the native basement membrane can profoundly affect epithelial cell behavior.

The ordering of liquid crystals (LCs) is known to be influenced by surfaces and contaminants. Here, we report that picogram per milliliter concentrations of endotoxin in water trigger ordering transitions in micrometer-size LC droplets. The ordering transitions, which occur at surface concentrations of endotoxin that are less than 10−5 Langmuir, are not due to adsorbate-induced changes in the interfacial energy of the LC. The sensitivity of the LC to endotoxin was measured to change by six orders of magnitude with the geometry of the LC (droplet versus slab), supporting the hypothesis that interactions of endotoxin with topological defects in the LC mediate the response of the droplets. The LC ordering transitions depend strongly on glycophospholipid structure and provide new designs for responsive soft matter.

We have previously shown that human corneal epithelial cells sense and react to nanoscale substrate topographic stimuli [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92; Karuri NW, Liliensiek S, Teixeira AI, Abrams G, Campbell S, Nealey PF, et al. Biological length scale topography enhances cell-substratum adhesion of human corneal epithelial cells. J Cell Sci 2004;117(15):3153-64]. Here we demonstrate that cellular responses to nanoscale substrate topographies are modulated by the context in which these stimuli are presented to cells. In Epilife medium, cells aligned preferentially in the direction perpendicular to nanoscale grooves and ridges. This is in contrast to a previous study where cells cultured in DMEM/F12 medium aligned in the direction parallel to nanoscale topographic features [Teixeira AI, Abrams GA, Bertics PJ, Murphy CJ, Nealey PF. Epithelial contact guidance on well-defined micro- and nanostructured substrates. J Cell Sci 2003;116(10):1881-92]. Additionally, cell alignment in Epilife medium was dependent on pattern pitch. Cells switched from perpendicular to parallel alignment when the pitch was increased from 400 to 4,000 nm. There was a transition region (between 800 and 1,600 nm pitch) where both parallel and perpendicular alignments were favored compared to all other cellular orientations. Cells formed focal adhesions parallel to the substrate topographies in this transition region. On the nano- and microscale patterns, 400 and 4,000 nm pitch, focal adhesions were almost exclusively oriented obliquely to the topographic patterns.

The nucleotide receptor P2X7 has been shown to modulate LPS-induced macrophage production of numerous inflammatory mediators. Although the C-terminal portion of P2X7 is thought to be essential for multiple receptor functions, little is known regarding the structural motifs that lie within this region. We show here that the P2X7 C-terminal domain contains several apparent protein-protein and protein-lipid interaction motifs with potential importance to macrophage signaling and LPS action. Surprisingly, P2X7 also contains a conserved LPS-binding domain. In this report, we demonstrate that peptides derived from this P2X7 sequence bind LPS in vitro. Moreover, these peptides neutralize the ability of LPS to activate the extracellular signal-regulated kinases (ERK1, ERK2) and to promote the degradation of the inhibitor of κB-α isoform (IκB-α) in RAW 264.7 macrophages. Collectively, these data suggest that the C-terminal domain of P2X7 may directly coordinate several signal transduction events related to macrophage function and LPS action.

Previous studies have suggested that the P2Z/P2X 7 purinergic receptor can participate in nucleotide-induced modulation of lipopolysaccharide (LPS) stimulated inflammatory mediator production. To test this hypothesis, we evaluated whether antagonism of the P2Z/P2X 7 receptor can influence LPS signaling and expression of the inducible form of nitric-oxide synthase (iNOS) in RAW 264.7 macrophages. In the present study, we demonstrate that pretreatment of RAW 264.7 macrophages with a P2Z/P2X 7 receptor antagonist, periodate oxidized adenosine 5-triphosphate (o-ATP), substantially inhibits LPS-stimulated NO production and iNOS expression without altering cell viability. This effect on LPS-induced iNOS expression is mimicked by a pyridoxal-phosphate-based antagonist (pyridoxal-phosphate-6-azophenyl-2,4-disulfonic acid) of the P2Z/P2X 7 purinergic receptor, indicating that these results are not unique to o-ATP. Additionally, o-ATP prevents cell death induced by P2Z/P2X 7 receptor agonists. To ascertain how P2Z/P2X 7 receptor antagonists influence LPS signaling, we evaluated the capacity of o-ATP to regulate LPSmediated activation of the transcription factor, nuclear factor-B, and the mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2. These experiments reveal that pretreatment of RAW 264.7 cells with o-ATP attenuates the LPS stimulation of a nuclear factor-B-like binding activity. Moreover, the activation of ERK1 and ERK2 by LPS, but not by the phorbol ester, phorbol 12-myristate 13-acetate, is also blocked in RAW 264.7 cells by o-ATP pretreatment. In summary, these data suggest that the P2Z/P2X 7 receptor modulates LPS-induced macrophage activation as assessed by iNOS expression and NO production. This report implicates the P2Z/P2X 7 receptor in the control of protein kinase cascades and transcriptional processes, and these observations are likely to be important for the development of selective purinergic receptor antagonists for the treatment of septic shock.

This paper reports a strategy for the oriented immobilization of protein receptors on gold films possessing nanometer-scale topographies and the detection of protein binding events to these receptors by using liquid crystals. The approach revolves around the use of self-assembled monolayers (SAMs) formed from nitrilotriacetic acid (NTA)-terminated alkanethiols, 1, and tri(ethylene glycol)-terminated alkanethiols, 2. The SAMs are formed on ultrathin gold films that are deposited from a vapor onto silica substrates oriented at an oblique angle of incidence. Single-component SAMs formed from 2 on these gold films resist nonspecific protein adsorption (using cell lysates) and promote uniform planar anchoring of the nematic liquid crystal, 4-cyano-4′-pentylbiphenyl (5CB). Surprisingly, the azimuthal orientation of nematic 5CB is parallel to the direction of maximum roughness within the gold film when using SAMs formed from 2 but perpendicular to the direction of maximum roughness when tetra(ethylene glycol)-terminated SAMs are formed on the gold films. Mixed SAMs formed from 1 and 2 bind the hexahistidine-tagged protein MEK via specific complexation of the hexahistidine tags of MEK to the Ni II -NTA complexes on the surface. When gold films are prepared by oblique deposition at an angle of 30°from the normal, we measure bound MEK to disrupt the uniform orientation of 5CB, thus leading to an easily visualized change in the optical appearance of the liquid crystal. However, by using gold films deposited at an angle of 40°from the normal, we report that bound MEK does not disrupt the alignment of the liquid crystal whereas anti-MEK IgG bound to the MEK does lead to a nonuniform alignment. These results, when combined with appropriate control experiments, suggest that nanostructured surfaces presenting NTA and ethylene glycol terminated SAMs form a useful interface for imaging proteins bound to histidine-tagged, surface-immobilized receptors.

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems. As cytoskeletal function is critical for macrophage behavior, we have tested the importance of these pathways in actin filament reorganization during P2X7 stimulation in RAW 264.7 macrophages. We observed that the P2X7 agonists adenosine 5'-triphosphate (ATP) and 3'-O-(4-benzoylbenzoyl) ATP (BzATP) stimulated actin reorganization and concomitant membrane blebbing within 5 min. Disruption of actin filaments with cytochalasin D attenuated membrane blebbing but not P2X7-dependent pore formation or extracellular-regulated kinase (ERK)1/ERK2 and p38 activation, suggesting that these latter processes do not require intact actin filaments. However, we provide evidence that p38 MAPK and Rho activation but not ERK1/ERK2 activation is important for P2X7-mediated actin reorganization and membrane blebbing. First, activation of p38 and Rho was detected within 5 min of BzATP treatment, which is coincident with membrane blebbing. Second, the p38 inhibitors SB202190 and SB203580 reduced nucleotide-induced blebbing and actin reorganization, whereas the MAPK kinase-1/2 inhibitor U0126, which blocks ERK1/ERK2 activation, had no discernable effect. Third, the Rho-selective inhibitor C3 exoenzyme and the Rho effector kinase, Rho-associated coiled-coil kinase, inhibitor Y-27632, markedly attenuated BzATP-stimulated actin reorganization and membrane blebbing. These data support a model wherein p38- and Rho-dependent pathways are critical for P2X7-dependent actin reorganization and membrane blebbing, thereby facilitating P2X7 involvement in macrophage inflammatory responses.

Macrophage activation is central to the progression of multiple diseases via the release of inflammatory mediators such as cytokines and nitric oxide. Despite the recognized overlap in the regulatory mechanisms involved in mediator production, little formation exists regarding receptor-initiated signaling pathways that coordinately control multiple end points, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production. In this study, the expression of inducible nitric oxide synthase (iNOS) in macrophages is shown to be regulated by calcium and by a purinoreceptor signaling system. The P2Y purinoreceptor partial agonist, 2-methylthio-ATP (2-MeS-ATP), inhibits the expression of iNOS induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in primary macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS in macrophages isolated from CD-1 mice challenged with LPS, and it inhibits LPS-induced TNF-alpha and interleukin-1 alpha (IL-1 alpha) release, thereby preventing endotoxic death. Thus, purinoreceptors and calcium are likely to be critical for macrophage activation and the production of inflammatory mediators stimulated by LPS.