immunoreactivity in the limbic system of the rat. Horm Metab Res 33: 525–531, 2001.[CrossRef][Web of Science][Medline]
  78. Wu HJ and Bondada S. Positive and negative roles of CD72 in B cell function. Immunol Res 25: 155–166, 2002.[CrossRef][Web of Science][Medline]
  79. Xu K and Geczy CL. IFN-gamma and TNF regulate macrophage expression of the chemotactic S100 protein S100A8. J Immunol 164: 4916–4923, 2000.[Abstract/Free Full Text]
  80. Yoshizawa T, Handa Y, Uematsu Y, Takeda S, Sekine K, Yoshihara Y, Kawakami T, Arioka K, Sato H, Uchiyama Y, Masushige S, Fukamizu A, Matsumoto T, and Kato S. Mice lacking the vitamin D receptor exhibit impaired bone formation, uterine hypoplasia and growth retardation after weaning. Nat Genet 16: 391–396, 1997.[CrossRef][Web of Science][Medline]
  81. Zehnder D, Bland R, Williams MC, McNinch RW, Howie AJ, Stewart PM, and Hewison M. Extrarenal expression of 25-hydroxyvitamin d(3)-1 alpha-hydroxylase. J Clin Endocrinol Metab 86: 888–894, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BioinformaticsHome page
J. R. Stevens and G. Nicholas
metahdep: meta-analysis of hierarchically dependent gene expression studies
Bioinformatics, October 1, 2009; 25(19): 2619 - 2620.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
F. E. Nashold, K. M. Spach, J. A. Spanier, and C. E. Hayes
Estrogen Controls Vitamin D3-Mediated Resistance to Experimental Autoimmune Encephalomyelitis by Controlling Vitamin D3 Metabolism and Receptor Expression
J. Immunol., September 15, 2009; 183(6): 3672 - 3681.
[Abstract] [Full Text] [PDF]

Home page
 Social Studies of ScienceHome page
A. Fausto-Sterling
The bare bones of race.
Social Studies of Science, October 1, 2008; 38(5): 657 - 694.
[Abstract] [PDF]

Home page
 Endocr. Rev.Home page
R. Bouillon, G. Carmeliet, L. Verlinden, E. van Etten, A. Verstuyf, H. F. Luderer, L. Lieben, C. Mathieu, and M. Demay
Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice
Endocr. Rev., October 1, 2008; 29(6): 726 - 776.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. M. Spach, F. E. Nashold, B. N. Dittel, and C. E. Hayes
IL-10 Signaling Is Essential for 1,25-Dihydroxyvitamin D3-Mediated Inhibition of Experimental Autoimmune Encephalomyelitis
J. Immunol., November 1, 2006; 177(9): 6030 - 6037.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
K. M. Spach and C. E. Hayes
Vitamin D3 Confers Protection from Autoimmune Encephalomyelitis Only in Female Mice
J. Immunol., September 15, 2005; 175(6): 4119 - 4126.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Renal Physiol.Home page
A. S. Dusso, A. J. Brown, and E. Slatopolsky
Vitamin D
Am J Physiol Renal Physiol, July 1, 2005; 289(1): F8 - F28.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
L. Chen, M. T. Cencioni, D. F. Angelini, G. Borsellino, L. Battistini, and C. F. Brosnan
Transcriptional Profiling of {gamma}{delta} T Cells Identifies a Role for Vitamin D in the Immunoregulation of the V{gamma}9V{delta}2 Response to Phosphate-Containing Ligands
J. Immunol., May 15, 2005; 174(10): 6144 - 6152.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Tables and Figures
Right arrow All Versions of this Article:
18/2/141    most recent
00003.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (17)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Spach, K. M.
Right arrow Articles by Hayes, C. E.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Spach, K. M.
Right arrow Articles by Hayes, C. E.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.