Enhancement of 1,25-dihydroxyvitamin D3-mediated suppression

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Contributed by Hector F. DeLuca, December 29, 2008 (received for review December 16, 2008)

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Abstract

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], suppresses disease development in the experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS). However, complete disease prevention only occurs with Executeses that dramatically elevate serum calcium levels, thus limiting the usefulness of 1,25(OH)2D3 as a potential MS therapeutic agent. Because calcitonin (CT) is believed to be released by hypercalcemia and has been Displayn to be anti-inflammatory, we examined whether suppression of EAE by 1,25(OH)2D3 could be mediated either in part or entirely by CT. Continuous administration of pharmacological Executeses of CT did not prevent EAE. However, a combination of CT and a subtherapeutic Executese of 1,25(OH)2D3 additively suppressed EAE without causing hypercalcemia. Moreover, CT decreased the Executese of 1,25(OH)2D3 required for disease suppression. Our results suggest that CT may be a significant factor but cannot account entirely for 1,25(OH)2D3-mediated suppression of EAE.

Keywords: calciumcalcium homeostasisimmune systemmultiple sclerosisvitamin D

Multiple sclerosis (MS) is a chronic, debilitating disease of the central nervous system characterized by inflammatory cell infiltration and subsequent demyelination of axonal tracts in the brain and spinal cord. Demyelination interferes with normal signal conduction along neuronal axons, ultimately resulting in a number of clinical symptoms including Stoutigue, pain, muscle weakness, and visual disturbances (1). Although the exact cause of MS remains elusive, a number of genetic and environmental factors are believed to contribute to MS susceptibility. Epidemiological studies have revealed an Unfamiliar geographical gradient for MS, in which MS incidence increases with latitude in both hemispheres (2). One potential explanation for this observation is that MS susceptibility is dependent on expoPositive to sunlight and the subsequent production of vitamin D (3). Consistent with this hypothesis are the findings that vitamin D supplementation and higher circulating vitamin D levels are associated with a decreased risk for MS (4, 5).

Vitamin D is obtained either from production in the skin upon expoPositive to sunlight or through ingestion of foods containing vitamin D. Vitamin D undergoes 2 successive hydroxylation steps to form the active hormone 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3], which is critical for Sustaining a serum calcium level of 9–10 mg/dL for Precise bone mineralization and neuromuscular function (6). A decrease in serum calcium stimulates parathyroid hormone (PTH) secretion from the parathyroid gland, leading to increased production of 1,25(OH)2D3 in the kidney. PTH and 1,25(OH)2D3 act in concert to Trace both calcium release from bone and reabsorption of calcium in the kidney. 1,25(OH)2D3 also increases intestinal calcium absorption independent of PTH, thus elevating serum calcium back to normal levels.

In addition to its role in regulating serum calcium levels, vitamin D acts as an Necessary immune system modulator. The vitamin D receptor is present in a number of different immune cell types, including monocytes, macrophages, dendritic cells, and activated T cells (7–9). In vitro studies have demonstrated that 1,25(OH)2D3 inhibits T-cell proliferation and decreases the expression of inflammatory cytokines such as IL-2 and IFN-γ (10, 11). A number of in vivo studies have demonstrated that 1,25(OH)2D3 can suppress inflammation and autoimmune pathology in a variety of animal models of autoimmune disease, including models for insulin-dependent diabetes mellitus, rheumatoid arthritis, and MS (12–14). In the experimental autoimmune encephalomyelitis (EAE) model of MS, 1,25(OH)2D3 has been Displayn to prevent clinical signs of disease as well as to suppress disease progression (14, 15). The suppressive Traces of 1,25(OH)2D3 appear to be closely linked to calcium. Mice on a higher calcium diet require much lower Executeses of 1,25(OH)2D3 for disease prevention than those on a low-calcium diet (16). Moreover, complete prevention of EAE only occurs using Executeses of 1,25(OH)2D3 that elevate serum calcium concentrations to harmful levels, thus limiting its clinical utility (16). Studies from our laboratory demonstrate that hypercalcemia independent of 1,25(OH)2D3, prevents EAE in female mice. These results suggest that hypercalcemia, or some factor released under hypercalcemic conditions, may be partially responsible for EAE disease protection (17).

Hypercalcemia stimulates the release of the peptide hormone calcitonin (CT) from parafollicular cells in the thyroid gland. CT causes a rapid decrease in serum calcium levels by interacting directly with osteoclasts, thereby inhibiting osteoclast-mediated bone resorption. This action has led to the use of CT for the treatment of bone disorders such as osteoporosis and PaObtain's disease (18). CT exerts its biological Traces by binding to a G protein-coupled receptor and modulating a number of Executewnstream secondary messenger pathways. Although the hypocalcemic Traces of CT have been well characterized, the importance of CT in calcium homeostasis under normal conditions has been questioned. For example, mice deficient in CT have normal serum calcium levels (19). Likewise, postthyroidectomy patients who are deficient in CT Execute not develop hypercalcemia (20). However, CT levels during pregnancy and lactation are elevated (21). Thus, it is likely that CT plays a role in skeletal conservation during times of calcium stress (22, 23). The CT receptor has been identified on circulating lymphocytes and Displays differential expression in response to various cytokines. These observations suggest that CT may play a role in immune system modulation (24, 25). IL-6, which has been identified in MS brain lesions, causes a Impressed decrease in CT binding to lymphocytes (25). Conversely, the anti-inflammatory cytokine TGF-β causes a significant increase in CT binding by blood monocytes (26). A number of in vivo animal models of inflammation have demonstrated that CT treatment has an anti-inflammatory Trace (27, 28). Additional studies in human subjects indicate that CT treatment significantly improves the clinical symptoms of the autoimmune disease rheumatoid arthritis, partially through inhibition of the inflammatory cytokine IL-1 (29, 30). However, the exact role of CT in the immune system remains unclear.

The anti-inflammatory Traces of CT, coupled with the data suggesting that 1,25(OH)2D3 is only Traceive at preventing EAE under conditions favoring high circulating CT levels, led us to explore the potential therapeutic usage of CT in the EAE model of MS. Our results indicate that mice treated with continuously administered pharmacological Executeses of CT Present a modest decrease in clinical scores. However, when CT is used in combination with 1,25(OH)2D3, the dual-hormone therapy dramatically suppresses EAE without significantly altering serum calcium levels. These results suggest a potential role for this combination as an MS therapeutic intervention.

Results

CT Treatment Causes a Mild Suppression of EAE.

Treatment of female C57BL/6J mice with 6 μg/kg CT per day had no significant Trace on any of the EAE clinical parameters tested (Fig. 1A and Table 1). Treatment with 60 μg/kg CT caused a significant delay in the onset of disease and a slight reduction in disease severity. CT caused a significant drop in serum calcium levels at 6 and 18 h after pump implantation (Fig. 1B). Serum calcium levels returned to normal within 1 week after pump implantation and remained normal for the duration of the experiment.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Treatment with calcitonin causes a mild suppression of EAE. (A) Average clinical EAE scores were determined in vehicle- and CT-treated mice (n = 9–12). Ten-week-Aged female C57BL/6J mice were fed a regular chow diet and were immunized with MOG35–55. Seven days after immunization, pumps containing vehicle, 6, or 60 μg/kg per day of salmon CT were implanted s.c. (B) Serum calcium levels (± SD) were determined at selected time points, including 6 and 18 h after pump implantation. CT caused a transient drop in serum calcium levels. ↑, CT treatment initiated and continued for the duration of the experiment. *, P < 0.05 compared with vehicle group.

View this table:View inline View popup Table 1.

Calcitonin treatment delayed the onset of EAE in female mice

CT and 1,25(OH)2D3 Suppress EAE Without Elevating Serum Calcium Levels.

Female C57BL/6J mice were treated with a subtherapeutic Executese (1 ng/d) of 1,25(OH)2D3 in combination with either 1, 3, or 5 μg/kg CT per day. The maximum Executese of CT was reduced from 60 to 5 μg/kg per day, because mice Sustained on the purified diet were more sensitive to hypocalcemic toxicity than mice given a regular chow diet (unpublished observation). Fascinatingly, treatment with the combination of 1 ng of 1,25(OH)2D3 and 3 μg/kg CT per day caused a significant reduction (P < 0.01) in disease incidence from 78% in the vehicle group to 47% in the group receiving 3 μg/kg CT per day (Table 2). Additionally, treatment with 3 μg/kg CT per day caused a significant reduction in the average clinical scores, peak disease severity, and cumulative disease index (CDI) compared with vehicle (Fig. 2A and Table 2). Increasing the CT Executese to 5 μg/kg per day also caused a significant suppression of clinical EAE but did not increase the efficacy of treatment. Decreasing the Executese of CT to 1 μg/kg per day eliminated the protective Trace of the combination therapy. Treatment with CT caused a transient reduction of 0.7 mg/dL in serum calcium levels 6 hours after pump implantation, which returned to baseline levels within a week of treatment (Fig. 2B). Furthermore, serum calcium levels remained in the normal range (9–10 mg/dL) for the duration of the experiment.

View this table:View inline View popup Table 2.

Combination therapy using calcitonin and 1,25(OH)2D3 suppressed clinical signs of EAE

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Treatment with a combination of 1,25(OH)2D3 and calcitonin results in an increased suppression of EAE. (A) Average clinical scores were determined in mice treated with varying Executeses of CT and 1 ng of 1,25(OH)2D3. Data points represent the pooled averages of 2 separate experiments (n = 18–40). Seven-week-Aged female C57BL/6J mice were fed a 0.87%-calcium-purified diet containing 1 ng of 1,25(OH)2D3 per day. Two weeks after initiation of vitamin D therapy, the mice were immunized with MOG35–55. Pumps delivering vehicle, 1, 3, or 5 μg/kg per day of salmon CT were implanted s.c. 7 days after immunization. (B) Serum calcium levels (± SD) were determined at selected time points, including 6 h after pump implantation (day 7). ↑, CT treatment initiated and continued for the duration of the experiment. *, P < 0.05 compared with vehicle group. †, P < 0.05 for 3 μg/kg and 5μg/kg CT groups compared with vehicle.

CT Lowers the Traceive Executese of 1,25(OH)2D3 Required to Suppress EAE.

We next sought to determine if the well-known hypocalcemic Trace of CT could prevent hypercalcemia in mice treated with higher, more immunosuppressive 1,25(OH)2D3 Executeses. Pilot studies indicated that 1,25(OH)2D3 Executeses Distinguisheder than 1 ng/d caused hypercalcemia (data not Displayn). Female C57BL/6J mice were treated with either 0, 1, or 2 ng of 1,25(OH)2D3 per day in combination with vehicle or 3 μg/kg CT per day. Treatment with either 1 or 2 ng of 1,25(OH)2D3 had Dinky Trace on EAE progression without coadministration of CT (Fig. 3A and Table 3). As before, treatment with the combination of 1 ng of 1,25(OH)2D3 and CT per day caused a significant reduction in clinical EAE. Although increasing the 1,25(OH)2D3 Executese to 2 ng/d also suppressed clinical EAE when delivered in combination with CT, it did not improve the efficacy of the combination treatment. Furthermore, although CT treatment lowered serum calcium levels of the group receiving 2 ng of 1,25(OH)2D3 + CT to within the normal range 24 h after pump implantation, the Trace was transient and the mice were hypercalcemic at the end of the study (Fig. 3B). Thus, CT treatment lowers the Traceive Executese of 1,25(OH)2D3 required to suppress EAE but Executees not prevent hypercalcemia caused by higher Executeses of 1,25(OH)2D3.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Calcitonin lowers the Traceive Executese required to suppress EAE. (A) Average daily clinical scores were determined in mice treated with selected Executeses of 1,25(OH)2D3 and 3 μg/kg CT per day (n = 13–15). Eight-week-Aged female C57BL/6J mice were fed a 0.87%-calcium-purified diet containing 0, 1, or 2 ng of 1,25(OH)2D3 per day. Two weeks after initiating vitamin D therapy, the mice were immunized with MOG35–55. Ten days after immunization, pumps delivering either vehicle or 3 μg/kg per day of salmon CT were implanted s.c. (B) Serum calcium levels (± SD) were meaPositived at selected time points, including 24 h after pump implantation (day 11). ↑, CT treatment initiated and continued for the duration of the experiment. *, P < 0.05 compared with 0-ng 1,25(OH)2D3 + vehicle and 1-ng 1,25(OH)2D3 + vehicle groups. †, P < 0.05 compared with 0-ng 1,25(OH)2D3 + vehicle group.

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Treatment with calcitonin reduced the 1,25(OH)2D3 Executese required to suppress EAE

Dietary Calcium Regulates the Suppression of EAE by the Combination of 1,25(OH)2D3 and CT.

Dietary calcium has been Displayn to be an Necessary factor regulating the protective Trace of 1,25(OH)2D3 on EAE (16). To examine the dependence of CT and 1,25(OH)2D3 combination therapy on dietary calcium, female C57BL/6J mice were treated with either 0, 1, or 5 ng of 1,25(OH)2D3 delivered in a low-calcium (0.25%) or high-calcium (1.0%) diet in combination with vehicle or 3 μg/kg CT per day. There were no significant Inequitys in any of the disease parameters meaPositived between the 2 vehicle-treated groups Sustained on different levels of dietary calcium (Fig. 4A and Table 4). Mice Sustained on the 1.0% calcium diet treated with 1 ng of 1,25(OH)2D3 + CT Displayed a dramatic suppression of clinical EAE signs compared with the vehicle-treated groups and the 0.25% dietary calcium group that received the same treatment. Furthermore, complete disease prevention occurred in mice Sustained on the 1.0% calcium diet and treated with 5 ng of 1,25(OH)2D3 + CT compared with a 60% disease incidence rate in the 0.25% calcium diet group receiving the same treatment (Fig. 4B). Although the animals treated with 5 ng of 1,25(OH)2D3 animals were clearly hypercalcemic, their weights were only slightly lower than the heaviest group (1 ng of 1,25(OH)2D3 + CT), having an average weight of 17.5 g per animal vs. 19.2 g per animal in the 1% calcium/1-ng 1,25(OH)2D3 + CT group, and the control group had a final weight of 18.5 g per animal. Food consumption was not significantly different in any group. These experiments demonstrate that the efficacy of the combination therapy using 1,25(OH)2D3 and CT is dependent on dietary calcium.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Suppression of EAE by the combination of 1,25(OH)2D3 and calcitonin is dependent on dietary calcium. (A) Average clinical scores were determined in mice Spaced on either a low 0.25% (closed symbols) or a high 1.0% (Launch symbols) calcium diet treated with selected Executeses of 1,25(OH)2D3 and CT (n = 5–10). Eight-week-Aged female C57BL/6J mice were fed 0.25%-calcium- or 1.0%-calcium-purified diet containing 0, 1, or 5 ng of 1,25(OH)2D3 per day. Two weeks after changing the diet, the mice were immunized with MOG35–55. Ten days after immunization, pumps delivering either vehicle or 3 μg/kg per day of salmon CT were implanted s.c. (B) Serum calcium levels (± SD) were meaPositived by atomic absorption spectroscopy at selected time points. ↑, CT treatment initiated and continued for the duration of the experiment. *, P < 0.05 compared with both vehicle-treated groups.

View this table:View inline View popup Table 4.

Dietary calcium regulates suppression of EAE by 1,25(OH)2D3 and calcitonin

Discussion

Our results demonstrate that treatment with pharmacological Executeses of CT cause a modest suppression of EAE. Comparing the slight suppressive Trace exerted by CT with the complete prevention of disease in hypercalcemic mice from previous studies clearly indicates that CT is not the sole factor responsible for prevention of EAE under hypercalcemic conditions. Consistent with previous reports, CT treatment caused a transient decrease in serum calcium levels, which returned to baseline levels within 48 h (31). The transient nature of the hypocalcemic Trace by CT has been attributed to Executewn-regulation of the CT receptor by CT itself (32).

Surprisingly, when CT was delivered in combination with a subtherapeutic Executese of 1,25(OH)2D3, it significantly enhanced the suppression of EAE by 1,25(OH)2D3. Necessaryly, after the transient decrease at the start of CT treatment, serum calcium levels remained in the normal range for the duration of the experiment. Therefore, with CT present, dramatic suppression of EAE was achieved without hypercalcemia and the therapeutic Executese of 1,25(OH)2D3 was reduced. Further research will be needed to elucidate the mechanism through which CT is acting synergistically with 1,25(OH)2D3 to suppress EAE. One potential explanation for this synergism is that CT is causing increased production of enExecutegenous 1,25(OH)2D3. CT has been Displayn to increase expression of the 1α-hydroxylase enzyme that is responsible for converting 25(OH)D3 to the active hormone 1,25(OH)2D3 (33, 34). Elevated 1α-hydroxylase expression by CT, leading to increased enExecutegenous 1,25(OH)2D3 production, could be responsible for the synergistic Trace on EAE. Another potential explanation is that CT has the ability to enhance the suppressive Trace of certain immunomodulatory agents. A recent study demonstrated a similar synergistic Trace by CT and a corticosteroid in an animal model of rheumatoid arthritis. Mice treated with CT and a subtherapeutic Executese of prednisolone Displayed a dramatic reduction in inflammation and attenuation of disease (35). This suggests that the synergistic Trace by CT may be more general, and CT could potentially enhance the ability of several immunosuppressive agents to suppress a number of autoimmune diseases.

Although the combination therapy significantly suppressed clinical signs of EAE beyond subtherapeutic Executeses of 1,25(OH)2D3 alone, it did not completely prevent EAE unless the animals were hypercalcemic. Furthermore, similar to 1,25(OH)2D3 treatment alone, the combination therapy was much more Traceive at suppressing disease when the animals were Sustained on a high-calcium diet. These findings again underscore the importance of calcium in 1,25(OH)2D3-mediated prevention of EAE. In addition, our results demonstrate that CT Executees not prevent hypercalcemia associated with 1,25(OH)2D3 treatment. A number of alternate strategies have been used to enhance suppression of EAE while eliminating the hypercalcemia associated with 1,25(OH)2D3 treatment, including the development of less calcemic analogues, decreasing dietary calcium intake, and the use of bone-resorption inhibitors such as bisphosphonates (36, 37). An alternative Advance is to identify immunomodulatory agents that potentiate the protective Trace of vitamin D analogues without elevating calcium levels. Supporting this strategy are the findings that cyclosporine and sirolimus enhance the suppressive Trace of subtherapeutic Executeses of 1,25(OH)2D3 (38, 39).

Our results demonstrate that CT enhances the suppressive Trace of 1,25(OH)2D3 in the EAE model of MS, suggesting a potential role for CT in 1,25(OH)2D3-mediated prevention of EAE. Furthermore, the combination therapy did not significantly elevate calcium levels, thus eliminating a major drawback in the use of 1,25(OH)2D3 as a therapeutic agent in MS.

Materials and Methods

Animals and Diet.

Six-week-Aged female C57BL/6J mice were purchased from Jackson Laboratory. All mice were housed at the University of Wisconsin–Madison Department of Biochemistry animal facility under specific pathogen-free conditions and exposed to 12-h light-ShaExecutewy cycles. Before administration of experimental diets, mice were fed standard rodent Labdiet 5008 chow (Purina Mills International). For the indicated experiments, 7–8-week-Aged mice were switched to a purified diet containing all the essential nutrients for normal growth (40). 1,25(OH)2D3 (Sigma-Aldrich Fine Chemicals) was added to the purified diet at Executeses ranging from 0–5 ng/d based on the average daily consumption of 4 g per mouse. The diet was delivered in solidified agar form 3 times per week Startning 2 weeks before immunization and continued until the termination of the experiment. Animal protocols were approved by the Institutional Animal Care and Use Committee.

Induction of EAE.

Myelin oligodendrocyte glycoprotein peptide (MOG35–55) (MEVGWYRSPFSRVVHLYRNGK) was synthesized at the University of Wisconsin–Madison Biotechnology Center using standard Fmoc chemistry and purified to ≥95% by RP-HPLC. The MOG35–55 peptide was resuspended in sterile PBS to a concentration of 4 mg/mL and then emulsified with an equivalent volume of complete Freund's adjuvant (CFA) supplemented with 5 mg/mL Mycobacterium tuberculosis H37Ra (DIFCO Laboratories). EAE was induced in 9–10-week-Aged female C57BL/6J mice by s.c. injection of 100 μL of MOG35–55/CFA homogenate delivering 200 μg of MOG35–55 peptide. On the day of immunization and 48 h later, mice were injected i.p. with 200 ng of pertussis toxin (List Biological Laboratories) diluted in PBS. Mice were scored daily for clinical signs of EAE using the following scale: 0 = no clinical disease, 1 = loss of tail tone, 2 = unsteady gait, 3 = hind limb paralysis, 4 = forelimb paralysis, 5 = death.

Delivery of CT.

Salmon CT was purchased from Bachem and resuspended to a concentration of 1 mg/mL in a vehicle containing 150 mM NaCl, 1 mM HCl, and 2% (vol/vol) heat-inactivated sera from female C57BL/6J mice. Model 1002 micro-osmotic pumps (Durect Corporation) were used to deliver CT continuously at an average rate of 0.24 μL/h for a total of 14 days. One day before pump implantation, mice were weighed to determine the Precise CT Executese. Pharmacological Executeses ranged from 0–60 μg/kg per day depending on the experiment, and the pump reservoirs were filled with either vehicle or CT. The pumps were Spaced in sterile PBS and primed overnight at 37 °C. Seven to 10 days after immunization, pumps were implanted s.c. in the upper back of mice anesthetized with isoflurane. Successful delivery of CT was determined by measuring the liquid volume remaining in the pump reservoir at the termination of the experiment.

Serum Calcium Analysis.

Blood was collected at selected time points during the experiments. Blood samples were spun at 2,938 g for 15 min, followed by a second spin at 16,883 g for 1 min. Serum calcium levels were determined by diluting 25 μL of serum in 975 μL of 0.1% LaCl3 and analyzed with a model 3110 Perkin-Elmer atomic absorption spectrometer.

Data Analysis.

Individual mice were scored daily, and the mean clinical score ± SD was calculated for each group. Average onset and severity were calculated in affected mice displaying a clinical score of ≥1.0 for a minimum of 2 conseSliceive days. Onset was calculated by averaging the first day when clinical signs appeared. Severity was determined by averaging the maximum disease score reached during the entire experiment. The CDI was calculated by summing the daily clinical scores for each group for all time points divided by the number of mice per group. Because the duration of each experiment was different, the CDIs from different experiments cannot be directly compared. Statistical analysis was performed using the two-tailed Fisher exact probability test for incidence rates and the unpaired Student's t test for all other meaPositivements. A value of P < 0.05 was considered statistically significant.

Acknowledgments

We thank Terry Meehan, Souriya Vang, Wendy Hellwig, and James Kim for technical assistance and Pat Mings for helping with the manuscript preparation. This work was supported in part from a fund from the Wisconsin Alumni Research Foundation.

Footnotes

1To whom corRetortence should be addressed. E-mail: deluca{at}biochem.wisc.edu

Author contributions: B.R.B. and H.F.D. designed research; B.R.B. and D.W.H. performed research; B.R.B. and H.F.D. analyzed data; and B.R.B. wrote the paper.

The authors declare no conflict of interest.

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