Human C-reactive protein accentuates macrophage activity in biobreeding diabetic rats

Abstract

Objective: Type 1 diabetes (T1DM) is a pro-inflammatory state characterized by high C-reactive protein (CRP) levels. However, there is a paucity of data examining the role of CRP in promoting the pro-inflammatory state of diabetes. Thus, we examined the pro-inflammatory effects of human CRP using spontaneously diabetic bio-breeding (BB) rats.

Methods: Diabetic rats (n=9/group) were injected with Human serum albumin (huSA) or Human CRP (hCRP, 20 mg/kg body weight; i.p.) for 3 consecutive days. Blood and peritoneal macrophages (MØ) were obtained following euthanasia. Peritoneal macrophages were used for measuring superoxide anion release, NF-κB DNA binding activity, proinflammatory mediator secretion.

Results: hCRP administration resulted in significantly increased superoxide anion production, along with increased release of cytokines/chemokines, and plasminogen activator inhibitor (PAI-1) and Tissue Factor (TF) activity in diabetic rats compared to huSA. hCRP-treated BB rat MØ showed significant induction of protein kinase C (PKC)-alpha, PKC-delta and p47 phox expression and NF-κB compared to huSA.

Conclusions: Thus, our data suggest that human CRP exacerbates in-vivo the pro-inflammatory, pro-oxidant and procoagulant states of diabetes predominantly via increased macrophage activity and this could have implications with respect to vascular complications and anti-inflammatory therapies.

Fig 1a

Circulating levels of pro-inflammatory cytokines and chemokines in sera following hCRP or huSA administration to diabetic BB rats (20mg/kg body weight for 3 days, n=9/group) were examined by multiplex assays as described in Methods. Values are expressed as ng/ml (mean ± SD). *P<0.005 vs. huSA. huSA=human serum albumin; hCRP=human C-reactive protein

Fig 1b

Pro-inflammatory cytokines and chemokines release by peritoneal macrophages of hCRP or huSA administration to BB rats (n=9/group) were measured by Multiplex assay as described in Methods. Values are expressed as ng/mg cell protein (mean ± SD). *P<0.005 vs. huSA. huSA=human serum albumin; hCRP=human C-reactive protein

Fig 2a

Superoxide anion release in hCRP or huSA (20 mg/kg b.w. for 3 days, n=9/group) administered by diabetic BB rat macrophages. Superoxide anion release was assessed by DHE fluorescence. Results are expressed as mean ± SD mean fluorescence intensity (MFI) *P< 0.005 vs. huSA. huSA=human serum albumin; hCRP=human C-reactive protein.

Fig 2b

Tissue factor activity in hCRP or huSA (20 mg/kg b.w. for 3 days, n=9/group) administered by the BB rat macrophages. The rats were sacrificed on the 4^th day and peritoneal macrophages were isolated and tissue factor was assessed as described in methods. Results are expressed as mean ± SD pg/mg cell protein. *P<0.005 vs. huSA. huSA=human serum albumin; hCRP=human C-reactive protein

Fig 2c

Plasminogen activator inhibitor-1 (PAI-1) antigen in hCRP or huSA (20 mg/kg b.w. for 3 days, n=9/group) administered diabetic BB rat macrophages. The rats were sacrificed on the 4^th day and peritoneal macrophages were isolated and PAI-1 levels were determined as described in methods. Results are expressed as mean ± SD pg/mg cell protein. *P<0.005 vs. huSA. huSA=human serum albumin; hCRP=human C-reactive protein.

Fig 3a

NF-κB DNA binding activity in hCRP or huSA (20mg/kg b.w. for 3 days, n=9/group) administered diabetic BB rat macrophages. The rats were sacrificed on the 4^th day and peritoneal macrophages were isolated and NF-κB DNA binding activity was assessed as described in methods. Results are expressed as mean ± SD ng/mg cell protein. *P<0.05 vs. huSA. huSA=human serum albumin; hCRP= human C-reactive protein; NF-κB=Nuclear factor-κB.

Fig 3b

Representative Western blot depicting PKC isoforms, and p47phox protein expression in peritoneal macrophage cell lysates of hCRP or huSA (20mg/kg b.w. for 3 days, n=9/group) administered diabetic BB rats. Beta-actin was used as internal loading control.