Antisense to protein kinase C-alpha and p47phox attenuates the pro-inflammatory effects of human C-reactive protein in macrophages of biobreeding diabetic rats

Abstract

Objective: Type 1 diabetes mellitus (T1DM) is a pro-inflammatory state characterized by high C-reactive protein (CRP) levels. Previously, we showed that CRP accentuated a macrophage (MO) activity in spontaneously diabetic biobreeding (BB) rats and increased the MO activity of protein kinase C-alpha (PKC-α) and p47phox. In this report, we tested the effects of molecular inhibition of CRP effects on MO activity using antisense oligonucleotide (ASO) to both PKC-α and p47phox.

Methods: Prior to administration of human C-reactive protein (hCRP) daily for 3 days, ASO or scrambled ASO to either PKC-α or p47phox was also delivered for 3 days and after killing on day 4, peritoneal MOs were isolated.

Results: The increase in the levels of superoxide anion, interleukin (IL)-1, monocyte chemoattractant protein-1 (MCP-1), tumour necrosis factor-alpha (TNF-α) and IL-6 release in MOs with hCRP compared to human albumin was significantly attenuated by antisense to either PKC-α and p47phox (p < 0.01 vs. scrambled ASO; n = 5 per group).

Conclusion: Our novel data suggest that antisense to either PKC-α or p47phox attenuates the pro-inflammatory effects of human CRP on MOs in diabetic rats.

Figure 1

Effect of PKC-α and p47phox ASO on (a) PKC and (b) p47phox expression in macrophages of BB rats: BB rats were treated with hCRP or huSA or hCRP + ASO to PKC-α or p47phox as described in section ‘Materials and methods’ (n = 5 per group). Scrambled ASOs were used as control. Representative Western blot of PKC-α and membrane p47phox from isolated macrophages are shown. *p < 0.05 compared to huSA and #p < 0.05 compared to hCRP + scrambled ASO. PKC-α: protein kinase C-alpha; ASO: antisense oligonucleotide; BB: biobreeding; hCRP: human C-reactive protein; huSA: human serum albumin.

Figure 2

Effect of PKC-α and p47phox ASO inhibition on superoxide anion and IL-1β, MCP-1, TNF-α and IL-6 release from macrophages of BB rats treated with hCRP or huSA (20 mg/kg body weight for 3 days, n = 5 per group). Superoxide anion release was assessed by DHE fluorescence. Scrambled ASOs were used as control. The results are expressed as mean ± SD MFI for superoxide and nanogram per milligram of cell protein for cytokines and MCP-1. *p < 0.01 versus huSA. #p < 0.01 versus hCRP + scrambled ASO. PKC-α: protein kinase C-alpha; ASO: antisense oligonucleotide; IL-1β: interleukin-1 beta; MCP-1: monocyte chemoattractant protein-1; TNF-α: tumour necrosis factor-alpha; IL-6: interleukin-6; BB: biobreeding; hCRP: human C-reactive protein; huSA: human serum albumin; DHE: dihydroethidium; SD: standard deviation; MFI: mean fluorescence intensity.