doi:10.1016/j.molimm.2005.07.023 
Copyright © 2005 Elsevier Ltd All rights reserved.
Selective inhibition of the C5a chemotactic cofactor function of the Vitamin D binding protein by 1,25(OH)2 Vitamin D3
Anisha B. Shah, Stephen J. DiMartino, Glenda Trujillo and Richard R. Kew
, 
Department of Pathology, Health Sciences Center, Stony Brook University School of Medicine, Stony Brook, NY 11794-8691, USA
Received 2 June 2005.
Available online 22 August 2005.
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Abstract
The Vitamin D binding protein (DBP) is a multifunctional plasma protein that can significantly enhance the chemotactic response to complement fragment C5a. The chemotactic cofactor function of DBP requires cell surface binding in order to mediate this process. The goal of this study was to investigate the effect of ligating DBP with its two primary physiological ligands, Vitamin D and G-actin, on both binding to neutrophils and the ability to enhance chemotaxis to C5a. There was no difference in neutrophil binding between of the holo (bound) forms versus the apo (unbound) form of radioiodinated DBP, indicating that the cell binding region of DBP is likely distinct from the Vitamin D sterol and G-actin binding sites. Likewise, G-actin, 25(OH)D3, and G-actin plus 25(OH)D3 bound to DBP did not alter its capacity to enhance chemotaxis toward C5a. However, the active form of Vitamin D (1,25(OH)2D3) completely eliminated the chemotactic cofactor function of DBP. Dose–response curves demonstrated that as little as 1 pM 1,25(OH)2D3 significantly inhibited chemotaxis enhancement. Moreover, at physiological concentrations 1,25(OH)2D3 needs to be bound to DBP to mediate the inhibitory effect. Neutrophil chemotaxis to optimal concentrations of C5a, formyl peptide, CXCL8 or leukotriene B4 was not altered by 1,25(OH)2D3, indicating that the active vitamin does not have a global inhibitory effect on neutrophil chemotaxis. Finally, inhibition of cell surface alkaline phosphatase (AP) with sodium orthovanadate completely reversed the inhibitory effect of 1,25(OH)2D3. These results indicate that the cell binding and co-chemotactic functions of DBP are not altered when the protein binds G-actin and/or Vitamin D. Furthermore, the co-chemotactic signal from DBP can be eliminated or counteracted by 1,25(OH)2D3.
Keywords: Complement; Chemotaxis; Neutrophils; Inflammation; Vitamin D
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Fig. 1. Effect of ligands on the binding of radioiodinated DBP to neutrophils and on the C5a co-chemotactic activity. (A) Purified radioiodinated DBP (100 nM) was treated for 5 min at 37 °C with and equimolar concentration of either G-actin, 25(OH)D3, 1,25(OH)2D3, or G-actin plus 25(OH)D3. Purified neutrophils (107) were incubated in HBSS containing 0.1% BSA with the 125I-DBP samples in a total volume of 0.1 ml. Samples were incubated at 37 °C for 60 min after which cells were placed on ice and then washed twice with ice-cold assay buffer (1.0 ml each) and centrifuged for 7 min at 200 × g at 2 °C. The cell pellets were then counted for total radioactivity in a gamma counter; data are presented as fmols radioiodinated DBP bound per 106 neutrophils (n = 3–4). (B) Purified DBP (100 nM) was treated for 5 min at 37 °C with and equimolar concentration of either G-actin, 25(OH)D3, 1,25(OH)2D3, or G-actin plus 25(OH)D3. Neutrophils (4 × 106/ml) were then preincubated for 40 min at 37 °C with either DBP alone or DBP plus ligands in HBSS plus 1% BSA. Neutrophil movement then was assayed to 10 pM C5a or buffer for 30 min at 37 °C. Numbers represent mean ± S.E.M. of three experiments using cells from different donors. DBP plus C5a samples were significantly greater than the corresponding C5a alone in all cases except as indicated by the asterisk where that sample is significantly (p < 0.001) less than all other C5a plus DBP samples.
Fig. 2. Dose–response curves of DBP bound to 25(OH)D3 or 1,25(OH)2D3. Purified DBP (100 nM) was pretreated with the indicated concentration of Vitamin D for 5 min at 37 °C. Neutrophils (4 × 106/ml) were then preincubated for 40 min at 37 °C with either DBP alone or DBP plus Vitamin D in HBSS plus 1% BSA. Neutrophil movement then was assayed to 10 pM C5a or buffer for 30 min at 37 °C. Numbers represent mean ± S.E.M. of three experiments using cells from different donors. Neutrophils treated with DBP bound to 1,25(OH)2D3 (10−6 to 10−12 M) migrated significantly less (p < 0.001) to C5a than the corresponding samples containing 25(OH)D3.
Fig. 3. Effect of bound vs. free 1,25(OH)2D3 on C5a co-chemotaxis by DBP. Purified DBP (1 nM) was either treated for 5 min at 37 °C with buffer (control), 100 pM 1,25(OH)2D3, 1 nM 25(OH)D3, or 1 nM 25(OH)D3 for 5 min followed by 100 pM 1,25(OH)2D3. Neutrophils (4 × 106/ml) were then preincubated for 40 min at 37 °C with either DBP alone or DBP plus Vitamin D in HBSS plus 1% BSA. Neutrophil movement then was assayed to 10 pM C5a or buffer for 30 min at 37 °C. Numbers represent mean ± S.E.M. of three experiments using cells from different donors. Asterisk indicates that the sample is significantly (p < 0.001) less than all others.
Fig. 4. Effect of 1,25(OH)2D3 on chemotaxis to C5a, LTB4, formyl peptide and CXCL8. Purified DBP (100 nM) was treated for 5 min at 37 °C with 10 nM 1,25(OH)2D3. Neutrophils (4 × 106/ml) were then preincubated for 40 min at 37 °C with either DBP plus 1,25(OH)2D3 or buffer. Neutrophil movement then was assayed for 30 min at 37 °C. Numbers represent mean ± S.E.M. of three experiments using cells from different donors.
Fig. 5. Effect of sodium orthovanadate on the inhibitory action of 1,25(OH)2D3. Purified DBP (100 nM) was treated for 5 min at 37 °C with 10 nM 1,25(OH)2D3. Neutrophils (4 × 106/ml) were first pretreated for 15 min at 37 °C with either 10 μM sodium orthovanadate (SOV) or buffer. Cells were then incubated for an additional 30 min at 37 °C with either DBP plus 1,25(OH)2D3 or DBP alone (control). Neutrophil movement to 10 pM C5a was assayed for 30 min at 37 °C. Numbers represent mean ± S.E.M. of three experiments using cells from different donors. Asterisk indicates that the sample is significantly (p < 0.001) less than all others.
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