Pentoxifylline, dexamethasone and azithromycin demonstrate distinct age-dependent and synergistic inhibition of TLR- and inflammasome-mediated cytokine production in human newborn and adult blood in vitro.

INTRODUCTION: Neonatal inflammation, mediated in part through Toll-like receptor (TLR) and inflammasome signaling, contributes to adverse outcomes including organ injury. Pentoxifylline (PTX), a phosphodiesterase inhibitor which potently suppresses cytokine production in newborn cord blood, is a candidate neonatal anti-inflammatory agent. We hypothesized that combinations of PTX with other anti-inflammatory agents, the steroid dexamethasone (DEX) or the macrolide azithromycin (AZI), may exert broader, more profound and/or synergistic anti-inflammatory activity towards neonatal TLR- and inflammasome-mediated cytokine production. METHODS: Whole newborn and adult blood was treated with PTX (50-200 µM), DEX (10-10-10-7 M), or AZI (2.5-20 µM), alone or combined, and cultured with lipopolysaccharide (LPS) (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/adenosine triphosphate (ATP) (inflammasome induction). Supernatant and intracellular cytokines, signaling molecules and mRNA were measured by multiplex assay, flow cytometry and real-time PCR. Drug interactions were assessed based on Loewe's additivity. RESULTS: PTX, DEX and AZI inhibited TLR- and/or inflammasome-mediated cytokine production in newborn and adult blood, whether added before, simultaneously or after TLR stimulation. PTX preferentially inhibited pro-inflammatory cytokines especially TNF. DEX inhibited IL-10 in newborn, and TNF, IL-1ß, IL-6 and interferon-α in newborn and adult blood. AZI inhibited R848-induced TNF, IL-1ß, IL-6 and IL-10, and LPS-induced IL-1ß and IL-10. (PTX+DEX) synergistically decreased LPS- and LPS/ATP-induced TNF, IL-1ß, and IL-6, and R848-induced IL-1ß and interferon-α, while (PTX+AZI) synergistically decreased induction of TNF, IL-1ß, and IL-6. Synergistic inhibition of TNF production by (PTX+DEX) was especially pronounced in newborn vs. adult blood and was accompanied by reduction of TNF mRNA and enhancement of IL10 mRNA. CONCLUSIONS: Age, agent, and specific drug-drug combinations exert distinct anti-inflammatory effects towards TLR- and/or inflammasome-mediated cytokine production in human newborn blood in vitro. Synergistic combinations of PTX, DEX and AZI may offer benefit for prevention and/or treatment of neonatal inflammatory conditions while potentially limiting drug exposure and toxicity.

Annexin A7 trafficking to alveolar type II cell surface: possible roles for protein insertion into membranes and lamellar body secretion.

A role for annexin A7 (A7) is postulated in the obligatory fusion between lamellar bodies and the plasma membrane during surfactant secretion in alveolar type II cells. This study investigated if surfactant secretagogues increase cell surface A7, which could support A7 insertion into plasma membrane as annexin proteins reportedly lack membrane penetration ability. In vivo trafficking of A7 to cell surface was determined by immuno-staining after non-permeabilizing fixation of alveolar type II cells. Stimulation with various secretagogues increased protein kinase-dependent staining for A7 and ABCA3 in comparison to control cells. Biotin-labeling of surface proteins showed ~4% of total A7 in control cells, which increased ~3-4 folds in stimulated type II cells. Increased cell surface A7 was also observed by protein cross-linking studies showing ~70kDa A7-adduct in the membranes but not in the cytosol fraction of PMA- or A23187-stimulated cells. In vitro phosphorylation increased the Ca(2+)-dependent binding of recombinant A7 to lung plasma membranes; and subsequent cross-linking showed increased levels of ~70kDa A7-adduct. PMA-stimulation of type II cells increased A7 trafficking to lipid rafts suggesting that the latter are involved in A7 trafficking to the cell surface. However, in vitro membrane insertion of recombinant A7 and its tryptophan mutants as determined by fluorescence quenching with doxylPC suggested only shallow membrane insertion by A7. Together, our studies support in vivo association between surfactant secretion and cell surface A7 occurring by insertion into plasma membrane and by fusion of A7 containing lamellar bodies.

Annexin A7 and SNAP23 interactions in alveolar type II cells and in vitro: a role for Ca(2+) and PKC.

Lung surfactant secretion involves lamellar body docking and fusion with the plasma membrane in alveolar type II cells. Annexin A7 (A7) is postulated to play a role in membrane fusion during exocytosis. Our recent studies demonstrated increased co-localization of A7 with ABCA3 in lamellar bodies in type II cells stimulated with established secretagogues of lung surfactant. In this study, we investigated in vivo and in vitro interactions of A7 with the t-SNARE protein, SNAP23. Immuno-fluorescence studies showed time-dependent increases in co-localization of A7 with SNAP23 in PMA- and in A23187-stimulated cells. PMA and A23187 also caused a time-dependent increase in co-localization of ABCA3 with SNAP23. The relocation of A7 to SNAP23 domains was inhibited in the presence of PKC inhibitor, similar to that previously reported for co-localization of A7 with ABCA3. The interaction of A7 and SNAP23 was confirmed by affinity binding and by in vitro interaction of recombinant A7 and SNAP23 proteins. The in vitro binding of recombinant A7 (rA7) to GST-SNAP23 fusion protein was calcium-dependent. Phosphorylation of rA7 with PKC increased its in vitro binding to SNAP23 suggesting that a similar mechanism may operate during A7 relocation to t-SNARE domains. Thus, our studies demonstrate that annexin A7 may function in co-ordination with SNARE proteins and that protein kinase activation may be required for annexin A7 trafficking to the interacting membranes (lamellar bodies and plasma membrane) to facilitate membrane fusion during surfactant secretion.

Secretagogues of lung surfactant increase annexin A7 localization with ABCA3 in alveolar type II cells.

Membrane fusion between the lamellar bodies and plasma membrane is an obligatory event in the secretion of lung surfactant. Previous studies have postulated a role for annexin A7 (A7) in membrane fusion during exocytosis in some cells including alveolar type II cells. However, the intracellular trafficking of A7 during such fusion is not described. In this study, we investigated association of endogenous A7 with lamellar bodies in alveolar type II cells following treatment with several secretagogues of lung surfactant. Biochemical studies with specific antibodies showed increased membrane-association of cell A7 in type II cells stimulated with agents that increase secretion through different signaling mechanisms. Immuno-fluorescence studies showed increased co-localization of A7 with ABCA3, the lamellar body marker protein. Because these agents increase surfactant secretion through activation of PKC and PKA, we also investigated the effects of PKC and PKA inhibitors, bisindolylmaleimideI (BisI) and H89, respectively, on A7 partitioning. Western blot analysis showed that these inhibitors prevented secretagogue-mediated A7 increase in the membrane fractions. These inhibitors also blocked increased co-localization of A7 with ABCA3 in secretagogue-treated cells, as revealed by immuno-fluorescence studies. In vitro studies with recombinant A7 showed phosphorylation with PKC and PKA. The cell A7 was also phosphorylated in cells treated with surfactant secretagogues. Thus, our studies demonstrate that annexin A7 relocates to lamellar bodies in a phosphorylation-dependent manner. We suggest that activation of protein kinase promotes phosphorylation and membrane-association of A7 presumably to facilitate membrane fusion during lung surfactant secretion.

Simulated transport alters surfactant homeostasis and causes dose-dependent changes in respiratory function in neonatal Sprague-Dawley rats.

OBJECTIVE: Forces transmitted to the neonate as a consequence of accelerations during transport have been associated with adverse neonatal outcomes including broncho-pulmonary dysplasia. In this study, we sought to determine the relationship between the duration of transport and respiratory performance in the rat model. METHODS: Four groups of Sprague-Dawley rat pups (10-12 pups/groups) were exposed to simulated medical transport on postnatal day of life 11 or 12. Each group was exposed to an average impulse of 27.4 m/s(2)/min for 0, 30, 60 or 90 min. During the exposure periods, impulse was monitored by computerized sampling using a digital accelerometer. Post-exposure, animals were immediately prepared, placed on mechanical ventilation and analyzed for elastance, tissue damping, airway resistance, ratio of damping to elastance (eta), hysteresivity, and inertance at positive end expiratory pressures (PEEPs) of 0, 3 and 6 cm(3) of H(2)O. Total phospholipid content and surfactant proteins A, B, and C mRNA levels in broncho-alveolar lavage fluid and lung tissue were obtained. RESULTS: Increased transport time resulted in a significant step-wise increase in airway resistance at all levels of PEEP (P<0.01). Static compliance decreased significantly after 60 min at PEEPs of 3 and 6 cm H(2)O (P<0.01). Eta significantly decreased with greater transport time at a PEEP of 6 cm H(2)O (P<0.05). Tissue damping increased with duration of transport time across all PEEP levels, but only exhibited statistical significance at a PEEP of 0 cm H(2)O (P<0.05). No differences were seen in hysteresivity or inertance. Compared with controls, transport was associated with significant reductions in total phospholipid content and mRNA levels of surfactant proteins B and C. CONCLUSION: Rat pups experienced significant deterioration of respiratory function with increasing duration of simulated transport.

Transient in utero disruption of cystic fibrosis transmembrane conductance regulator causes phenotypic changes in alveolar type II cells in adult rats.

BACKGROUND: Mechanicosensory mechanisms regulate cell differentiation during lung organogenesis. We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) was integral to stretch-induced growth and development and that transient expression of antisense-CFTR (ASCFTR) had negative effects on lung structure and function. In this study, we examined adult alveolar type II (ATII) cell phenotype after transient knock down of CFTR by adenovirus-directed in utero expression of ASCFTR in the fetal lung. RESULTS: In comparison to (reporter gene-treated) Controls, ASCFTR-treated adult rat lungs showed elevated phosphatidylcholine (PC) levels in the large but not in the small aggregates of alveolar surfactant. The lung mRNA levels for SP-A and SP-B were lower in the ASCFTR rats. The basal PC secretion in ATII cells was similar in the two groups. However, compared to Control ATII cells, the cells in ASCFTR group showed higher PC secretion with ATP or phorbol myristate acetate. The cell PC pool was also larger in the ASCFTR group. Thus, the increased surfactant secretion in ATII cells could cause higher PC levels in large aggregates of surfactant. In freshly isolated ATII cells, the expression of surfactant proteins was unchanged, suggesting that the lungs of ASCFTR rats contained fewer ATII cells. Gene array analysis of RNA of freshly isolated ATII cells from these lungs showed altered expression of several genes including elevated expression of two calcium-related genes, Ca2+-ATPase and calcium-calmodulin kinase kinase1 (CaMkk1), which was confirmed by real-time PCR. Western blot analysis showed increased expression of calmodulin kinase I, which is activated following phosphorylation by CaMkk1. Although increased expression of calcium regulating genes would argue in favor of Ca2+-dependent mechanisms increasing surfactant secretion, we cannot exclude contribution of alternate mechanisms because of other phenotypic changes in ATII cells of the ASCFTR group. CONCLUSION: Developmental changes due to transient disruption of CFTR in fetal lung reflect in altered ATII cell phenotype in the adult life.

Small interfering peptide (siP) for in vivo examination of the developing lung interactonome.

To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein-protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch-induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno-associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides (siPs) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation. In all cases only siPs with sequences necessary for a predicted normal function were found to interfere with assembly of the multimeric enzyme. A noninterfering control siP to nonessential regions or reporter genes alone had no effect. Physiologically, it was shown that siPs that interfered with the NOX-CFTR-CK2 complex that we call an "interactonome" affected markers of stretch-induced lung organogenesis including Wnt/beta-catenin signaling.

A role for diacylglycerol in annexin A7-mediated fusion of lung lamellar bodies.

Lung surfactant secretion in alveolar type II cells occurs following lamellar body fusion with plasma membrane. Annexin A7 is a Ca2+-dependent membrane-binding protein that is postulated to promote membrane fusion during exocytosis in some cell types including type II cells. Since annexin A7 preferably binds to lamellar body membranes, we postulated that specific lipids could modify the mode of annexin A7 interaction with membranes and its membrane fusion activity. Initial studies with phospholipid vesicles containing phosphatidylserine and other lipids showed that certain lipids affected protein interaction with vesicle membranes as determined by change in protein tryptophan fluorescence, protein interaction with trans membranes, and by protein sensitivity to limited proteolysis. The presence of signaling lipids, diacylglycerol or phosphatidylinositol-4,5-bisphosphate, as minor components also modified the lipid vesicle effect on these characteristics and membrane fusion activity of annexin A7. In vitro incubation of lamellar bodies with diacylglycerol or phosphatidylinositol-4,5-bisphosphate caused their enrichment with either lipid, and increased the annexin A7 and Ca2+-mediated fusion of lamellar bodies. Treatment of isolated lung lamellar bodies with phosphatidylinositol- or phosphatidylcholine phospholipase C to increase diacylglycerol, without or with preincubation with phosphatidylinositol-4,5-bisphosphate, augmented the fusion activity of annexin A7. Thus, increased diacylglycerol in lamellar bodies following cell stimulation with secretagogues may enhance membrane fusion activity of annexin A7.

Leptin enhances lung maturity in the fetal rat.

Pulmonary alveolar type II cells synthesize and secrete phospholipids and surfactant proteins. In most mammalian species, the synthesis of phospholipids and proteins of lung surfactant increases with fetal lung maturation, which occurs late in gestation. Factors that may promote lung maturation and surfactant production include the placental hormone, leptin, whose expression increases with advancing gestational age. We demonstrate that physiologic concentrations of leptin (1 and 10 ng/mL) increase the levels of surfactant proteins (SP) A, B, and C mRNA as well as SP-A and SP-B protein in d-17 fetal rat lung explants in vitro. To determine whether leptin exerts similar effects in vivo, we administered leptin antenatally to pregnant rats and compared its effects to that of dexamethasone, a known mediator of fetal lung development. Antenatal treatment with leptin for 2 d significantly increased the average weight of the fetal lungs in relation to their body weight. Histologic analysis revealed that the increase in fetal lung weight was accompanied by an increase in the number and maturation of type II alveolar cells and the expression of surfactant proteins B and C in these cells. Collectively, these results suggest that leptin is a cytokine regulator of rat fetal lung maturity.

A ten-residue domain (Y11-A20) in the NH2-terminus modulates membrane association of annexin A7.

Annexin A7 (synexin, annexin VII) is postulated to promote membrane fusion during surfactant secretion in alveolar type II cells and catecholamine secretion in adrenal chromaffin cells. Recently, we demonstrated that the 1-29 residues in the NH(2)-terminus could, possibly by interaction with the COOH-terminus, influence the Ca(2+)-dependent membrane binding, aggregation, and fusion properties of annexin A7 (A7). In this study, we further investigated this 29-residue domain by evaluating several deletion and point mutations for membrane-associated functions of A7. In comparison to A7, the mutants lacking 1-29 residues (A7Delta(1-29)) or 1-21 residues (A7Delta(1-21)), but not those lacking 1-10 residues (A7Delta(1-10)) or 21-29 residues (A7Delta(21-29)), showed diminished membrane binding. Segmental deletion of 10-20 residues (A7Delta(10-20)) also decreased the protein binding to membranes. The Ca(2+)-dependent membrane aggregation of PLV with A7Delta(1-29) was maximally diminished but less so with A7Delta(10-20) or A7Delta(1-21) in comparison to that with A7. However, phospholipid vesicle (PVL) aggregation was unaffected with A7Delta(1-10) or A7Delta(21-29). The Ca(2+)-dependent membrane fusion of PLV was also diminished with A7Delta(10-20) and A7Delta(1-29), but not with A7Delta(1-10). Since the mode of annexin A7 association and function with biological membranes could be different, we also evaluated these proteins for functional changes with isolated lung lamellar bodies. In comparison to A7, the binding to lamellar bodies was diminished for A7Delta(1-29) and A7Delta(1-21) but not for A7Delta(1-10). The Ca(2+)-dependent fusion of isolated lamellar bodies with PLV was also diminished with A7Delta(1-29), but not with A7Delta(10-20) or A7Delta(1-21). Taken together, our studies suggest that the 10-residue domain (Y(11)-A(20)) in the NH(2)-terminus modifies the phospholipid binding and aggregation properties of annexin A7. For binding and fusion of biological membranes, the 10-29-residue domain may be required although the annexin A7 properties are primarily modulated through the Y(11)-A(20) domain.

Partial truncation of the NH2-terminus affects physical characteristics and membrane binding, aggregation, and fusion properties of annexin A7.

Annexin A7 (synexin, annexin VII), a member of the annexin family of proteins, causes aggregation of membranes in a Ca2+-dependent manner and has been suggested to promote membrane fusion during exocytosis of lung surfactant, catecholamines, and insulin. Although annexin A7 (A7) was one of the first annexin proteins described, limited studies of its physical characteristics or of structural domains affecting any of its proposed functions have been conducted. As postulated for other annexin proteins, the unique NH2-domain possibly determines the functional specificity of A7. Therefore, we evaluated the effects of segmental deletions in the NH2-terminus on several characteristics associated with the COOH-terminus of A7. The COOH-terminus contains the only tryptophan residue, and all potential trypsin sites, and the Ca2+ and phospholipid binding sites. Recombinant rat A7 and its deletion mutants were expressed using constructs based on the cDNA sequence obtained by screening a rat lung cDNA library. Ca2+ increased the tryptophan fluorescence of A7 and caused a small red shift in the emission maximum (lambdamax), which was further increased in presence of phospholipid vesicles (PLV). NH2-terminal deletions of 29, 51, and 109 residues affected the peak width of fluorescence and lambdamax, surface-exposure of tryptophan residue, and caused a smaller Ca2+-dependent red shift in lambdamax of membrane-bound protein in comparison to A7. Limited proteolysis with trypsin showed that Ca2+ increased the proteolysis of all proteins, but the deletions also affected the pattern of proteolysis. The presence of PLV protected against Ca2+-dependent increase in proteolysis of all proteins. The deletion of first 29 residues also caused decreased membrane binding, aggregation, and fusion, when compared with A7. Collectively, these results suggest that specific NH2-terminus domains can alter those properties of A7 that are normally associated with the COOH-terminus. We speculate that interactions between the NH2- and COOH-termini are required for membrane binding, and aggregation and fusion properties of annexin A7.

Calcium ionophore and phorbol ester increase membrane binding of annexin a7 in alveolar type II cells.

The fusion of lamellar body with plasma membrane, a distal obligatory step in exocytosis of lung surfactant, may be mediated by annexin a7 (anx a7; synexin). To understand the mechanism of anx a7 action, we tested the hypothesis that anx a7 binding to membranes would increase in order to facilitate membrane fusion during stimulation of lung surfactant secretion. Isolated rat alveolar type II cells were treated with established secretagogues of lung surfactant and the membrane and cytosol fractions were analyzed for in vitro binding of anx a7. In cells treated with calcium ionophore (A23187) or phorbol 12-myristate 13-acetate (PMA), anx a7 binding to the membrane fraction was increased by 120%, while that to the cytosol fraction was decreased by 40%, when compared with binding to corresponding fractions from control cells. Protein kinase inhibitors prevented the PMA effects on anx a7 binding. The lamellar body and plasma membrane fractions of A23187-treated cells also showed increased binding of anx a7. The lamellar bodies of A23187-treated cells showed lower K(m) for Ca(2+) and higher maximal binding of anx a7, when compared with those from control cells. Collectively, our findings suggest that these two agents modify membrane proteins to regulate anx a7 binding, which may facilitate increased membrane fusion activity during stimulation of surfactant secretion.