Vitamin D3 Suppresses Class II Invariant Chain Peptide Expression on Activated B-Lymphocytes: A Plausible Mechanism for Downregulation of Acute Inflammatory Conditions

Abstract

Class II invariant chain peptide (CLIP) expression has been demonstrated to play a pivotal role in the regulation of B cell function after nonspecific polyclonal expansion. Several studies have shown vitamin D3 helps regulate the immune response. We hypothesized that activated vitamin D3 suppresses CLIP expression on activated B-cells after nonspecific activation or priming of C57BL/6 mice with CpG. This study showed activated vitamin D3 actively reduced CLIP expression and decreased the number of CLIP(+) B-lymphocytes in a dose and formulation dependent fashion. Flow cytometry was used to analyze changes in mean fluorescent intensity (MFI) based on changes in concentration of CLIP on activated B-lymphocytes after treatment with the various formulations of vitamin D3. The human formulation of activated vitamin D (calcitriol) had the most dramatic reduction in CLIP density at an MFI of 257.3 [baseline of 701.1 (P value = 0.01)]. Cholecalciferol and alfacalcidiol had no significant reduction in MFI at 667.7 and 743.0, respectively. Calcitriol seemed to best reduce CLIP overexpression in this ex vivo model. Bioactive vitamin D3 may be an effective compliment to other B cell suppression therapeutics to augment downregulation of nonspecific inflammation associated with many autoimmune disorders. Further study is necessary to confirm these findings.

Figure 1

This bar graph shows the effects of various vitamin D3 compounds at a low concentration of 0.1 ng/mL on the mean fluorescent intensity (MFI) of CLIP⁺ B cells after TLR-engagement. This figure demonstrates the changes in MFI of CLIP and CLIP⁺ B220 subsets of B cells after CpG-induced TLR-9 activation. These results suggest that the active formulation of vitamin D3 may lead to a statistically significant reduction in CLIP expression on polyclonally activated splenic B cells. Administration of the human calcitriol of vitamin D3 (P = 0.01) resulted in the greatest overall reduction in the MFI (257 versus a baseline of 701.1) for CLIP expression on stimulated B cells.

Figure 2

Bar graph showing the effects of 1 ng/mL of various vitamin D3 compounds on the distribution of TLR-activated CLIP⁺ and B220 subsets of B cells. This figure demonstrates the changes in MFI of CLIP and CLIP⁺ B220 subsets of B cells 15 hrs after administration of different compounds of vitamin D3 versus no treatment or DMSO (control). These results suggest that the active formulation of vitamin D3 may lead to a statistically significant reduction in CLIP expression on polyclonally activated splenic B cells. Administration of the human calcitriol of vitamin D3 (P = 0.048) resulted in the greatest overall reduction in the MFI (482.3 versus a baseline of 701.1) for CLIP expression on stimulated B cells. Administration of the active form of vitamin D3 again resulted in statistically significant reduction in CLIP expression on CpG-activated B cells.

Figure 3

This bar graph also shows the effects of different vitamin D3 compounds at a concentration of 10 ng/mL on the MFI of CLIP expression and CLIP⁺ B220 subsets of B cells after TLR-activation. This graph compares the changes in MFI of CLIP and CLIP⁺ B220 subsets of B cells 15 hrs after administration of different compounds of vitamin D3 versus no treatment or DMSO (control). These results suggest that exogenously produced bioactive 1,25(OH)²D3 may be needed in higher doses of activated vitamin D3 in order to reduce the level of CLIP expression on polyclonally activated B cells (P = 0.055). Administration of human calcitriol again resulted in most significant reduction in CLIP expression on polyclonally activated B-lymphocytes.

Figure 4

This figure compares the impact of the various vitamin D compounds at 0.1 mg/dL on the density of cell surface CLIP expression and B220 subsets of B cells 15 hrs after treatment using the vitamin D3 compounds versus nontreatment or DMSO. These results suggest that the active formulation of vitamin D3 once again leads to a statistically significant reduction in CLIP expression on polyclonally activated splenic B cells even at a higher concentration of 10 mg/dL. Administration of the human calcitriol of vitamin D3 (P = 0.0181) resulted in the greatest overall reduction in the MFI (257.3 versus a baseline of 701.1 and 667.7 for the control) of CLIP expression on mitogen stimulated B cells.

Figure 5

This figure compares the impact of the various vitamin D compounds at 1.0 mg/dL on the density of cell surface CLIP expression and B220 subsets of B cells 15 hrs after treatment using the vitamin D3 compounds versus nontreatment or DMSO. These results suggest that the active formulation of vitamin D3 once again leads to a statistically significant reduction in CLIP expression on polyclonally activated splenic B cells even at a higher concentration of 10 mg/dL. Administration of the human calcitriol of vitamin D3 (P = 0.0181) resulted in the greatest overall reduction in the MFI (482.3 versus a baseline of 701.1 and 667.7 for the control) of CLIP expression on mitogen stimulated B cells.

Figure 6

This linear graph demonstrates the impact of the various vitamin D compounds at 10.0 mg/dL on the density of cell surface CLIP expression and B220 subsets of B cells 15 hrs after treatment using the vitamin D3 compounds versus nontreatment or DMSO. These results suggest that the active formulation of vitamin D3 once again leads to a statistically significant reduction in CLIP expression on polyclonally activated splenic B cells even at a higher concentration of 10 mg/dL. Administration of the human calcitriol of vitamin D3 (P = 0.0181) resulted in the greatest overall reduction in the MFI (443.3) versus a baseline of 701.1 for no treatment and 667.7 for the control of CLIP expression on mitogen stimulated B cells.