Macrophage migration inhibitory factor deficiency protects pancreatic islets from cytokine-induced apoptosis in vitro

Abstract

During pathogenesis of diabetes, pancreatic islets are exposed to high levels of cytokines and other inflammatory mediators that induce deterioration of insulin-producing beta cells. Macrophage migration inhibitory factor (MIF) plays a key role in the onset and development of several immunoinflammatory diseases and also controls apoptotic cell death. Because the occurrence of apoptosis plays a pathogenetic role in beta cell death during type 1 diabetes development and MIF is expressed in beta cells, we explored the influence of MIF deficiency on cytokine-induced apoptosis in pancreatic islets. The results indicated clearly that elevated MIF secretion preceded C57BL/6 pancreatic islets death induced by interferon (IFN)-γ + tumour necrosis factor (TNF)-α + interleukin (IL)-1β. Consequently, MIF-deficient [MIF-knock-out (KO)] pancreatic islets or islet cells showed significant resistance to cytokine-induced death than those isolated from C57BL/6 mice. Furthermore, upon exposure to cytokines pancreatic islets from MIF-KO mice maintained normal insulin expression and produced less cyclooxygenase-2 (COX-2) than those from wild-type C57BL6 mice. The final outcome of cytokine-induced islet apoptosis in islets from wild-type mice was the activation of mitochondrial membrane pore-forming protein Bcl-2-associated X protein and effector caspase 3. In contrast, these apoptotic mediators remained at normal levels in islets from MIF-KO mice suggesting that MIF absence prevented initiation of the mitochondrial apoptotic pathway. Additionally, the protection from apoptosis was also mediated by up-regulation of prosurvival kinase extracellular-regulated kinase 1/2 in MIF-KO islets. These data indicate that MIF is involved in the propagation of pancreatic islets apoptosis probably via nuclear factor-κB and mitochondria-related proteins.

Fig. 1

Macrophage migration inhibitory factor (MIF) is a mediator of islet dysfunction and death. Pancreatic islets from C57BL/6 mice were incubated in groups of 20, in the presence or absence of cytokine mixture (interleukin-1β + interferon-γ + tumour necrosis factor-α, 10 ng/ml each) for 24 h and MIF concentration from cell-free supernatants was determined by enzyme-linked immunosorbent assay (a). Islet cells (1 × 10⁴/well) (b) or pancreatic islets (groups of 20) (c) isolated from C57BL/6 and MIF-knock-out (KO) mice were incubated in the presence or absence of cytokine mixture and their viability was assessed by MTT assay after 24 h or 48 h, respectively. Data are presented as % viable cells or islets compared to untreated cells or islets (100%). (d) Relative insulin mRNA expression was determined by real-time polymerase chain reaction in C57BL/6 and MIF-KO islets either left untreated or treated with cytokine mixture for 6 h. Results from three experiments were presented as mean ± standard deviation. *P < 0·05 cytokine-treated versus untreated (0). #P < 0·05 MIF-KO versus C57BL/6 islet cells or islets.

Fig. 2

Macrophage migration inhibitory factor (MIF) absence confers resistance of islets through impairment of nuclear factor-κB and cyclooxygenase (COX)-2 activation. Pancreatic islets (in groups of 100) from C57BL/6 or MIF-knock-out (KO) mice were incubated in the presence or absence of cytokine mixture (interleukin-1β + interferon-γ + tumour necrosis factor-α, 10 ng/ml each) for determination of p-IκB protein content by Western blot after 24 h (a), COX-2 mRNA expression by polymerase chain reaction (PCR) after 6 h (b), COX-2 content by Western blot after 24 h (c), inducible nitric oxide synthase mRNA expression after 6 h (d) or nitric oxide production after 48 h (e); n.d. = not detectable. Data are presented as mean ± standard deviation from three separate experiments. *P < 0·05 cytokine-treated versus untreated (0); #P < 0·05 MIF-KO versus C57BL/6 islets. Photographs of PCR amplicons and Western blot are displayed above the adequate graphs; lower lane: β-actin, upper lane: gene or protein in question.

Fig. 3

Macrophage migration inhibitory factor (MIF) absence protects islets through down-regulation of mitochondrial apoptotic pathway. Pancreatic islets from C57BL/6 or MIF-knock-out (KO) mice were incubated in the presence or absence of cytokine mixture (interleukin-1β + interferon-γ + tumour necrosis factor-α, 10 ng/ml each) for determination of BAX mRNA expression (in groups of 100) by real-time polymerase chain reaction after 6 h (a) or BAX protein content after 24 h (b), or caspase 3 activity (in groups of 10) after 24 h (c). Photograph of the blot is displayed above the adequate graph; lower lane: β-actin, upper lane: BAX protein. Data are presented as mean ± standard deviation from three experiments. *P < 0·05 cytokine-treated versus untreated (0); #P < 0·05 MIF-KO versus C57BL/6 islets.

Fig. 4

Prosurvival p-extracellular-regulated kinase (ERK)1/2 is up-regulated in cytokine-stimulated macrophage migration inhibitory factor knock-out (MIF-KO) islets. Pancreatic islets (in groups of 100) from C57BL/6 or MIF-KO mice were incubated in the presence or absence of cytokine mixture (interleukin-1β + interferon-γ + tumour necrosis factor-α, 10 ng/ml each) for determination of phosphorylated ERK subunits (p-p42 and p-p44) after 24 h. Data are presented as mean ± standard deviation from two experiments. *P < 0·05 cytokine-treated versus untreated (0); #P < 0·05 MIF-KO versus C57BL/6 islets.