Immune Netw.  2011 Oct;11(5):281-287. 10.4110/in.2011.11.5.281.

Nanoliposomes of L-lysine-conjugated poly(aspartic acid) Increase the Generation and Function of Bone Marrow-derived Dendritic Cells

• Im SA
• Kim KH
• Ji HG
• Yu HG
• Park SK
• Lee CK

• KMID: 2168000
• DOI: http://doi.org/10.4110/in.2011.11.5.281

Abstract

BACKGROUND
Biodegradable polymers have increasingly been recognized for various biological applications in recent years. Here we examined the immunostimulatory activities of the novel poly(aspartic acid) conjugated with L-lysine (PLA).
METHODS
PLA was synthesized by conjugating L-lysine to aspartic acid polymer. PLA-nanoliposomes (PLA-NLs) were prepared from PLA using a microfluidizer. The immunostimulatory activities of PLA-NLs were examined in mouse bone marrow-derived dendritic cells (BM-DCs).
RESULTS
PLA-NLs increased the number of BM-DCs when added to cultures of GM-CSF-induced DC generation on day 4 after the initiation of cultures. Examination of the phenotypic properties showed that BM-DCs generated in the presence of PLA-NLs are more mature in terms of the expression of MHC class II molecules and major co-stimulatory molecules than BM-DCs generated in the absence of PLA-NLs. In addition, the BM-DCs exhibited enhanced capability to produce cytokines, such as IL-6, IL-12, TNF-alpha and IL-1beta. Allogeneic mixed lymphocyte reactions also confirmed that the BMDCs were more stimulatory on allogeneic T cells. PLA- NL also induced further growth of immature BM-DCs that were harvested on day 8.
CONCLUSION
These results show that PLA-NLs induce the generation and functional activities of BM-DCs, and suggest that PLA-NLs could be immunostimulating agents that target DCs.

Keyword

Nanoliposome; Poly(aspartic acid); L-Lysine; Dendritic cell; Immunomodulation

MeSH Terms

Animals
Aspartic Acid
Cytokines
Dendritic Cells
Immunomodulation
Interleukin-12
Interleukin-6
Lymphocyte Culture Test, Mixed
Lysine
Mice
Polymers
T-Lymphocytes
Tumor Necrosis Factor-alpha
Aspartic Acid
Cytokines
Interleukin-12
Interleukin-6
Lysine
Polymers
Tumor Necrosis Factor-alpha

Figure

• 

  Figure 1 PLA-NLs increase the generation of BM-DCs. Mouse BM-DCs were generated from C57BL/6 mouse BM cells. BM cells were cultured with the indicated concentration of PLA-NLs and GM-CSF (40 ng/ml). PLA-NLs were added to the culture on day 4 after the initiation of the BM cell culture, and the resultant BM-DCs were harvested on day 8 and counted using a hemocytometer. Results are mean±SD. *p<0.05 compared with untreated control.

• 

  Figure 2 PLA-NLs induce phenotypic maturation of BM-DCs. PLA-NLs (0.1 mg/ml) were added to BM cell culture together with GM- CSF (40 ng/ml) on day 4 after the initiation of cultures, and the resultant BM-DCs were harvested on day 8, washed, and then used for immunophenotypic analysis. Levels of expression (thin line) in BM-DCs generated in the presence of GM-CSF and PLA-NLs are illustrated in comparison with those in BM-DCs generated in the presence of GM-CSF only (shaded line).

• 

  Figure 3 DCs generated in the presence of PLA-NLs exert increased capability to produce cytokines. PLA-NLs (0.1 mg/ml) were added to BM cell cultures together with GM-CSF (40 ng/ml) on day 4 after the initiation of cultures, and the resultant BM-DCs were harvested on day 8 and then cultured with OVA-containing nanoparticles (50 µg/ml as OVA). After 24 h, cytokines were measured by ELISA. Results are mean±SD. *p<0.05, **p<0.01 compared with untreated control.

• 

  Figure 4 DCs generated in the presence of PLA-NLs exert increased allostimulatory capacity. PLA-NLs (0.1 mg/ml) were added to BM cell cultures together with GM-CSF (40 ng/ml) on day 4 after the initiation of cultures, and the resultant BM-DCs were harvested on day 8. All BM-DCs were treated with mitomycin C, washed, and then cultured with allogeneic mouse lymphocytes. After 3 days, cell growth was measured by [3H]-thymidine incorporation. Results are mean±SD. *p<0.01 compared with untreated control.

• 

  Figure 5 PLA-NLs induce the proliferation of GM-CSF-generated BM-DCs. The DCs generated in the presence of 40 ng/ml of GM-CSF only were harvested on day 8, and then cultured with the indicated concentration of PLA-NLs. After 2 days, the growth of BM-DCs was measured by [3H]-thymidine incorporation. Results are mean±SD. *p<0.05, **p<0.01 compared with untreated control.

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