Monique R. Heitmeier

IL-1 produced and released endogenously within human islets inhibits beta cell function.

Resident macrophages have been suggested to participate in the initiation of beta cell damage during the development of autoimmune diabetes. The purpose of this study was to determine if the endogenous production and release of interleukin 1 (IL-1) in human islets of Langerhans by resident macrophages results in the inhibition of beta cell function. Treatment of human islets with a combination of tumor necrosis factor (TNF) + lipopolysaccharide (LPS) + interferon-gamma (IFN-gamma) stimulates inducible nitric oxide synthase (iNOS) expression, nitric oxide production, and inhibits glucose-stimulated insulin secretion. The IL-1 receptor antagonist protein (IRAP) prevents TNF + LPS + IFN-gamma-induced iNOS expression and nitrite production, and attenuates the inhibitory effects on glucose-stimulated insulin secretion by human islets. Inhibition of iNOS activity by aminoguanidine also attenuates TNF + LPS + IFN-gamma-induced inhibition of insulin secretion by human islets. These results indicate that the inhibitory effects of TNF + LPS + IFN-gamma are mediated by nitric oxide, produced by the actions of IL-1 released endogenously within human islets. Reverse transcriptase polymerase chain reaction was used to confirm that TNF + LPS + IFN-gamma stimulates the expression of both IL-1alpha and IL-1beta in human islets. Two forms of evidence indicate that resident macrophages are the human islet cellular source of IL-1: culture conditions that deplete islet lymphoid cells prevent TNF + LPS + IFN-gamma-induced iNOS expression, nitric oxide production, and IL-1 mRNA expression by human islets; and IL-1 and the macrophage surface marker CD69 colocalize in human islets treated with TNF + LPS + IFN-gamma as determined by immunohistochemical analysis. Lastly, nitric oxide production is not required for TNF + LPS + IFN-gamma-induced IL-1 release in human islets. However, cellular damage stimulates IL-1 release by islet macrophages. These findings support the hypothesis that activated islet macrophages may mediate beta cell damage during the development of insulin-dependent diabetes by releasing IL-1 in human islets followed by cytokine-induced iNOS expression by beta cells.

Double-stranded RNA-induced Inducible Nitric-oxide Synthase Expression and Interleukin-1 Release by Murine Macrophages Requires NF-κB Activation

The effects of double-stranded RNA (synthetic polyinosinic-polycytidylic acid; poly(I-C)) on macrophage expression of inducible nitric-oxide synthase (iNOS), production of nitric oxide, and release of interleukin-1 (IL-1) were investigated. Individually, poly(I-C), interferon-γ (IFN-γ), and lipopolysaccharide (LPS) stimulate nitrite production and iNOS expression by RAW 264.7 cells. In combination, the effects of poly(I-C) + IFN-γ are additive, while poly(I-C) does not further potentiate LPS-induced nitrite production. These results suggest that poly(I-C) and LPS may stimulate iNOS expression by similar signaling pathways, which may be independent of pathways activated by IFN-γ. LPS-induced iNOS expression is associated with the activation of NF-κB. We show that inhibition of NF-κB by pyrrolidinedithiocarbamate prevents poly(I-C) + IFN-γ-, poly(I-C) + LPS-, and LPS-induced iNOS expression, nitrite production and IκB degradation by RAW 264.7 cells. The effects of poly(I-C) on iNOS expression appear to be cell-type specific. Poly(I-C), alone or in combination with IFN-γ, does not stimulate, nor does poly(I-C) potentiate, IL-1-induced nitrite production by rat insulinoma RINm5F cells. In addition, we show that the combination of poly(I-C) + IFN-γ stimulates iNOS expression, nitrite production, IκB degradation, and the release of IL-1 by primary mouse macrophages, and these effects are prevented by pyrrolidinedithiocarbamate. These findings indicate that double-stranded RNA, in the presence of IFN-γ, is a potent activator of macrophages, stimulating iNOS expression, nitrite production, and IL-1 release by a mechanism which requires the activation of NF-κB.

Potential role of PKR in double-stranded RNA-induced macrophage activation.

In this study, the role of the double‐stranded (ds) RNA‐dependent protein kinase (PKR) in macrophage activation was examined. dsRNA [polyinosinic:polycytidylic acid (poly IC)]‐stimulated inducible nitric oxide synthase, interleukin (IL)‐1α and IL‐1β mRNA expression, nitrite formation and IL‐1 release are attenuated in RAW264.7 cells stably expressing dominant negative (dn) mutants of PKR. The transcriptional regulator nuclear factor (NF)‐κB is activated by dsRNA, and appears to be required for dsRNA‐induced macrophage activation. While dnPKR mutants prevent macrophage activation, they fail to attenuate dsRNA‐induced IκB degradation or NF‐κB nuclear localization. The inhibitory actions of dnPKR on dsRNA‐induced macrophage activation can be overcome by treatment with interferon (IFN)‐γ, an event associated with PKR degradation. Furthermore, dsRNA + IFN‐γ stimulate inducible nitric oxide synthase expression, IκB degradation and NF‐κB nuclear localization to similar levels in macrophages isolated from PKR−/− and PKR+/+ mice. These findings indicate that both NF‐κB and PKR are required for dsRNA‐induced macrophage activation; however, dsRNA‐induced NF‐κB activation occurs by PKR‐independent mechanisms in macrophages. In addition, the PKR dependence of dsRNA‐induced macrophage activation can be overcome by IFN‐γ.

Prolonged STAT1 activation is associated with interferon-gamma priming for interleukin-1-induced inducible nitric-oxide synthase expression by islets of Langerhans.

In this study, the ability of interferon-γ (IFN-γ) to prime rat and nonobese diabetic (NOD) mouse islets for interleukin-1 (IL-1)-stimulated expression of inducible nitric-oxide synthase (iNOS) has been examined. IL-1-induced iNOS expression by rat islets is concentration-dependent with maximal expression occurring in response to 1.0 unit/ml. Individually, neither 0.1 unit/ml IL-1 nor 150 units/ml IFN-γ stimulates iNOS expression or nitrite production by rat islets. However, a 30–60-min pulse of rat islets with IFN-γ, followed by washing to remove the cytokine and continued culture with 0.1 unit/ml IL-1 for 40 h, results in iNOS expression and nitrite production to levels similar in magnitude to the individual effects of 1.0 unit/ml IL-1. A 1-h pulse with IFN-γ primes for IL-1-induced islet degeneration that is mediated by the expression of iNOS and increased production of nitric oxide. IFN-γ also primes for IL-1-induced iNOS expression and nitrite formation by NOD mouse islets. The priming actions of IFN-γ appear to be selective for β-cells, as IFN-γ primes for IL-1-induced nitrite formation by primary β-cells and RINm5F insulinoma cells, but not primary α-cells. The priming actions of IFN-γ for IL-1-induced iNOS expression do not requirede novo protein synthesis as preincubation of RINm5F cells with cycloheximide does not inhibit iNOS mRNA accumulation under priming conditions. The priming actions of IFN-γ on IL-1-induced iNOS expression persists for extended periods of up to 7 days and are associated with persistent signal transducers and activators of transcription (STAT)-1 activation. A 30-min pulse of rat islets with IFN-γ stimulates STAT1 phosphorylation, and STAT1 remains phosphorylated for up to 7 days following IFN-γ removal. In addition, STAT1 remains nuclear for up to 7 days after IFN-γ removal. These results indicate that IFN-γ primes for IL-1-induced islet degeneration via a nitric oxide-dependent mechanism. These findings also provide evidence that the priming actions of IFN-γ for IL-1-induced iNOS expression by islets are associated with the prolonged phosphorylation and activation of STAT1.

Mechanisms of β-Cell Death in Response to Double-Stranded (ds) RNA and Interferon-γ: dsRNA-Dependent Protein Kinase Apoptosis and Nitric Oxide-Dependent Necrosis

Viral infection is one environmental factor that has been implicated as a precipitating event that may initiate β-cell damage during the development of diabetes. This study examines the mechanisms by which the viral replicative intermediate, double-stranded (ds) RNA impairs β-cell function and induces β-cell death. The synthetic dsRNA molecule polyinosinic-polycytidylic acid (poly IC) stimulates β-cell DNA damage and apoptosis without impairing islet secretory function. In contrast, the combination of poly IC and interferon (IFN)-γ stimulates DNA damage, apoptosis, and necrosis of islet cells, and this damage is associated with the inhibition of glucose-stimulated insulin secretion. Nitric oxide mediates the inhibitory and destructive actions of poly IC + IFN-γ on insulin secretion and islet cell necrosis. Inhibitors of nitric oxide synthase, aminoguanidine, and NG-monomethyl-l-arginine, attenuate poly IC + IFN-γ-induced DNA damage to levels observed in response to poly IC alone, prevent islet cell necrosis, and prevent the inhibitory actions on glucose-stimulated insulin secretion. NG-monomethyl-l-arginine fails to prevent poly IC- and poly IC + IFN-γ-induced islet cell apoptosis. PKR, the dsRNA-dependent protein kinase that mediates the antiviral response in infected cells, is required for poly IC- and poly IC + IFN-γ-induced islet cell apoptosis, but not nitric oxide-mediated islet cell necrosis. Alone, poly IC fails to stimulate DNA damage in islets isolated from PKR-deficient mice; however, nitric oxide-dependent DNA damage induced by the combination of poly IC + IFN-γ is not attenuated by the genetic absence of PKR. These findings indicate that dsRNA stimulates PKR-dependent islet cell apoptosis, an event that is associated with normal islet secretory function. In contrast, poly IC + IFN-γ-induced inhibition of glucose-stimulated insulin secretion and islet cell necrosis are events that are mediated by islet production of nitric oxide. These findings suggest that at least one IFN-γ-induced antiviral response (islet cell necrosis) is mediated through a PKR-independent pathway.

Irreversible Inhibition of Metabolic Function and Islet Destruction after a 36-Hour Exposure to Interleukin-1β1

The purpose of this study was to identify the duration of exposure of islets to interleukin 1β (IL-1β) that results in irreversible damage. Treatment of rat islets for 18 h with IL-1β results in an inhibition of glucose-stimulated insulin secretion, mitochondrial aconitase activity, and total protein synthesis. The addition of NG-monomethyl-l-arginine (NMMA) or aminoguanidine to islets preincubated for 18 h with IL-1β, followed by continued culture for 8 h (with both NMMA and IL-1β), results in the recovery of islet secretory function, aconitase activity, and protein synthesis. However, islet metabolic function is irreversibly inhibited after a 36-h incubation with IL-1β, as an additional 8-h incubation with NMMA or aminoguanidine does not stimulate the recovery of insulin secretion, aconitase activity, or protein synthesis. The irreversible inhibition of metabolic function correlates with the commitment of islets to destruction. Treatment of islets for 96 h with IL-1β results in islet degeneration. NMMA,...

Role of cyclooxygenase-2 in cytokine-induced β-cell dysfunction and damage by isolated rat and human islets

Type I diabetes mellitus is an autoimmune disease characterized by the selective destruction of the insulin-secreting β-cell found in pancreatic islets of Langerhans. Cytokines such as interleukin-1 (IL-1), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) mediate β-cell dysfunction and islet degeneration, in part, through the induction of the inducible isoform of nitric-oxide synthase and the production of nitric oxide by β-cells. Cytokines also stimulate the expression of the inducible isoform of cyclooxygenase, COX-2, and the production of prostaglandin E2 (PGE2) by rat and human islets; however, the role of increased COX-2 expression and PGE2 production in mediating cytokine-induced inhibition of islet metabolic function and viability has been incompletely characterized. In this study, we have shown that treatment of rat islets with IL-1β or human islets with a cytokine mixture containing IL-1β + IFN-γ ± TNF-α stimulates COX-2 expression and PGE2 formation in a time-dependent manner. Co-incubation of rat and human islets with selective COX-2 inhibitors SC-58236 and Celecoxib, respectively, attenuated cytokine-induced PGE2 formation. However, these inhibitors failed to prevent cytokine-mediated inhibition of insulin secretion or islet degeneration. These findings indicate that selective inhibition of COX-2 activity does not protect rat and human islets from cytokine-induced β-cell dysfunction and islet degeneration and, furthermore, that islet production of PGE2 does not mediate these inhibitory and destructive effects.

Tamm-Horsfall protein-deficient thick ascending limbs promote injury to neighboring S3 segments in an MIP-2-dependent mechanism

Tamm-Horsfall protein (THP) is a glycoprotein expressed exclusively in thick ascending limbs (TAL) of the kidney. We recently described a novel protective role of THP against acute kidney injury (AKI) via downregulation of inflammation in the outer medulla. Our current study investigates the mechanistic relationships among the status of THP, inflammation, and tubular injury. Using an ischemia-reperfusion model in wild-type and THP-/- mice, we demonstrate that it is the S3 proximal segments but not the THP-deficient TAL that are the main targets of tubular injury during AKI. The injured S3 segments that are surrounded by neutrophils in THP-/- mice have marked overexpression of neutrophil chemoattractant MIP-2 compared with wild-type counterparts. Neutralizing macrophage inflammatory protein-2 (MIP-2) antibody rescues S3 segments from injury, decreases neutrophil infiltration, and improves kidney function in THP-/- mice. Furthermore, using immunofluorescence volumetric imaging of wild-type mouse kidneys, we show that ischemia alters the intracellular translocation of THP in the TAL cells by partially shifting it from its default apical surface domain to the basolateral domain, the latter being contiguous to the basolateral surface of S3 segments. Concomitant with this is the upregulation, in the basolateral surface of S3 segments, of the scavenger receptor SRB-1, a putative receptor for THP. We conclude that TAL affects the susceptibility of S3 segments to injury at least in part by regulating MIP-2 expression in a THP-dependent manner. Our findings raise the interesting possibility of a direct role of basolaterally released THP on regulating inflammation in S3 segments.

Tamm-Horsfall protein translocates to the basolateral domain of thick ascending limbs, interstitium, and circulation during recovery from acute kidney injury.

Tamm-Horsfall protein (THP) is a glycoprotein normally targeted to the apical membrane domain of the kidneys thick ascending limbs (TAL). We previously showed that THP of TAL confers protection to proximal tubules against acute kidney injury (AKI) via a possible cross talk between the two functionally distinct tubular segments. However, the extent, timing, specificity, and functional effects of basolateral translocation of THP during AKI remain unclear. Using an ischemia-reperfusion (IRI) model of murine AKI, we show here that, while THP expression in TAL is downregulated at the peak of injury, it is significantly upregulated 48 h after IRI. Confocal immunofluorescence and immunoelectron microscopy reveal a major redirection of THP during recovery from the apical membrane domain of TAL towards the basolateral domain, interstitium, and basal compartment of S3 segments. This corresponds with increased THP in the serum but not in the urine. The overall epithelial polarity of TAL cells does not change, as evidenced by correct apical targeting of Na(+)-K(+)-2Cl cotransporter (NKCC2) and basolateral targeting of Na(+)-K(+)-ATPase. Compared with the wild-type, THP(-/-) mice show a significantly delayed renal recovery after IRI, due possibly to reduced suppression by THP of proinflammatory cytokines and chemokines such as monocyte chemoattractant protein-1 during recovery. Taken together, our data suggest that THP redistribution in the TAL after AKI is a protein-specific event and its increased interstitial presence negatively regulates the evolving inflammatory signaling in neighboring proximal tubules, thereby enhancing kidney recovery. The increase of serum THP may be used as a prognostic biomarker for recovery from AKI.

Double-stranded RNA inhibits beta-cell function and induces islet damage by stimulating beta-cell production of nitric oxide.

Viral infection has been implicated as a triggering event that may initiate β-cell damage during the development of autoimmune diabetes. In this study, the effects of the viral replicative intermediate, double-stranded RNA (dsRNA) (in the form of synthetic polyinosinic-polycytidylic acid (poly IC)) on islet expression of inducible nitric oxide synthase (iNOS), production of nitric oxide, and islet function and viability were investigated. Treatment of rat islets with poly(IC) + interferon-γ (IFN-γ) stimulates the time- and concentration-dependent expression of iNOS and production of nitrite by rat islets. iNOS expression and nitrite production by rat islets in response to poly(IC) + IFN-γ correlate with an inhibition of insulin secretion and islet degeneration, effects that are prevented by the iNOS inhibitor aminoguanidine (AG). We have previously shown that poly(IC) + IFN-γ activates resident macrophages, stimulating iNOS expression, nitric oxide production and interleukin-1 (IL-1) release. In addition, in response to tumor necrosis factor-α (TNF-α) + lipopolysaccharide, activated resident macrophages mediate β-cell damage via intraislet IL-1 release followed by IL-1-induced iNOS expression by β-cells. The inhibitory and destructive effects of poly(IC) + IFN-γ, however, do not appear to require resident macrophages. Treatment of macrophage-depleted rat islets for 40 h with poly(IC) + IFN-γ results in the expression of iNOS, production of nitrite, and inhibition of insulin secretion. The destructive effects of dsRNA + IFN-γ on islets appear to be mediated by a direct interaction with β-cells. Poly IC + IFN-γ stimulates iNOS expression and inhibits insulin secretion by primary β-cells purified by fluorescence-activated cell sorting. In addition, AG prevents the inhibitory effects of poly(IC) + IFN-γ on glucose-stimulated insulin secretion by β-cells. These results indicate that dsRNA + IFN-γ interacts directly with β-cells stimulating iNOS expression and inhibiting insulin secretion in a nitric oxide-dependent manner. These findings provide biochemical evidence for a novel mechanism by which viral infection may directly mediate the initial destruction of β-cells during the development of autoimmune diabetes.