Plasminogen and plasmin can bind to human T cells and generate truncated CCL21 that increases dendritic cell chemotactic responses.

Plasmin is a broad-spectrum protease and therefore needs to be tightly regulated. Active plasmin is formed from plasminogen, which is found in high concentrations in the blood and is converted by the plasminogen activators. In the circulation, high levels of α2-antiplasmin rapidly and efficiently inhibit plasmin activity. Certain myeloid immune cells have been shown to bind plasmin and plasminogen on their cell surface via proteins that bind to the plasmin(ogen) kringle domains. Our earlier work showed that T cells can activate plasmin but that they do not themselves express plasminogen. Here, we demonstrate that T cells express several known plasminogen receptors and that they bind plasminogen on their cell surface. We show T cell-bound plasminogen was converted to plasmin by plasminogen activators upon T cell activation. To examine functional consequences of plasmin generation by activated T cells, we investigated its effect on the chemokine, C-C motif chemokine ligand 21 (CCL21). Video microscopy and Western blotting confirmed that plasmin bound by human T cells cleaves CCL21 and increases the chemotactic response of monocyte-derived dendritic cells toward higher CCL21 concentrations along the concentration gradient by increasing their directional migration and track straightness. These results demonstrate how migrating T cells and potentially other activated immune cells may co-opt a powerful proteolytic system from the plasma toward immune processes in the peripheral tissues, where α2-antiplasmin is more likely to be absent. We propose that plasminogen bound to migrating immune cells may strongly modulate chemokine responses in peripheral tissues.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Site-specific glycation of Aß1-42 affects fibril formation and is neurotoxic.

Aß1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). Nϵ-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of Aß1-42. Methyglyoxal is commonly used for the unspecific glycation of Aß1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on Aß1-42 at Lys-16 (Aß-CEL16), Lys-28 (Aß-CEL28), and Lys-16 and -28 (Aß-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of Aß1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that Aß-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of Aß1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of Aß1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only Aß-CEL16 and Aß-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas Aß-CEL16&28 showed no neurotoxicity. Interestingly, Aß-CEL16&28 had depolarized the mitochondrial membrane potential, whereas Aß-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated Aß1-42.

Physiological and pathological functions of neuroserpin: Regulation of cellular responses through multiple mechanisms.

It is 27 years since neuroserpin was first discovered in the nervous system and identified as a member of the serpin superfamily. Since that time potential roles for this serine protease inhibitor have been identified in neuronal and non-neuronal systems. Many are linked to inhibition of neuroserpin's principal enzyme target, tissue plasminogen activator (tPA), although some have been suggested to involve alternate non-inhibitory mechanisms. This review focuses mainly on the inhibitory roles of neuroserpin and discusses the evidence supporting tPA as the physiological target. While the major sites of neuroserpin expression are neural, endocrine and immune tissues, most progress on characterizing functional roles for neuroserpin have been in the brain. Roles in emotional behaviour, synaptic plasticity and neuroprotection in stroke and excitotoxicity models are discussed. Current knowledge on three neurological diseases associated with neuroserpin mutation or activity, Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), Alzheimer's disease and brain metastasis is presented. Finally, we consider mechanistic studies that have revealed a distinct inhibitory mechanism for neuroserpin and its possible implications for neuroserpin function.

Efficient synthesis and characterisation of the amyloid beta peptide, Aß1-42, using a double linker system.

The amyloidogenic Aß42 peptide was efficiently prepared using a double linker system, markedly improving solubility and chromatographic peak resolution, thus enabling full characterisation using standard techniques. The tag was readily cleaved with sodium hydroxide and removed by aqueous extraction, affording Aß42 in high purity and yield for biophysical characterisation studies.

Plasmin and regulators of plasmin activity control the migratory capacity and adhesion of human T cells and dendritic cells by regulating cleavage of the chemokine CCL21.

The homeostatic chemokine CCL21 has a pivotal role in lymphocyte homing and compartment localisation within the lymph node, and also affects adhesion between immune cells. The effects of CCL21 are modulated by its mode of presentation, with different cellular responses seen for surface-bound and soluble forms. Here we show that plasmin cleaves surface-bound CCL21 to release the C-terminal peptide responsible for CCL21 binding to glycosaminoglycans on the extracellular matrix and cell surfaces, thereby generating the soluble form. Loss of this anchoring peptide enabled the chemotactic activity of CCL21 and reduced cell tethering. Tissue plasminogen activator did not cleave CCL21 directly but enhanced CCL21 processing through generation of plasmin from plasminogen. The tissue plasminogen activator inhibitor neuroserpin prevented processing of CCL21 and blocked the effects of soluble CCL21 on cell migration. Similarly, the plasmin-specific inhibitor α2-antiplasmin inhibited CCL21-mediated migration of human T cells and dendritic cells and tethering of T cells to APCs. We conclude that the plasmin system proteins plasmin, tissue plasminogen activator and neuroserpin regulate CCL21 function in the immune system by controlling the balance of matrix- and cell-bound CCL21.

An analysis approach to identify specific functional sites in orthologous proteins using sequence and structural information: application to neuroserpin reveals regions that differentially regulate inhibitory activity.

The analysis of sequence conservation is commonly used to predict functionally important sites in proteins. We have developed an approach that first identifies highly conserved sites in a set of orthologous sequences using a weighted substitution-matrix-based conservation score and then filters these conserved sites based on the pattern of conservation present in a wider alignment of sequences from the same family and structural information to identify surface-exposed sites. This allows us to detect specific functional sites in the target protein and exclude regions that are likely to be generally important for the structure or function of the wider protein family. We applied our method to two members of the serpin family of serine protease inhibitors. We first confirmed that our method successfully detected the known heparin binding site in antithrombin while excluding residues known to be generally important in the serpin family. We next applied our sequence analysis approach to neuroserpin and used our results to guide site-directed polyalanine mutagenesis experiments. The majority of the mutant neuroserpin proteins were found to fold correctly and could still form inhibitory complexes with tissue plasminogen activator (tPA). Kinetic analysis of tPA inhibition, however, revealed altered inhibitory kinetics in several of the mutant proteins, with some mutants showing decreased association with tPA and others showing more rapid dissociation of the covalent complex. Altogether, these results confirm that our sequence analysis approach is a useful tool that can be used to guide mutagenesis experiments for the detection of specific functional sites in proteins.

Chemicals eluting from disposable plastic syringes and syringe filters alter neurite growth, axogenesis and the microtubule cytoskeleton in cultured hippocampal neurons.

Cultures of dissociated hippocampal neurons are often used to study neuronal cell biology. We report that the development of these neurons is strongly affected by chemicals leaching from commonly used disposable medical-grade syringes and syringe filters. Contamination of culture medium by bioactive substance(s) from syringes and filters occurred with multiple manufacturing lots and filter types under normal use conditions and resulted in changes to neurite growth, axon formation and the neuronal microtubule cytoskeleton. The effects on neuronal morphology were concentration dependent and significant effects were detected even after substantial dilution of the contaminated medium. Gas chromatography-mass spectrometry analyses revealed many chemicals eluting from the syringes and filters. Three of these chemicals (stearic acid, palmitic acid and 1,2-ethanediol monoacetate) were tested but showed no effects on neurite growth. Similar changes in neuronal morphology were seen with high concentrations of bisphenol A and dibutyl phthalate, two hormonally active plasticisers. Although no such compounds were detected by gas chromatography­mass spectrometry, unknown plasticisers in leachates may affect neurites. This is the first study to show that leachates from laboratory consumables can alter the growth of cultured hippocampal neurons. We highlight important considerations to ensure leachate contamination does not compromise cell biology experiments.

MicroRNA regulation in human CD8+ T cell subsets--cytokine exposure alone drives miR-146a expression.

BACKGROUND: microRNAs (miRNAs) are emerging as key regulators of the immune system, but their role in CD8+ T cell differentiation is not well explored. Some evidence suggests that signals from cell surface receptors influence the expression of miRNAs in CD8+ T cells, and may have consequent effects on cell phenotype and function. We set out to investigate whether common gamma chain cytokines modulated human CD8+ T cell expression of miR-146a, which previous studies have associated with different stages of CD8+ differentiation. We also investigated how changes in miR-146a related to other miRNAs that alter with CD8+ differentiation status. METHODS: We treated human CD8+ T cells with the cytokines IL-2, IL-7 or IL-15 either at rest or after stimulation with anti-CD3 and anti-CD28. For some experiments we also purified human CD8+ T cell subsets ex vivo. Flow cytometry was used in parallel to assess cell surface memory marker expression. Total RNA from these cells was subjected to microarray analysis and real-time PCR for miRNA expression. Nucleofection studies were performed to assess potential mRNA targets of miR-146a. RESULTS: We find that miR-146a is up-regulated in naïve CD8+ T cells exposed to IL-2 or IL-15, even in the absence of an activating T cell receptor stimulus, but not when IL-7 is also present. miR-146a expression correlates with a memory phenotype in both ex vivo and in vitro cultured cells although in our hands overexpression of miR-146a was not sufficient alone to drive a full memory phenotype. In ex vivo analysis, miR-146a was one of a small number of miRNAs that was differentially expressed between naïve and memory CD8+ T cells. CONCLUSIONS: miR-146a is emerging as a critical regulator of immune system. Our data shows that miR-146a expression is strongly influenced by the cytokine milieu even in the absence of a T cell receptor stimulus. Our results have implications for studies designed to assess the function of miR-146a, help to define a fingerprint of miRNA expression in CD8+ T cell subsets and may be useful when designing optimal protocols for T cell expansion as efficacy of T cell immunotherapy is correlated with an 'early' memory phenotype.

The serine protease inhibitor neuroserpin regulates the growth and maturation of hippocampal neurons through a non-inhibitory mechanism.

Neuroserpin is a brain-specific serine protease inhibitor that is expressed in the developing and adult nervous system. Its expression profile led to suggestions that it played roles in neuronal growth and connectivity. In this study, we provide direct evidence to support a role for neuroserpin in axon and dendritic growth. We report that axon growth is enhanced while axon and dendrite diameter are reduced following neuroserpin treatment of hippocampal neurons. More complex effects are seen on dendritic growth and branching with neuroserpin-stimulating dendritic growth and branching in young neurons but switching to an inhibitory response in older neurons. The protease inhibitory activity of neuroserpin is not required to activate changes in neuronal morphology and a proportion of responses are modulated by an antagonist to the LRP1 receptor. Collectively, these findings support a key role for neuroserpin as a regulator of neuronal development through a non-inhibitory mechanism and suggest a basis for neuroserpin's effects on complex emotional behaviours and recent link to schizophrenia.

Live-Cell Microscopy Reveals That Human T Cells Primarily Respond Chemokinetically Within a CCL19 Gradient That Induces Chemotaxis in Dendritic Cells.

The ability to study migratory behavior of immune cells is crucial to understanding the dynamic control of the immune system. Migration induced by chemokines is often assumed to be directional (chemotaxis), yet commonly used end-point migration assays are confounded by detecting increased cell migration that lacks directionality (chemokinesis). To distinguish between chemotaxis and chemokinesis we used the classic "under-agarose assay" in combination with video-microscopy to monitor migration of CCR7+ human monocyte-derived dendritic cells and T cells in response to a concentration gradient of CCL19. Formation of the gradients was visualized with a fluorescent marker and lasted several hours. Monocyte-derived dendritic cells migrated chemotactically towards the CCL19 gradient. In contrast, T cells exhibited a biased random walk that was largely driven by increased exploratory chemokinesis towards CCL19. This dominance of chemokinesis over chemotaxis in T cells is consistent with CCR7 ligation optimizing T cell scanning of antigen-presenting cells in lymphoid tissues.

Expression of titin-linked putative mechanosensing proteins in skeletal muscle after power resistance exercise in resistance-trained men.

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex + Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P ≤ 0.05) pHSP27-Ser15 (∼6-fold) and creatine kinase (∼50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (∼15-fold) and MLP (∼2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (∼3-fold, Ex) and MLPb (∼20-fold, Ex + Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.NEW & NOTEWORTHY This is the first study to assess the early changes in the expression of titin-linked putative mechanosensing proteins and associated myogenic regulatory factors in skeletal muscle after power resistance exercise in fed, resistance-trained men. We report that power resistance exercise induces subtle early responses in the expression of Ankrd 1 and MLP, suggesting these proteins play a role in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.

Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neurons.

Creatine uptake by neurons requires a specific creatine transporter (CRT). The purpose of the present work was to investigate the activity and localization of the CRT in primary cultures of hippocampal neurons obtained from 18-day rat embryos. Creatine uptake increased as the neurons differentiated in culture. Immunofluorescence microscopy showed most of the CRT was associated with dendrites, although some CRT was present in axons and axon terminals. Neurons contained high levels of Na(+)-dependent creatine transport activity (K(m) = 45.5 µM; V(max), 1719 pmol creatine/min/mg protein) which was inhibited by competitive inhibitors of the CRT. The IC(50) for guanidinoacetate, a precursor of creatine, was 712 µM, ∼ 15-fold higher than the K(m) for creatine. Incubation of neurons with 1 mM creatine resulted in the accumulation of high levels of creatine which affected the V(max) but not the K(m) for creatine transport. The rate of creatine release from neurons increased in the absence of Na(+) showing the importance of the electrochemical gradient for creatine retention. This is the first detailed study of the CRT in neurons and identifies primary cultures of rat hippocampal neurons as a good model for future studies of the CRT in relation to the effects of creatine on neuronal function and viability.

Neuroserpin regulates the density of dendritic protrusions and dendritic spine shape in cultured hippocampal neurons.

Neuroserpin is a member of the serpin superfamily that is expressed principally in neurons of the central and peripheral nervous systems. Neuroserpin's spatial-temporal expression during development and in the adult brain suggests possible roles in synaptogenesis and synaptic plasticity. This is supported by behavioral changes in transgenic mice overexpressing neuroserpin. We have used an embryonic rat primary hippocampal neuron culture model to investigate whether neuroserpin can regulate elements of synaptic morphology that may be involved in these changes in cognitive function. Neuroserpin localized to axonal and dendritic compartments in cultured neurons and accumulated in synapsin-positive presynaptic terminals. Increased expression of neuroserpin resulted in an increase in the density of dendritic protrusions and alterations in dendritic spine shape. Our results identify neuroserpin as a new regulator of structural plasticity and suggest a cellular mechanism that may contribute to neuroserpin's effects on cognition.

Neuroserpin regulates human T cell-T cell interactions and proliferation through inhibition of tissue plasminogen activator.

T cells play a key role in mounting an adaptive immune response. T cells are activated upon recognition of cognate Ag presented by an APC. Subsequently, T cells adhere to other activated T cells to form activation clusters, which lead to directed secretion of cytokines between communicating cells. T cell activation clusters have been implicated in regulating activation, proliferation, and memory formation in T cells. We previously reported the expression of the protease inhibitor neuroserpin by human T cells and showed that expression and intracellular localization is regulated following T cell activation. To gain a better understanding of neuroserpin in the proteolytic environment postactivation we assessed its role in human T cell clustering and proliferation. Neuroserpin knockdown increased T cell proliferation and cluster formation following T cell activation. This increased cluster formation was dependent on the proteases tissue plasminogen activator (tPA) and plasmin. Furthermore, neuroserpin knockdown or plasmin treatment of T cells increased the cleavage of annexin A2, a known plasmin target that regulates the actin cytoskeleton. Live cell imaging of activated T cells further indicated a role of the actin cytoskeleton in T cell clustering. The inhibition of actin regulators myosin ATPase and Rho-associated protein kinase signaling completely reversed the neuroserpin knockdown-induced effects. The results presented in this study reveal a novel role for neuroserpin and the proteolytic environment in the regulation of T cell activation biology.

Neuroserpin regulates N-cadherin-mediated cell adhesion independently of its activity as an inhibitor of tissue plasminogen activator.

Neuroserpin is an inhibitor of tissue plasminogen activator (tPA) that is expressed in developing and adult nervous systems. Spatial and temporal analysis of neuroserpin expression suggests that it is involved in regulating the proteolytic balance associated with axonogenesis and synaptogenesis during development and synaptic plasticity in the adult. Here we demonstrate that altered expression of neuroserpin modulates the degree of cell-cell adhesion in pheochromocytoma PC12 cells independently of its role as an inhibitor of tPA. Levels of the homophilic cell-cell adhesion molecule N-cadherin are increased in neuroserpin-overexpressing cell lines. N-cadherin immunoreactivity was detected in a Triton X-100-insoluble fraction and localized to regions of cell contact, consistent with a role in enhancing cell surface adhesion. PC12 cell lines expressing neuroserpin mutants that lack tPA inhibitory activity also showed increased cell-cell adhesion and N-cadherin expression. Our results identify neuroserpin as a novel regulator of cell-cell adhesion and the synaptic adhesion molecule N-cadherin as a key effecter in this response. In nerve cells, neuroserpin may regulate the levels of N-cadherin available for construction, maintenance, and control of synapses and synaptic dynamics.

Expression of the serine protease inhibitor neuroserpin in cells of the human myeloid lineage.

Myeloid progenitors in the bone marrow differentiate into most of the major cell types of the immune system, including macrophages and dendritic cells. These cells play important roles in both innate and adaptive immunity. They express a number of proteases and protease inhibitors including members of the serine proteinase inhibitor or serpin superfamily. In this study we report the differential expression of neuroserpin in cells of the human myeloid lineage. Neuroserpin was highly expressed and secreted following the differentiation of monocytes to macrophages and dendritic cells. Activation of dendritic cells with lipopolysaccharide resulted in increased neuroserpin mRNA levels but no neuroserpin secretion. Confocal immunofluorescence microscopy showed neuroserpin was differentially localised in human myeloid cells. In macrophages and dendritic cells it was concentrated in vesicles located in close proximity to the plasma membrane. The majority of activated dendritic cells also exhibited an intracellular focal concentration of neuroserpin which co-localised with the lysosomal/late endosomal marker LAMP-1. As neuroserpin inhibits tissue plasminogen activator, a comparative analysis of tPA and plasminogen activator inhibitor-1 (PAI-1) expression was undertaken. This analysis revealed differential expression of PAI-1 and neuroserpin suggesting they may have different functions in human immune cells.

The transmembrane domain of the prohormone convertase PC3: a key motif for targeting to the regulated secretory pathway.

The biosynthesis of hormones and neuropeptides involves post-translational cleavage of precursors at basic amino acids by prohormone convertases (PCs) predominantly in secretory granules that bud from the trans-Golgi Network. This study reports that the amino acid sequence of PC3 (aa617-638), previously identified as a novel transmembrane (TM) domain, confers lipid raft association and facilitates sorting of the enzyme to the secretory granules of Neuro2A cells for prohormone cleavage. Floatation analysis on sucrose density gradients showed that a proportion of full length (PC3-FL) and carboxyl terminus-truncated PC3(1-638) (PC3-638) containing the TM domain were associated with lipid rafts in Neuro2A cells, while PC3(1-616) (PC3-616) and PC3-DeltaTM lacking the TM domain were not. Secondly, PC3-FL and PC3-638 underwent stimulated secretion and were shown to be colocalized with a secretory granule marker, chromogranin A, by immunocytochemistry. In contrast, PC3-616 and PC3-DeltaTM were constitutively secreted and primarily localized in the Golgi. These data indicate that the transmembrane domain of PC3 plays a key role in sorting the enzyme to the regulated secretory pathway.

Neuroserpin Attenuates H2O2-Induced Oxidative Stress in Hippocampal Neurons via AKT and BCL-2 Signaling Pathways.

Oxidative stress plays a critical role in neuronal injury and is associated with various neurological diseases. Here, we explored the potential protective effect of neuroserpin against oxidative stress in primary cultured hippocampal neurons. Our results show that neuroserpin inhibits H2O2-induced neurotoxicity in hippocampal cultures as measured by WST, LDH release, and TUNEL assays. We found that neuroserpin enhanced the activation of AKT in cultures subjected to oxidative stress and that the AKT inhibitor Ly294002 blocked this neuroprotective effect. Neuroserpin increased the expression of the anti-apoptotic protein BCL-2 and blocked the activation of caspase-3. Neuroserpin did not increase the level of neuroprotection over levels seen in neurons transduced with a BCL-2 expression vector, and an inhibitor of Trk receptors, K252a, did not block neuroserpin's effect. Taken together, our study demonstrates that neuroserpin protects against oxidative stress-induced dysfunction and death of primary cultured hippocampal neurons through the AKT-BCL-2 signaling pathway through a mechanism that does not involve the Trk receptors and leads to inhibition of caspase-3 activation.

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport.

Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10-8 cm/s vs (738 ± 190) ×10-8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI-H441 cells as a model for investigating ion and water transport in the human alveolar epithelium and also identify the pathways of sodium and chloride transport.