Scavenger receptor-A (CD204): a two-edged sword in health and disease.

Scavenger receptor A (SR-A), also known as the macrophage scavenger receptor and cluster of differentiation 204 (CD204), plays roles in lipid metabolism, atherogenesis, and a number of metabolic processes. However, recent evidence points to important roles for SR-A in inflammation, innate immunity, host defense, sepsis, and ischemic injury. Herein, we review the role of SR-A in inflammation, innate immunity, host defense, sepsis, cardiac and cerebral ischemic injury, Alzheimer's disease, virus recognition and uptake, bone metabolism, and pulmonary injury. Interestingly, SR-A is reported to be host protective in some disease states, but there is also compelling evidence that SR-A plays a role in the pathophysiology of other diseases. These observations of both harmful and beneficial effects of SR-A are discussed here in the framework of inflammation, innate immunity, and endoplasmic reticulum stress.

Scavenger receptor class a plays a central role in mediating mortality and the development of the pro-inflammatory phenotype in polymicrobial sepsis.

Sepsis is a frequent complication in critical illness. The mechanisms that are involved in initiation and propagation of the disease are not well understood. Scavenger receptor A (SRA) is a membrane receptor that binds multiple polyanions such as oxidized LDL and endotoxin. Recent studies suggest that SRA acts as a pattern recognition receptor in the innate immune response. The goal of the present study was to determine the role of SRA in polymicrobial sepsis. SRA deficient (SRA(-/-)) and C57BL/6JB/6J (WT) male mice were subjected to cecal ligation and puncture (CLP) to induce polymicrobial sepsis. NFκB activity, myeloperoxidase activity, and co-association of SRA with toll like receptor (TLR) 4 and TLR2 was analyzed in the lungs. Spleens were analyzed for apoptosis. Serum cytokines and chemokines were assayed. Blood and peritoneal fluid were cultured for aerobic and anaerobic bacterial burdens. Long-term survival was significantly increased in SRA(-/-) septic mice (53.6% vs. 3.6%, p < 0.05) when compared to WT mice. NFκB activity was 45.5% lower in the lungs of SRA(-/-) septic mice versus WT septic mice (p < 0.05). Serum levels of interleukin (IL)-5, IL-6, IL-10 and monocyte chemoattractant protein -1 were significantly lower in septic SRA(-/-) mice when compared to septic WT mice (p < 0.05). We found that SRA immuno-precipitated with TLR4, but not TLR2, in the lungs of WT septic mice. We also found that septic SRA(-/-) mice had lower bacterial burdens than WT septic mice. SRA deficiency had no effect on pulmonary neutrophil infiltration or splenocyte apoptosis during sepsis. We conclude that SRA plays a pivotal, and previously unknown, role in mediating the pathophysiology of sepsis/septic shock in a murine model of polymicrobial sepsis. Mechanistically, SRA interacts with TLR4 to enhance the development of the pro-inflammatory phenotype and mediate the morbidity and mortality of sepsis/septic shock.

Scavenger receptor A (SR-A) is required for LPS-induced TLR4 mediated NF-κB activation in macrophages.

Recent evidence suggests that the macrophage scavenger receptor class A (SR-A, aka, CD204) plays a role in the induction of innate immune and inflammatory responses. We investigated whether SR-A will cooperate with Toll-like receptors (TLRs) in response to TLR ligand stimulation. Macrophages (J774/a) were treated with Pam2CSK4, (TLR2 ligand), Polyinosinic:polycytidylic acid (Poly I:C) (TLR3 ligand), and Lipopolysaccharides (LPS) (TLR4 ligand) for 15 min in the presence or absence of fucoidan (the SR-A ligand). The levels of phosphorylated IκBα (p-IκBα) were examined by Western blot. We observed that Poly I:C and LPS alone, but not Pam2CSK4 or fucoidan increased the levels of p-IκBα. However, LPS-induced increases in p-IκBα levels were further enhanced when presence of the fucoidan. Immunoprecipitation and double fluorescent staining showed that LPS stimulation promotes SR-A association with TLR4 in the presence of fucoidan. To further confirm our observation, we isolated peritoneal macrophages from SR-A deficient (SR-A(-/-)), TLR4(-/-) and wild type (WT) mice, respectively. The peritoneal macrophages were treated with LPS for 15min in the presence and absence of fucoidan. We observed that LPS-stimulated TNFα and IL-1ß production was further enhanced in the WT macrophages, but did not in either TLR4(-/-) or SR-A(-/-) macrophages, when fucoidan was present. Similarly, in the presence of fucoidan, LPS-induced IκBα phosphorylation, NF-κB binding activity, and association between TLR4 and SR-A were significantly enhanced in WT macrophages compared with LPS stimulation alone. The data suggests that SR-A is needed for LPS-induced inflammatory responses in macrophages.

TLR2 ligand induces protection against cerebral ischemia/reperfusion injury via activation of phosphoinositide 3-kinase/Akt signaling.

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB-binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3ß/GSK-3ß compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.

Toll-like receptor 3 plays a central role in cardiac dysfunction during polymicrobial sepsis.

OBJECTIVE: To determine the role of Toll-like receptor 3 in cardiac dysfunction during polymicrobial sepsis. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Male C57BL/6, wild-type, Toll-like receptor 3-/-. INTERVENTION: Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Toll-like receptors (TLRs) play a critical role in the pathophysiology of sepsis/septic shock. TLR3 is located in intracellular endosomes, and recognizes double-stranded RNA. This study examined the role of TLR3 in cardiac dysfunction following cecal ligation and puncture (CLP)-induced sepsis. TLR3 knockout (TLR3-/-, n=12) and age-matched wild-type (n=12) mice were subjected to CLP. Cardiac function was measured by echocardiography before and 6 hrs after CLP. MEASUREMENTS AND MAIN RESULTS: CLP resulted in significant cardiac dysfunction as evidenced by decreased ejection fraction by 25.7% and fractional shortening by 29.8%, respectively. However, TLR3-/- mice showed a maintenance of cardiac function at pre-CLP levels. Wild-type mice showed 50% mortality at 58 hrs and 100% mortality at 154 hrs after CLP. In striking contrast, 70% of TLR3-/- mice survived indefinitely, that is, >200 hrs. TLR3 deficiency significantly decreased CLP-induced cardiac-myocyte apoptosis and attenuated CLP-induced Fas and Fas ligand expression in the myocardium. CLP-activation of TLR4-mediated nuclear factor-κB and Toll/IL-1 receptor-domain-containing adapter-inducing interferon-ß-dependant interferon signaling pathways was prevented by TLR3 deficiency. In addition, CLP-increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression, and neutrophil and macrophage sequestration in the myocardium were also attenuated in septic TLR3-/- mice. More significantly, adoptive transfer of wild-type bone-marrow stromal cells to TLR3-/- mice abolished the cardioprotective effect in sepsis. CONCLUSIONS: These data indicate that TLR3 plays a deleterious role in mediating cardiac dysfunction in sepsis. Thus, modulation of the TLR3 activity may be useful in preventing cardiac dysfunction in sepsis.

Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.

The TIR/BB-loop mimetic AS-1 prevents cardiac hypertrophy by inhibiting IL-1R-mediated MyD88-dependent signaling.

Activation of NF-κB contributes to cardiac hypertrophy and the interleukin-1 receptor (IL-1R)-mediated MyD88-dependent signaling pathway predominately activates NF-κB. Recent studies have shown that the TIR/BB-Loop mimetic (AS-1) disrupted the interaction of MyD88 with the IL-1R, resulting in blunting of NF-κB activation. We have examined the effects of AS-1 on the IL-1ß-induced hypertrophic response using cultured neonatal cardiac myocytes in vitro and transverse aortic constriction (TAC) pressure overload-induced cardiac hypertrophy in vivo. Neonatal cardiac myocytes were treated with AS-1 15 min prior to IL-1ß stimulation for 24 h. AS-1 treatment significantly attenuated IL-1ß-induced hypertrophic responses of cardiac myocytes. In vivo experiments showed that AS-1 administration prevented cardiac hypertrophy and dysfunction induced by pressure overload. AS-1 administration disrupted the interaction of IL-1R with MyD88 in the pressure overloaded hearts and prevented activation of NF-κB. In addition, AS-1 prevented increases in activation of the MAPK pathway (p38 and p-ERK) in TAC-induced hypertrophic hearts. Our data suggest that the IL-1R-mediated MyD88-dependent signaling pathway plays a role in the development of cardiac hypertrophy and AS-1 attenuation of cardiac hypertrophy is mediated by blocking the interaction between IL-1R and MyD88, resulting in decreased NF-κB binding activity and decreased MAPK activation.

TLR2 ligands attenuate cardiac dysfunction in polymicrobial sepsis via a phosphoinositide 3-kinase-dependent mechanism.

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. In the present study, we examined the effect of Toll-like receptor 2 (TLR2) ligands, peptidoglycan (PGN), and Pam3CSK4 (Pam3) on cardiac function in cecal ligation and puncture (CLP)-induced sepsis in mice. We also investigated whether the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway is involved in the effect of TLR2 ligands on cardiac function in CLP mice. PGN was administered to C57B6/L mice 1 h before the induction of CLP. Sham surgically operated mice served as a control. Cardiac function indexes (rate of change in left ventricular pressure, stroke work, cardiac output, and ejection fraction) were examined by a microconductance pressure catheter. Cardiac function was significantly decreased 6 h after CLP-induced sepsis compared with sham-operated control. In contrast, PGN administration attenuated CLP-induced cardiac dysfunction. Importantly, the therapeutic treatment with Pam3 1 h after CLP also significantly attenuated cardiac dysfunction in CLP mice. However, the beneficial effect of TLR2 ligands on cardiac dysfunction in CLP-mice was abolished in TLR2-deficient mice. PGN administration significantly increased the levels of phospho-Akt and phospho-GSK-3beta in the myocardium compared with the levels in untreated CLP mice. PI3K inhibition abolished the PGN-induced attenuation of cardiac dysfunction in CLP mice. In conclusion, these data demonstrate that the administration of TLR2 ligands, PGN, or Pam3 attenuates cardiac dysfunction in septic mice via a TLR2/PI3K-dependent mechanism. More significantly, Pam3 therapeutic treatment will have a potential clinical relevance.

Pacing lead implantation without live fluoroscopy: feasibility of acute success in the live canine model.

INTRODUCTION: Fluoroscopic visualization for transvenous pacing lead placement necessitates lead shielding to minimize radiation exposure. An electromagnetic (EM) navigation system that integrates real-time intracardiac tracking within an anatomic navigation environment may provide an effective alternative for lead delivery that obviates live fluoroscopy. We assessed feasibility of pacing lead implantation with electromagnetic tracking guided solely by radiographic virtual navigation and compared this to fluoroscopy-guided implants in a canine model. METHODS: Seven mongrel dogs with normal hearts were randomized to 47 pacing lead placements in the right atrium (RA) or right ventricle (RV) guided by single-plane fluoroscopy, or an experimental EM navigation system guided by registered fluoroscopic snapshots obtained before implant (EMN). Ability to achieve successful lead delivery acutely was assessed, and pacing parameters as well as fluoroscopy and implant times were measured. Means were compared using a paired t-test. RESULTS: All lead delivery attempts were acutely successful. One atrial lead dislodged with EMN, resulting in 46 successful pacing attempts. There was no statistical difference in pacing parameters and time for lead placement between the approaches (EMN vs fluoroscopic navigation [mean +/- SD]: RA threshold 1.15 V +/- 0.98 V vs 1.95 V +/- 0.98 V [P = NS], RV threshold 1.18 V +/- 0.58 V vs 1.42 V +/- 0.63 V [P = NS], implant time 4:38 +/- 2:37 minutes vs 4:44 +/- 2:38 minutes [P = NS]). No live fluoroscopy was required for EMN implants. CONCLUSION: Pacing lead placement with an EM system guided by preprocedural fluoroscopic views is feasible and comparable to fluoroscopic navigation, and avoids the use of live fluoroscopy.

Lipopolysaccharide-induced myocardial protection against ischaemia/reperfusion injury is mediated through a PI3K/Akt-dependent mechanism.

AIMS: The ability of lipopolysaccharide (LPS) pre-treatment to induce cardioprotection following ischaemia/reperfusion (I/R) has been well documented; however, the mechanisms have not been fully elucidated. LPS is a Toll-like receptor 4 (TLR4) ligand. Recent evidence indicates that there is cross-talk between the TLR and phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathways. We hypothesized that activation of PI3K/Akt signalling plays a critical role in LPS-induced cardioprotection. METHODS AND RESULTS: To evaluate this hypothesis, we pre-treated mice with LPS 24 h before the hearts were subjected to ischaemia (45 min) and reperfusion (4 h). We examined activation of the PI3K/Akt/GSK-3beta signalling pathway. The effect of PI3K/Akt inhibition on LPS-induced cardioprotection was also evaluated. LPS pre-treatment significantly reduced infarct size (71.25%) compared with the untreated group (9.3+/-1.58 vs. 32.3+/-2.92%, P<0.01). Cardiac myocyte apoptosis and caspase-3 activity in LPS-pre-treated mice were significantly reduced following I/R. LPS pre-treatment significantly increased the levels of phospho-Akt, phospho-GSK-3beta, and heat shock protein 27 in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase-defective Akt transgenic mice abolished the cardioprotection induced by LPS. CONCLUSION: These results indicate that LPS-induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

Preconditioning with a TLR2 specific ligand increases resistance to cerebral ischemia/reperfusion injury.

The brain's resistance to ischemic injury can be transiently augmented by prior exposure to a sub-lethal stress stimulus, i.e. preconditioning. It has been reported that Toll-like receptors (TLRs) are involved in the preconditioning-induced protective effect against ischemic brain injury. In this study, we investigated the effect of preconditioning with a TLR2 specific ligand, Pam3CSK4, on focal cerebral ischemia/reperfusion (I/R) injury in mice. Pam3CSK4 was administered systemically 24 h before the mice were subjected to focal cerebral ischemia (1 h) followed by reperfusion. Cerebral infarct size was determined, blood brain barrier (BBB) permeability was evaluated, and expression of tight-junction proteins were examined after focal cerebral I/R. Results showed that pre-treatment with Pam3CSK significantly reduced brain infarct size (1.9+/-0.5% vs 9.4+/-2.2%) compared with the untreated I/R group. Pam3CSK4 pre-treatment also significantly reduced acute mortality (4.3% vs 24.2%), preserved neurological function (8.22+/-0.64 vs 3.91+/-0.57), and attenuated brain edema (84.61+/-0.08% vs 85.29+/-0.09%) after cerebral I/R. In addition, Pam3CSK4 pre-treatment preserved BBB function as evidenced by decreased leakage of serum albumin (0.528+/-0.026 vs 0.771+/-0.059) and Evans Blue (9.23+/-0.72 microg/mg vs 12.56+/-0.65 microg/mg) into brain tissue. Pam3CSK4 pre-treatment also attenuated the loss of the tight junction protein occludin in response to brain I/R injury. These results suggest that TLR2 is a new target of ischemic preconditioning in the brain and preconditioning with a TLR2 specific ligand will protect the brain from I/R injury.

Activation of Toll-like receptor 4 signaling contributes to hippocampal neuronal death following global cerebral ischemia/reperfusion.

Toll-like receptors (TLRs) play a critical role in the induction of innate immune responses which have been implicated in neuronal death induced by global cerebral ischemia/reperfusion (GCI/R). The present study investigated the role and mechanisms-of-action of TLR4 signaling in ischemia-induced hippocampal neuronal death. Neuronal damage, activation of the TLR4 signaling pathway, expression of pro-inflammatory cytokines and activation of the PI3K/Akt signaling pathway in the hippocampal formation (HF) were assessed in wild type (WT) mice and TLR4 knockout (TLR4(-/-)) mice after GCI/R. GCI/R increased expression of TLR4 protein in the hippocampal formation (HF) and other brain structures in WT mice. Phosphorylation of the inhibitor of kappa B (p-IkappaB) as well as activation of nuclear factor kappa B (NFkappaB) increased in the HF of WT mice. In contrast, there were lower levels of p-IkappaB and NFkappaB binding activity in TLR4(-/-) mice subjected to GCI/R. Pro-inflammatory cytokine expression was also decreased, while phosphorylation of Akt and GSK3beta were increased in the HF of TLR4(-/-) mice after GCI/R. These changes correlated with decreased neuronal death/apoptosis in TLR4(-/-) mice following GCI/R. These data suggest that activation of TLR4 signaling contributes to ischemia-induced hippocampal neuronal death. In addition, these data suggest that modulation of TLR4 signaling may attenuate ischemic injury in hippocampal neurons.

The development of a novel mouse model of transient global cerebral ischemia.

A reproducible model of global cerebral ischemia in mice is essential for elucidating the molecular mechanism(s) of neuronal damage induced by cerebral ischemia/reperfusion injury. In the present study, we developed a mouse model of transient global ischemia induced by occlusion of the bilateral common carotid arteries and the left subclavian artery together with right subclavian artery (RSA) stenosis (CSOSS) under controlled ventilation in C57BL/10ScSn mice. The mean arterial blood pressure was maintained in the physiological range. The cortical cerebral blood flow was reduced to less than 10% of the pre-ischemic value. Twelve minutes of global ischemia induced brain damage in several brain structures. The neuropathological score in the hippocampus CA1 region was 1.7, 3.5 and 3.7 following reperfusion for 24, 48 and 72 h, respectively. Less extensive damage was seen in the dentate gyrus and cortical regions, compared with the CA1 region. Damage was observed in these regions 24h after ischemia and was not different between 48 and 72 h post-ischemia. Results indicated that this global ischemia model possessed several advantages, including reproducible cerebral ischemic insult, sufficient reperfusion and low mortality rate (10%), and could be used for studies on cerebral ischemia/reperfusion injury in mice.

Activation of nuclear factor-kappaB.

Nuclear factor-kappaB (NF-kappaB) is a key transcription factor that regulates the expression of genes involved in immune and inflammatory responses and in cell death and survival. This chapter describes in detail the method for measuring the NF-kappaB binding activity in the cultured human mast cell line HMC-1 using the electrophoretic mobility shift assay: the activation of NF-kappaB was illustrated by adding lipopolysaccharide to mast cells, nuclear proteins were isolated, and the NF-kappaB binding activity was determined. We also demonstrate the specificity of the NF-kappaB binding activity using a competition and supershift assay with specific antibodies that recognize NF-kappaB subunits p50 and p65. Lipopolysaccharide caused a rapid and significant increase in NF-kappaB binding activity.

Mast cell activation by lipoproteins.

Mast cells are activated by a number of agents that act independently from immunoglobulin E (IgE)-mediated hypersensitivity. One of these agents is oxidized low-density lipoprotein (oxLDL). OxLDL has been implicated in the pathogenesis of atherosclerosis and has been shown to induce microvascular dysfunction by the activation of mast cells. In this chapter, we describe the method for isolation of human LDL, oxidation of LDL, and demonstrate that oxLDL activates mast cells by measuring messenger ribonucleic acid (mRNA) levels and protein levels of interleukin (IL)-8 an inflammatory cytokine. IL-8 is a potent chemoattractant for neutrophils and monocytes, which would result in a chronic inflammatory response. IL-8 mRNA levels were measured by reverse-transcription polymerase chain reaction and protein levels by enzyme-linked immunoassay.

Reduced cardiac hypertrophy in toll-like receptor 4-deficient mice following pressure overload.

OBJECTIVE: We have previously demonstrated that nuclear factor kappa B (NFkappaB) activation is needed for the development of cardiac hypertrophy in vivo. NFkappaB is a downstream transcription factor in the Toll-like receptor (TLR)-mediated signaling pathway; therefore, we investigated a role of TLR4 in cardiac hypertrophy in vivo. METHODS: TLR4-deficient mice (C.C3H-Tlr4(lps-d), n = 6), wild-type (WT) genetic background mice (BALB/c, n = 6), TLR4-deleted strain (C57BL/10ScCr, n = 8), and WT controls (C57BL/10ScSn, n = 8) were subjected to aortic banding for 2 weeks. Age-matched surgically operated mice served as controls. In a separate experiment, rapamycin (2 mg/kg, daily) was administered to TLR4-deficient mice and WT mice immediately following aortic banding. The ratio of heart weight/body weight (HW/BW) was calculated, and cardiac myocyte size was examined by FITC-labeled wheat germ agglutinin staining of membranes. NFkappaB binding activity and the levels of phospho-p70S6K in the myocardium were also examined. RESULTS: Aortic banding significantly increased the ratio of HW/BW by 33.9% (0.601 +/- 0.026 vs. 0.449 +/- 0.004) and cell size by 68.4% in WT mice and by 10.00% (0.543 +/- 0.011 vs. 0.495 +/- 0.005) and by 11.8% in TLR4-deficient mice, respectively, compared with respective sham controls. NFkappaB binding activity and phospho-p70S6K levels were increased by 182.6% and 115.2% in aortic-banded WT mice and by 78.0% and 162.0% in aortic-banded TLR4-deficient mice compared with respective sham controls. In rapamycin-treated aortic-banded mice, the ratio of HW/BW was increased by 18.0% in WT mice and by 3.5% in TLR4-deficient mice compared with respective sham controls. CONCLUSION: Our results demonstrate that TLR4 is a novel receptor contributing to the development of cardiac hypertrophy in vivo and that both the TLR4-mediated pathway and PI3K/Akt/mTOR signaling are involved in the development of cardiac hypertrophy in vivo.

Attenuation of cardiac hypertrophy by inhibiting both mTOR and NFkappaB activation in vivo.

A role for the PI3K/Akt/mTOR pathway in cardiac hypertrophy has been well documented. We reported that NFkappaB activation is needed for cardiac hypertrophy in vivo. To investigate whether both NFkappaB activation and PI3K/Akt/mTOR signaling participate in the development of cardiac hypertrophy, two models of cardiac hypertrophy, namely, induction in caAkt-transgenic mice and by aortic banding in mice, were employed. Rapamycin (2 mg/kg/daily), an inhibitor of the mammalian target of rapamycin, and the antioxidant pyrrolidine dithiocarbamate (PDTC; 120 mg/kg/daily), which can inhibit NFkappaB activation, were administered to caAkt mice at 8 weeks of age for 2 weeks. Both rapamycin and PDTC were also administered to the mice immediately after aortic banding for 2 weeks. Administration of either rapamycin or PDTC separately or together to caAkt mice reduced the ratio of heart weight/body weight by 21.54, 32.68, and 42.07% compared with untreated caAkt mice. PDTC administration significantly reduced cardiac NFkappaB activation by 46.67% and rapamycin significantly decreased the levels of p70S6K by 34.20% compared with untreated caAkt mice. Similar results were observed in aortic-banding-induced cardiac hypertrophy in mice. Our results suggest that both NFkappaB activation and the PI3K/Akt signaling pathway participate in the development of cardiac hypertrophy in vivo.

Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans.

Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

Modulating Toll-like receptor mediated signaling by (1-->3)-beta-D-glucan rapidly induces cardioprotection.

OBJECTIVE: Immune and inflammatory signaling pathways, initiated by the innate response, are involved in myocardial ischemia/reperfusion (I/R) injury. Toll-like receptor (TLR) mediated MyD88-dependent NFkappaB pathways play a role in the induction of innate immunity. We have reported that glucan phosphate (GP) improved survival in experimental sepsis, which correlated with decreased tissue NFkappaB activation. In the present study, we report that GP rapidly induced cardioprotection against I/R injury in vivo. METHODS: Sprague-Dawley rats were pretreated with GP (40 mg/kg, i.p) 1 h before 45 min of ligation of the left anterior descending coronary followed by reperfusion for 4 and 24 h. Infarction size was examined by triphenyltetrazolium chloride (TTC) staining. NFkappaB activation was analyzed by electrophoretic mobility shift assay (EMSA). IkappaB kinase-beta (IKKbeta), IL-1 receptor-associated kinase (IRAK) and Phosphoinositide 3-kinase (PI3K) activities were determined by kinase assay with appropriate substrates. Association of TLR4 with MyD88 or with PI3K p85 was assessed by immunoprecipitation with anti-TLR4 followed by immunoblotting with anti-MyD88 or anti-p85. RESULTS: GP treatment reduced infarct size by 47% in rat hearts subjected to reperfusion for 4 h and by 50% following reperfusion for 24 h. The same protective effect was observed when GP was administrated 5 min after initiation of ischemia. The mechanisms of GP induced cardioprotection involve decreased association of TLR4 with MyD88, inhibition of I/R induced IRAK and IKKbeta activity and decreased NFkappaB activity. In addition, GP increased TLR4 phosphotyrosine, resulting in increasing PI3K/Akt activity in the myocardium, which correlated with decreased cardiac myocyte apoptosis following I/R. CONCLUSION: The results suggest that activation of the TLR mediated MyD88-dependent NFkappaB signaling pathway may play an important role in myocardial I/R injury, while stimulation of the PI3K/Akt signaling could serve a protective role. The data indicates that GP treatment shifts the TLR mediated activation signal in I/R from a predominantly NFkappaB pathway to a predominant PI3K/Akt signaling pathway.

Regulation of eosinophil-active cytokine production from human cord blood-derived mast cells.

Human mast cells are multifunctional tissue-dwelling cells that play a crucial role in eosinophil-dependent disorders, such as asthma and parasitic diseases, by the secretion of eosinophil-active mediators. Mast cell-derived cytokines, generated in response to cross-linking of the high-affinity IgE receptor, can regulate eosinophil activation, survival, and chemotaxis. In this study, mast cells generated from human cord blood progenitors (stem cells) were studied for eosinophil-active inflammatory cytokine expression. Cord blood-derived mast cells (CBDMC) expressed typical intracellular scroll granules and microvilli-like structures on their cell surfaces, demonstrated the presence of tryptase, and elaborated prostaglandin D2 (PGD2) after cross-linkage of the high-affinity receptor for IgE (FcepsilonRI). CBDMC expressed tumor necrosis factor-alpha (TNF-alpha) and the eosinophil-active growth factors, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after activation. (IL-1beta greatly enhanced IgE-dependent production of these cytokines in response to FcepsilonRI cross-linkage, suggesting a role for bystander/phagocytic cells in modulating mast cell function. In contrast, interferon-alpha (IFN-alpha) inhibited IL-5 and GM-CSF generation, and the glucocorticoid, dexamethasone (Dex), inhibited production of IL-5 and GM-CSF from CBDMC. A macrophage-mast cell-eosinophil axis may exist in vivo that may be susceptible to pharmacologic manipulation.