The Aldosterone-Mineralocorticoid Receptor Pathway Exerts Anti-Inflammatory Effects in Endotoxin-Induced Uveitis

We have previously shown that the eye is a mineralocorticoid-sensitive organ and we now question the role of mineralocorticoid receptor (MR) in ocular inflammation. The endotoxin-induced uveitis (EIU), a rat model of human intraocular inflammation, was induced by systemic administration of lipopolysaccharide (LPS). Evaluations were made 6 and 24 hours after intraocular injection of aldosterone (simultaneous to LPS injection). Three hours after onset of EIU, the MR and the glucocorticoid metabolizing enzyme 11-beta hydroxysteroid dehydrogenase type 2 (11β-HSD2) expression were down-regulated in iris/ciliary body and the corticosterone concentration was increased in aqueous humor, altering the normal MR/glucocorticoid receptor (GR) balance. At 24 hours, the GR expression was also decreased. In EIU, aldosterone reduced the intensity of clinical inflammation in a dose-dependent manner. The clinical benefit of aldosterone was abrogated in the presence of the MR antagonist (RU26752) and only partially with the GR antagonist (RU38486). Aldosterone reduced the release of inflammatory mediators (6 and 24 hours: TNF-α, IFN-γ, MIP-1α) in aqueous humor and the number of activated microglia/macrophages. Aldosterone partly prevented the uveitis-induced MR down-regulation. These results suggest that MR expression and activation in iris/ciliary body could protect the ocular structures against damages induced by EIU.     

Factors Influencing Aqueous Proinflammatory Cytokines and Growth Factors in Uveitic Glaucoma

Purpose

To analyze the effects of factors on aqueous humor proinflammatory cytokine and growth factor levels in patients with uveitic glaucoma (UG).

Methods

In this cross-sectional study, we enrolled 143 participants: 1) UG patients (n = 39); 2) primary open-angle glaucoma (POAG) patients (n = 36); and 3) cataract surgery patients, as a comparative group (n = 68). Aqueous humor samples were obtained at the start of surgery. Aqueous cytokine levels were determined using a multiplex immunoassay (xMAP and the Human Cytokine/Chemokine Panel I).

Results

In UG cases, mean interleukin (IL)-6, IL-8, monocyte chemotactic protein (MCP)-1, tumor necrosis factor (TNF)-α, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, and VEGF levels were 171.1, 214.5, 2791.7, 3.5, 23.9, 5.4, and 168.9 pg/mL, respectively, and were higher than those in cataract (non-glaucomatous) cases except PDGF. Levels of IL-6, MCP-1, and VEGF were higher in UG cases than in POAG cases. UG cases with a history of phacoemulsification displayed significantly higher levels of IL-6 (P = 0.0164), IL-8 (P = 0.0003), MCP-1 (P = 0.0465), and PDGF-AB/BB (P = 0.0062) compared to the phakic cases. The presence of cells in the anterior chamber was related to higher levels of IL-8 (P = 0.0002), TNF-α (P = 0.0037), and PDGF-AB/BB (P = 0.0009). The level of PDGF-AB/BB was higher in infectious uveitis than in non-infectious uveitis (P = 0.0211). The level of transforming growth factor (TGF)-β2 was negatively correlated with the levels of MCP-1 (adjusted R² = 0.28, t = -2.45, P = 0.031) and TNF-α (adjusted R² = 0.27, t = -2.43, P = 0.032).

Conclusion

A history of phacoemulsification, the presence of cells in the anterior chamber, and infectious uveitis were related to aqueous proinflammatory cytokine levels in patients with UG. TGF-β2 might be an anti-inflammatory factor in aqueous humor of UG patients.     

The Features of Inflammation Factors Concentrations in Aqueous Humor of Polypoidal Choroidal Vasculopathy

Purpose

To investigate the cytokine concentrations in the aqueous humor of patients with refractory polypoidal choroidal vasculopathy (PCV).

Methods

Three separate groups of patients were studied–refractory PCV (Group A, 41 eyes), stable PCV (Group B, 39 eyes) and senile cataract (Group C, 44 eyes). Aqueous humor samples were collected at two time points for Groups A and B–before the first intravitreal ranibizumab injection and before the last injection. Aqueous humor samples were collected prior to phacoemulsification in Group C. The cytokine concentrations of interleukin 2, 6, and 8 (IL-2, IL-6, and IL-8), tumor necrosis factor α (TNF-α), monocyte chemotactic protein 1 (MCP-1), and vascular endothelial growth factor (VEGF) were measured by cytometric bead array and flow cytometry.

Results

Before the first treatment, the MCP-1, VEGF, and TNF-α levels in Group A were significantly higher than those in Group C (P < 0.05), and the MCP-1 and VEGF levels in Group A were significantly higher than those in Group B (P < 0.05). Significantly higher MCP-1 and VEGF levels were seen in Group B compared to Group C (P < 0.05). Before the final treatment, the MCP-1, VEGF, and TNF-α concentrations in Group A were significantly higher than those in Group B (P < 0.05) and Group C (P < 0.05). IL-2 levels were significantly lower in Group A compared to Group B (P < 0.05) and Group C (P < 0.05).

Conclusion

Inflammatory cytokines such as MCP-1, VEGF, and TNF-α may be associated with the pathogenesis of both stable and refractory PCV.     

A Pseudopterane Diterpene Isolated From the Octocoral Pseudopterogorgia acerosa Inhibits the Inflammatory Response Mediated by TLR-Ligands and TNF-Alpha in Macrophages

Several diterpenoids isolated from terrestrial and marine environments have been identified as important anti-inflammatory agents. Although considerable progress has been made in the area of anti-inflammatory treatment, the search for more effective and safer compounds is a very active field of research. In this study we investigated the anti-inflammatory effects of a known pseudopterane diterpene (referred here as compound 1) isolated from the octocoral Pseudopterogorgia acerosa on the tumor necrosis factor- alpha (TNF-α) and TLRs- induced response in macrophages. Compound 1 inhibited the expression and secretion of the inflammatory mediators TNF-α, interleukin (IL)-6, IL-1β, nitric oxide (NO), interferon gamma-induced protein 10 (IP-10), ciclooxygenase (COX)-2, inducible nitric oxide synthase (iNOS) and monocyte chemoattractant protein-1 (MCP-1) induced by LPS in primary murine macrophages. This effect was associated with the inhibition of IκBα degradation and subsequent activation of NFκB. Compound 1 also inhibited the expression of the co-stimulatory molecules CD80 and CD86, which is a hallmark of macrophage activation and consequent initiation of an adaptive immune response. The anti-inflammatory effect was not exclusive to LPS because compound 1 also inhibited the response of macrophages to TNF-α and TLR2 and TLR3 ligands. Taken together, these results indicate that compound 1 is an anti-inflammatory molecule, which modulates a variety of processes occurring in macrophage activation.     

High-density Lipoprotein Increases the Uptake of Oxidized Low Density Lipoprotein via PPARγ/CD36 Pathway in Inflammatory Adipocytes

Aim: Previous studies have demonstrated that the dysregulated-secretion of adipokines by adipocytes may contribute to obesity-associated atherosclerosis (As) and high density lipoprotein (HDL) may protect against atherogenesis through multiple pathways. This study was to explore the effect of HDL on the oxLDL uptake in inflammatory adipocytes stimulated by endotoxin lipopolysaccharide (LPS) and the possible mechanism.Methods and Results: 3T3-L1 adipocytes were cultured and induced to differentiation and maturation. Acute inflammation in adipocytes was induced by LPS (100 ng/ml) for 6 hours. The adipocytes were pretreated with HDL in various concentrations (10, 50, 100 μg/ml) for 16 hours or with specific PPARγ antagonist (GW9662, 10 μM) or agonist (Rosiglitazone, 10 μM) for 30 min before administration of LPS. The results showed that LPS significantly increased the release of inflammation-related adipokines, such as monocyte chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor 1 (PAI-1), tumor necrosis factor-alpha (TNF-α), interleukin (IL)-8 and IL-6, while decreasing the release of leptin and adiponectin. Meanwhile, LPS reduced the uptake and degradation of ¹²⁵I-oxLDL, and down-regulated the expression of PPARγ and CD36. Pretreatment with HDL dose-dependently affected the release of IL-8 and IL-6 and the reduced uptake and degradation of oxLDL of adipocytes stimulated by LPS, accompanied with marked upregulation of PPARγ and CD36 expression. Pretreatment with GW9662 markedly inhibited the upregulation of CD36 expression mediated by HDL (100 μg/ml), while the effects of Rosiglitazone were opposite to GW9662.Conclusions: HDL may increase oxLDL uptake of inflammatory adipocytes stimulated by LPS via upregulation of PPARγ/CD36 pathway, which may be a new mechanism of anti-atherosclerosis mediated by HDL.     

Morin,a Bioflavonoid Suppresses Monosodium Urate Crystal-Induced Inflammatory Immune Response in RAW 264.7 Macrophages through the Inhibition of Inflammatory Mediators,Intracellular ROS Levels and NF-κB Activation

Our previous studies had reported that morin, a bioflavanoid exhibited potent anti-inflammatory effect against adjuvant-induced arthritic rats. In this current study, we investigated the anti-inflammatory mechanism of morin against monosodium urate crystal (MSU)-induced inflammation in RAW 264.7 macrophage cells, an in vitro model for acute gouty arthritis. For comparison purpose, colchicine was used as a reference drug. We have observed that morin (100–300 μM) treatment significantly suppressed the levels of inflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1 and VEGF), inflammatory mediators (NO and PEG₂), and lysosomal enzymes (acid phosphatase, β-galactosidase, N-acetyl glucosamindase and cathepsin D) in MSU-crystals stimulated macrophage cells. The mRNA expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1), inflammatory enzymes (iNOS and COX-2), and NF-κBp65 was found downregulated in MSU crystal stimulated macrophage cells by morin treatment, however, the mRNA expression of hypoxanthine phospho ribosyl transferse (HPRT) was found to be increased. The flow cytometry analysis revealed that morin treatment decreased intracellular reactive oxygen species levels in MSU crystal stimulated macrophage cells. The western blot analysis clearly showed that morin mainly exerts its anti-inflammatory effects by inhibiting the MSU crystal-induced COX-2 and TNF-α protein expression through the inactivation of NF-κB signaling pathway in RAW 264.7 macrophage cells similar to that of BAY 11–7082 (IκB kinase inhibitor). Our results collectively suggest that morin can be a potential therapeutic agent for inflammatory disorders like acute gouty arthritis.     

Direct, real-time, simultaneous monitoring of intravitreal nitric oxide and oxygen in endotoxin-induced uveitis in rabbits

Nitric oxide (NO) plays a critical role in the pathogenesis of endotoxin-induced uveitis (EIU). Since NO is a labile free radical, it is difficult to examine the dynamics of NO directly in vivo. In this study, we established a system for direct monitoring of the dynamics of NO and partial pressure of oxygen (pO(2)) in EIU in rabbits. The currents (calculated concentrations) of NO and pO(2) in the vitreous were monitored after the intravitreal injection of lipopolysaccharide (LPS). In addition, the protein concentrations and nitrite levels in the aqueous humor were analyzed. The eyes were enucleated, and a histologic study was performed on their posterior segments. The tissue slices were also immunostained with anti-nitrotyrosine as a marker of peroxinitrite and/or nitrogen-related oxidants. The NO level decreased temporarily after LPS injection and then increased from 1 to 7 h. pO(2) increased temporarily for about 30 min after LPS injection. The change in NO current was inversely proportional to pO(2) after LPS injection and vice versa. The protein concentration and nitrite level after LPS injection increased significantly. These changes were suppressed by pretreatment with N(G)-nitro-l-arginine-methyl-ester. Immunohistochemical study showed enhanced immunoreactivity of nitrotyrosine in the inflamed retina. Since nitrotyrosine was detected, it appears that NO readily reacts with oxygen to produce cytotoxic species, peroxynitrite, and/or nitrogen-related oxidants. This process may be related to the retinal injury in EIU. This monitoring system can provide useful information on dynamic changes in intravitreal NO and pO(2) for understanding EIU.     

Localization and ontogeny of aquaporin-1 and -4 expression in iris and ciliary epithelial cells in rats

The precise localization of aquaporin (AQP)1 and AQP4 was studied in iris and ciliary epithelial cells, in both mature and developing rats, to elucidate the molecular mechanisms underlying aqueous humor balance. Anterior segments of eyes dissected from embryonic day (E)13, E15, E18, and E20, postnatal day (P)0, P7, and P14, and postnatal week 8 rats were subjected to immunofluorescence analysis with AQP isoform-specific antibodies. In adult rat eye, AQP1 was localized to the apical and basolateral plasma membranes of iris epithelial cell layers and of anterior ciliary non-pigmented epithelial (NPE) cells. Conversely, AQP4 was localized to the basolateral plasma membrane of NPE cells in ciliary epithelium and the posterior iris. Developmentally, AQP1 was detected as early as E15 in immature iris and ciliary epithelial cells, and expression persisted throughout development up to adulthood. In contrast, AQP4 was first observed at P7 in the developing pars plicata, and the AQP4-positive area gradually spread to cover the entire pars plicata as development proceeded. These findings indicate that both AQP1 and AQP4 contribute to aqueous humor secretion in the rat eye, thereby maintaining proper intraocular pressure. Moreover, AQP appears to play a major role in aqueous humor secretion in early eye development. This study thus provides a basis for understanding the molecular mechanisms of aqueous humor secretion in pathological and physiological conditions.     

Anti-inflammatory effect of the alpha-melanocyte stimulating hormonein animal eyes undergoing extracapsular lens extraction

In this study anti-inflammatory effects of the alpha-melanocyte stimulating hormone (α-MSH) on ocular inflammation caused by extracapsular lens extraction (ECLE) have been investigated and the potential mechanism of an anti-inflammatory effect is discussed. Pigmented rabbit eyes after ECLE were treated locally with α-MSH, dexamethasone, diclofenac, or saline 4 times a day (q.i.d.) for 4 weeks. The inhibitory effect of α-MSH on infiltrating cells in the aqueous humor (AqH) was almost twice as good as that of dexamethasone or diclofenac for 3 days, 1 week, and 2 weeks after the operation. The eyes of Sprague-Dawley rats were treated with an intravenous injection of α-MSH or saline immediately after ECLE. Six hours postoperatively, the iris/ciliary body exhibited increased expression of TNF-α and IL-6 mRNAs, which were significantly decreased after α-MSH treatment. The number of activated NF-kappa B (NFκB)-positive cells in the iris/ciliary body was also significantly reduced by the α-MSH treatment. These results suggested that α-MSH could effectively reduce ocular inflammation after ECLE, and the potential mechanism for this is by down-regulating the expression of proinflammatory cytokines and inhibiting the NFκB-dependent signaling pathway.     

Study of 27 Aqueous Humor Cytokines in Type 2 Diabetic Patients with or without Macular Edema

The aim of the present study was to compare the changes in the levels of 27 aqueous humor cytokines between diabetic patients with macular edema (ME) and diabetic patients without ME. Undiluted aqueous humor samples were obtained from 68 consecutive type 2 diabetic patients without ME and 56 consecutive type 2 diabetic patients with ME. The concentrations of 27 cytokines in the aqueous humor samples were measured using a multiplex bead immunoassay. Compared with diabetic patients without ME, diabetic patients with ME had significantly higher concentrations of IL-1β, IL-6, IL-8, IP-10, MCP-1, and VEGF in the aqueous humor. However, the concentrations of IL-10 and IL-12 were significantly lower in the diabetic patients with ME. The aqueous humor levels of IL-1β, IL-6, IL-8, MCP-1, IP-10, and VEGF were closely correlated with retinal macular thickness, retinal macular volume and the severity of ME. In addition, the aqueous humor levels of IL-10 and IL-12 decreased with increasing the severity of ME. A variety of cytokines associated with inflammation and angiogenesis may contribute to the pathogenesis of diabetic macular edema, and both anti-inflammatory and antiangiogenic agents should be included in the treatment of ME simultaneously.     

S100-β aggravates spinal cord injury via activation of M1 macrophage phenotype

Objectives:S100-β has been identified as a sensitive biomarker in central nervous system injuries. However, the functions and mechanisms of S100-β are unknown in spinal cord injury.Methods:Spinal cord injury (SCI) mouse model was generated by surgical operation, microglia activation model was established by inducing BV-2 cells with LPS. The SCI model was evaluated by Basso-Beattie-Bresnahan (BBB) behavioral score, HE staining, and Nissl staining. The expression level of S100-β was detected by qRT-PCR, western blot, and immunofluorescence. qRT-PCR and western blot were used to detect the expression of iNOS and CD16. Pro-inflammatory cytokines TNF-α and IL-1β levels were detected by qRT-PCR and ELISA.Results:The expression of IL-1β, TNF-α, iNOS, and CD16 increased at 3^rd day after SCI. In BV2 microglia, LPS treatment promoted the expression of S100-β, IL-1β, TNF-α, iNOS, and CD16. Knockdown of S100-β reduced the expression of iNOS stimulated by LPS. Over-expression of S100-β increased IL-1β and TNF-α, and S100-β inhibition suppressed IL-1β and TNF-α. In SCI mice, knockdown of S100-β attenuated the spinal cord injury and inhibited the expression of iNOS, IL-1β, and TNF-α.Conclusions:Down-regulation of S100-β could inhibit the pathogenesis of SCI and inhibit the activation of M1 macrophages. S100-β may be a useful diagnostic biomarker or therapeutic target for SCI.     

Effects of Benzalkonium Chloride on THP-1 Differentiated Macrophages In Vitro

Purpose

To characterize the effects of benzalkonium chloride (BAK) in THP-1 differentiated cells in vitro.

Methods

Macrophages were obtained after differentiation of THP-1 cells, a human monocytic leukemia cell line. Macrophages were exposed for 24 h to 33 nM (10⁻⁵%) benzalkonium chloride (BAK), 10 nM dinitrochlorobenzene (DNCB), 100 ng/mL lipopolysaccharide (LPS), 5 ng/mL tumor necrosis factor alpha (TNF-α) or phosphate buffered saline (PBS) as controls. The expression of CD11b, CD11c, CD33 and CD54 was evaluated using immunohistochemistry and flow cytometry (FCM). Phagocytosis function was analyzed using carboxylate-modified fluorescent microspheres and quantified by FCM. Migration was evaluated in cocultures with conjunctival epithelial cells. Cytokine production was detected and quantified in culture supernatants using a human cytokine array.

Results

Stimulation of THP-1-derived macrophages with a low concentration of BAK increased CD11b and CD11c expression and decreased CD33. Macrophages exposed to BAK, LPS and TNF-α had increased phagocytosis. In contrast to LPS, BAK and TNF-α increased macrophage migration. Cytokines in supernatants of macrophages exposed to BAK revealed an increased release of CCL1, CCL4/MIP-1β, TNF-α, soluble CD54/ICAM-1 and IL-1β.

Conclusion

In vitro, BAK has a direct stimulating effect on macrophages, increasing phagocytosis, cytokine release, migration and expression of CD11b and CD11c. Long-term exposure to low concentrations of BAK should be considered as a stimulating factor responsible for inflammation through macrophage activation.     

Aqueous Cytokine Changes Associated with Posner-Schlossman Syndrome with and without Human Cytomegalovirus

Aim

To study the differences in aqueous cytokines in Posner-Schlossman Syndrome (PSS) patients with and without human cytomegalovirus (CMV) DNA in the aqueous humor.

Methods

This is a prospective study. Fifty-three uveitis patients with clinical signs of PSS were enrolled and aqueous humor samples were collected. Fourteen PSS patients were positive of CMV DNA in the aqueous by polymerase chain reaction (PCR) analysis. These eyes were negative of common ocular pathogens such as herpes simplex virus, varicella-zoster virus, rubella virus and toxoplasma. Twenty-five otherwise healthy cataract patients were enrolled as controls. Cytokine concentration was measured by a magnetic color-bead-based multiplex assay and analyzed using statistical and classification approaches.

Results

The average age of 53 PSS patients was 48.74±13.43 years (yrs) (mean ± standard deviation) and 66.3±15.0 yrs for the controls. The median CMV viral DNA copy number was 26000/mL aqueous (range 1400 to 85000 copies/mL) in 14 CMV positive patients as determined by quantitative PCR. PSS aqueous had significantly higher Interleukin (IL)-8 (CXCL8), monocyte chemotactic protein-1 (CCL2), macrophage inhibitory protein 1-β (CCL4), granulocyte colony-stimulating factor (GCSF) and transforming growth factor-β (TGF-β) levels than controls after adjusted by age and gender. IL-2, IL-12, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α) levels were significantly lower in PSS aqueous than controls. No difference between CMV positive PSS and CMV negative PSS aqueous was observed. Over 97% of PSS samples were distinguished from controls by elevated CXCL10 (>500 ng/mL), CXCL8 (>30 ng/mL) and CCL2 (>60 ng/mL) levels.

Conclusion

PSS eyes were characterized by elevated aqueous chemokine concentration. The presence of CMV viral DNA was not associated with significant change of the type of cytokine expression in PSS patients.     

Melittin-loaded Iron Oxide Nanoparticles Prevent Intracranial Arterial Dolichoectasia Development through Inhibition of Macrophage-mediated Inflammation

Rationale: In intracranial arterial dolichoectasia (IADE) development, the feedback loop between inflammatory cytokines and macrophages involves TNF-α and NF-κB signaling pathways and leads to subsequent MMP-9 activation and extracellular matrix (ECM) degeneration. In this proof-of-concept study, melittin-loaded L-arginine-coated iron oxide nanoparticle (MeLioN) was proposed as the protective measure of IADE formation for this macrophage-mediated inflammation and ECM degeneration.Methods: IADE was created in 8-week-old C57BL/6J male mice by inducing hypertension and elastase injection into a basal cistern. Melittin was loaded on the surface of ION as a core-shell structure (hydrodynamic size, 202.4 nm; polydispersity index, 0.158). Treatment of MeLioN (2.5 mg/kg, five doses) started after the IADE induction, and the brain was harvested in the third week. In the healthy control, disease control, and MeLioN-treated group, the morphologic changes of the cerebral arterial wall were measured by diameter, thickness, and ECM composition. The expression level of MMP-9, CD68, MCP-1, TNF-α, and NF-κB was assessed from immunohistochemistry, polymerase chain reaction, and Western blot assay.Results: MeLioN prevented morphologic changes of cerebral arterial wall related to IADE formation by restoring ECM alterations and suppressing MMP-9 expression. MeLioN inhibited MCP-1 expression and reduced CD68-positive macrophage recruitments into cerebral arterial walls. MeLioN blocked TNF-α activation and NF-κB signaling pathway. In the Sylvian cistern, co-localization was found between the CD68-positive macrophage infiltrations and the MeLioN distributions detected on Prussian Blue and T2* gradient-echo MRI, suggesting the role of macrophage harboring MeLioN.Conclusions: The macrophage infiltration into the arterial wall plays a critical role in the MMP-9 secretion. MeLioN, designed for ION-mediated melittin delivery, effectively prevents IADE formation by suppressing macrophage-mediated inflammations and MMP activity. MeLioN can be a promising strategy preventing IADE development in high-risk populations.     

Liposomal Lipopolysaccharide Initiates TRIF-Dependent Signaling Pathway Independent of CD14

Lipopolysaccharide (LPS) is recognized by CD14 with Toll-like receptor 4 (TLR4), and initiates 2 major pathways of TLR4 signaling, the MyD88-dependent and TRIF-dependent signaling pathways. The MyD88-dependent pathway induces inflammatory responses such as the production of TNF-α, IL-6, and IL-12 via the activation of NFκB and MAPK. The TRIF-dependent pathway induces the production of type-I IFN, and RANTES via the activation of IRF-3 and NFκB, and is also important for the induction of adaptive immune responses. CD14 plays a critical role in initiating the TRIF-dependent signaling pathway response to LPS, to support the internalization of LPS via endocytosis. Here, we clearly demonstrate that intracellular delivery of LPS by LPS-formulated liposomes (LPS-liposomes) initiate only TRIF-dependent signaling via clathrin-mediated endocytosis, independent of CD14. In fact, LPS-liposomes do not induce the production of TNF-α and IL-6 but induce RANTES production in peritoneal macrophages. Additionally, LPS-liposomes could induce adaptive immune responses effectively in CD14-deficient mice. Collectively, our results strongly suggest that LPS-liposomes are useful as a TRIF-dependent signaling-based immune adjuvant without inducing unnecessary inflammation.     

TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells.