Limits...
Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

Wang M, Mukherjee PK, Chandra J, Lattif AA, McCormick TS, Ghannoum MA - BMC Microbiol. (2008)

Bottom Line: The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml.CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio, USA. mingyuewang@gmail.com

ABSTRACT

Background: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.

Results: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.

Conclusion: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

Show MeSH
Glycoprotein fraction of CA-SIIF demonstrated higher inhibitory effect on human monocytes IL-12 production. CA-SIIF was passed through a ConA lectin affinity column. Both resulting fractions and CA-SIIF crude were tested for the effect on IL-12 production of 1 × 106/ml human monocytes. (A) glycoprotein fraction (CA-SIIF-GP) successfully achieved higher inhibitory effect than the non-glycoprotein (CA-SIIF-NGP) fraction (P < 0.01). (B) Inhibition efficiency of CA-SIIF-GP fraction is significantly higher compared to CA-SIIF crude and CA-SIIF-NGP (P < 0.01). Fold decrease was calculated by decrease of the IL-12 level by percentage; inhibition efficiency was determined by evaluating the fold decrease of IL-12 level/protein dose ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2289826&req=5

Figure 6: Glycoprotein fraction of CA-SIIF demonstrated higher inhibitory effect on human monocytes IL-12 production. CA-SIIF was passed through a ConA lectin affinity column. Both resulting fractions and CA-SIIF crude were tested for the effect on IL-12 production of 1 × 106/ml human monocytes. (A) glycoprotein fraction (CA-SIIF-GP) successfully achieved higher inhibitory effect than the non-glycoprotein (CA-SIIF-NGP) fraction (P < 0.01). (B) Inhibition efficiency of CA-SIIF-GP fraction is significantly higher compared to CA-SIIF crude and CA-SIIF-NGP (P < 0.01). Fold decrease was calculated by decrease of the IL-12 level by percentage; inhibition efficiency was determined by evaluating the fold decrease of IL-12 level/protein dose ratio.

Mentions: In the previous study, we showed that CA-SIIF contains a carbohydrate component [8]. In this study, we determined whether CA-SIIF is a glycoprotein using affinity chromatography based on binding to ConA (a lectin that binds specifically mannosyl/glucosyl residues) [14]. We separated ConA-binding glycoprotein fraction (CA-SIIF-GP) and non-ConA-binding non-glycoprotein fraction (CA-SIIF-NGP) from CA-SIIF, and determined the effect of these fractions on IL-12 production by activated monocytes. Our results showed that activated monocytes grown in presence of CA-SIIF-GP produced significantly less IL-12 than those grown in its absence (IL-12 level = 446 ± 29 pg/ml vs. 705 ± 31 pg/ml, respectively, P < 0.01). In contrast, addition of CA-SIIF-NGP was unable to induce a similar reduction in monocytic IL-12 levels (IL-12 level = 641 ± 22 pg/ml vs. 705 ± 31 pg/ml; P < 0.01, Figure 6A). To further demonstrate the enhanced effect of CA-SIIF after purification, inhibition efficiencies were calculated based on the fold decrease of monocyte IL-12 level (per mg protein). As can be seen in Figure 6B, CA-SIIF-GP exhibited a significantly higher IL-12 inhibition efficiency (fold decrease = 8.2 ± 0.9) compared to crude CA-SIIF supernatant or CA-SIIF-NGP (fold decrease = 3.2 ± 0.2 or 2.0 ± 0.7 respectively; P < 0.01). These studies demonstrated that partial purification of CA-SIIF based on glycoprotein properties increased CA-SIIF activity and suggested that this inhibitory activity of CA-SIIF is mediated by its glycoprotein fraction. Preliminary SDS-PAGE analysis suggested that CA-SIIF protein has a molecular weight of around 70 kDa (data not shown). Further purification and identification of this CA-SIIF glycoprotein is currently underway in our group.


Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

Wang M, Mukherjee PK, Chandra J, Lattif AA, McCormick TS, Ghannoum MA - BMC Microbiol. (2008)

Glycoprotein fraction of CA-SIIF demonstrated higher inhibitory effect on human monocytes IL-12 production. CA-SIIF was passed through a ConA lectin affinity column. Both resulting fractions and CA-SIIF crude were tested for the effect on IL-12 production of 1 × 106/ml human monocytes. (A) glycoprotein fraction (CA-SIIF-GP) successfully achieved higher inhibitory effect than the non-glycoprotein (CA-SIIF-NGP) fraction (P < 0.01). (B) Inhibition efficiency of CA-SIIF-GP fraction is significantly higher compared to CA-SIIF crude and CA-SIIF-NGP (P < 0.01). Fold decrease was calculated by decrease of the IL-12 level by percentage; inhibition efficiency was determined by evaluating the fold decrease of IL-12 level/protein dose ratio.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2289826&req=5

Figure 6: Glycoprotein fraction of CA-SIIF demonstrated higher inhibitory effect on human monocytes IL-12 production. CA-SIIF was passed through a ConA lectin affinity column. Both resulting fractions and CA-SIIF crude were tested for the effect on IL-12 production of 1 × 106/ml human monocytes. (A) glycoprotein fraction (CA-SIIF-GP) successfully achieved higher inhibitory effect than the non-glycoprotein (CA-SIIF-NGP) fraction (P < 0.01). (B) Inhibition efficiency of CA-SIIF-GP fraction is significantly higher compared to CA-SIIF crude and CA-SIIF-NGP (P < 0.01). Fold decrease was calculated by decrease of the IL-12 level by percentage; inhibition efficiency was determined by evaluating the fold decrease of IL-12 level/protein dose ratio.
Mentions: In the previous study, we showed that CA-SIIF contains a carbohydrate component [8]. In this study, we determined whether CA-SIIF is a glycoprotein using affinity chromatography based on binding to ConA (a lectin that binds specifically mannosyl/glucosyl residues) [14]. We separated ConA-binding glycoprotein fraction (CA-SIIF-GP) and non-ConA-binding non-glycoprotein fraction (CA-SIIF-NGP) from CA-SIIF, and determined the effect of these fractions on IL-12 production by activated monocytes. Our results showed that activated monocytes grown in presence of CA-SIIF-GP produced significantly less IL-12 than those grown in its absence (IL-12 level = 446 ± 29 pg/ml vs. 705 ± 31 pg/ml, respectively, P < 0.01). In contrast, addition of CA-SIIF-NGP was unable to induce a similar reduction in monocytic IL-12 levels (IL-12 level = 641 ± 22 pg/ml vs. 705 ± 31 pg/ml; P < 0.01, Figure 6A). To further demonstrate the enhanced effect of CA-SIIF after purification, inhibition efficiencies were calculated based on the fold decrease of monocyte IL-12 level (per mg protein). As can be seen in Figure 6B, CA-SIIF-GP exhibited a significantly higher IL-12 inhibition efficiency (fold decrease = 8.2 ± 0.9) compared to crude CA-SIIF supernatant or CA-SIIF-NGP (fold decrease = 3.2 ± 0.2 or 2.0 ± 0.7 respectively; P < 0.01). These studies demonstrated that partial purification of CA-SIIF based on glycoprotein properties increased CA-SIIF activity and suggested that this inhibitory activity of CA-SIIF is mediated by its glycoprotein fraction. Preliminary SDS-PAGE analysis suggested that CA-SIIF protein has a molecular weight of around 70 kDa (data not shown). Further purification and identification of this CA-SIIF glycoprotein is currently underway in our group.

Bottom Line: The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml.CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Medical Mycology, Department of Dermatology, University Hospitals of Cleveland and Case Western Reserve University, Cleveland, Ohio, USA. mingyuewang@gmail.com

ABSTRACT

Background: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed.

Results: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature.

Conclusion: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

Show MeSH