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Harvesting Murine Alveolar Macrophages and Evaluating Cellular Activation Induced by Polyanhydride Nanoparticles
Published on: June 8, 2012
The activation of murine macrophages and natural killer cells by the partially thiolated double stranded RNA
P F Cavanaugh1, Y K Ho, T J Bardos
1Department of Biochemical Pharmacology, School of Pharmacy, State University of New York at Buffalo, 14260, USA.
Abstract:
Partially thiolated analogs of the biological response modifier poly I.poly C (pI.pC) were synthesized. Each of these analogs (pI.MPC) contained a partially thiolated polycytidylate (MPC) strand containing either 1.2%, 4.6%, or 17% 5-mercaptocytosine (%SH) randomly introduced throughout the polynucleotide. The ability of these double stranded RNAs (dsRNAs) to activate murine peritoneal macrophages in vitro and augment natural killer (NK) cell activity in mice following intraperitoneal (i.p.) administration was determined. Macrophages were treated in vitro for 24 hours with pI.pC, or pI.MPC, washed, and then overlayed with exponentially growing L1210 leukemia cells at an effector to target (E:T) ratio of 25:1. The cytostatic effect of the macrophages on the L1210 cells was determined by 3H.thymidine pulse labelling. The rank order of potency for macrophage activation was determined to be: pI.pC>pI.MPC(1.2% SH)>pI.MPC(4.6% SH)pI.MPC(17% SH). Twenty hours following i.p. administration of 5 mg/kg of each pI.MPC analog, splenic NK cell activity was assessed in a standard 51Cr release assay using the murine tumor target cell line YAC- 1. The rank order of potency observed for NK cell activation was determined to be; pI.pCpI MPC(1.2% SH)>pI.MPC(4.6% SH)>pI.MPC(17% SH). These dsRNAs activated NK cells in a dose dependent manner. The efficacy and time course for NK cell activation following i.p. administration of pI.MPC (1.2% SH) at a dose of 10 mg/kg was directly compared to an equivalent 10 mg/kg i.p. dose of pI.pC. NK cell activation took place within three hours following treatment with pI-pC whereas the onset of NK cell activation by pI.MPC (1.2%) occurred between 8 and 20 hours post treatment. NK cell activity steadily declined from 24 to 50 hours post treatment at which time the NK activity in both treatment groups was similar. There was a significant correlation between the immunostimulatory potency of these dsRNAs and their experimentally determined melting temperatures (r2 = 0.88) and percent hyperchromicity upon thermal denaturation (r2 = 0.99). At the lower %SH, pI.MPC retains most of the immunostimulatory activities of p1.pC and may serve as a useful and potent biological response modifier.
Insights
Partially thiolated analogs of poly I.poly C (pI.pC) retain significant biological response modifier activity. These novel dsRNA compounds show potential for immune system modulation and cancer therapy applications.
Area of Science:
- Immunology
- Biochemistry
- Molecular Biology
Background:
- Poly I.poly C (pI.pC) is a biological response modifier known to activate immune cells.
- Developing novel analogs of pI.pC with modified properties is crucial for enhancing therapeutic potential.
- Understanding the structure-activity relationship of these analogs is key to optimizing their immunostimulatory effects.
Purpose of the Study:
- To synthesize and characterize partially thiolated analogs of pI.pC (pI.MPC).
- To evaluate the ability of these dsRNA analogs to activate murine peritoneal macrophages in vitro.
- To assess the in vivo augmentation of natural killer (NK) cell activity in mice.
Main Methods:
- Synthesis of pI.MPC analogs with varying percentages (1.2%, 4.6%, 17%) of 5-mercaptocytosine (%SH).
- In vitro macrophage activation assay using L1210 leukemia cells and 3H-thymidine pulse labeling.
- In vivo NK cell activity assessment via 51Cr release assay in mice treated intraperitoneally.
Main Results:
- Macrophage activation potency followed the order: pI.pC > pI.MPC (1.2% SH) > pI.MPC (4.6% SH) > pI.MPC (17% SH).
- NK cell activation potency followed the order: pI.pC > pI.MPC (1.2% SH) > pI.MPC (4.6% SH) > pI.MPC (17% SH).
- A strong correlation was observed between immunostimulatory potency and thermal denaturation properties (melting temperature and hyperchromicity).
Conclusions:
- Partially thiolated analogs (pI.MPC) retain significant immunostimulatory activities of pI.pC, especially at lower %SH.
- These dsRNA analogs demonstrate potential as potent biological response modifiers.
- The findings suggest pI.MPC analogs could be valuable in immunotherapy and as adjuncts in cancer treatment.
