Hyperoxia enhances brain-derived neurotrophic factor and tyrosine kinase B receptor expression in peribronchial smooth muscle of neonatal rats

Airway hyperreactivity is one of the hallmarks of hyperoxic lung injury in early life. As neurotrophins such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) are potent mediators of neuronal plasticity, we hypothesized that neurotrophin levels in the pulmonary system may be disturbed by hyperoxic exposure. We therefore evaluated the effects of hyperoxia on the expression of BDNF, NGF, and their corresponding high-affinity receptors, TrkB and TrkA, respectively, in the lung of rat pups. Five-day-old Sprague-Dawley rat pups were randomized to hyperoxic or control groups and then continuously exposed to hyperoxia (>95% oxygen) or normoxia over 7 days. At both mRNA and protein levels, BDNF was detected in lung but not in trachea; its level was substantially enhanced in lungs from the hyperoxia-exposed rat pups. Distribution of BDNF mRNA by in situ hybridization indicates that peribronchial smooth muscle was the major source of increased BDNF production in response to hyperoxic exposure. Interestingly, hyperoxia-induced elevation of BDNF was not accompanied by any changes of NGF levels in lung. Furthermore, hyperoxic exposure increased the expression of TrkB in peribronchial smooth muscle but had no effect on the distribution of the specific NGF receptor TrkA. These findings indicate that hyperoxic stress not only upregulates BDNF at mRNA and protein levels but also enhances TrkB within peribronchial smooth muscle. However, there was no corresponding effect on NGF and TrkA receptors. We speculate that the increased level of BDNF may contribute to hyperoxia-induced airway hyperresponsiveness in early postnatal life.     

Neurobehavioural responses to hypergravity environment in the CD-1 mouse

Behavioural responses of CD-1 mice exposed to 2 g hypergravity (HG; 60 or 120 min) were investigated during adolescence and at adulthood. To characterise motion sickness (MS), pica behaviour, a reliable MS index measured through kaolin consumption, and spontaneous activity were evaluated before, during and after HG exposure. Short- and/or long-lasting effects on emotional responses, exploratory behaviour and spatial learning performances were also investigated and brain levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) assessed. An increased pica behaviour during post rotational days and a reduction in spontaneous activity during rotation indicated a mild sickness associated with HG, and susceptibility to MS was sex and age related. Short- and long-lasting effects of exposure were also observed, rotated mice showing altered emotional/anxiety behavioural profiles and impaired spatial learning performances. Moreover, central NGF levels were markedly increased after rotation, while minor changes were observed in BDNF levels.     

Long-lasting depression-like behavior and epigenetic changes of BDNF gene expression induced by perinatal exposure to methylmercury

Substantial evidence indicates that predisposition to diseases can be acquired during early stages of development and interactions between environmental and genetic factors may be implicated in the onset of many pathological conditions. Data collected over several decades have shown that chemicals are among the relevant factors that can endanger CNS. We previously showed that perinatal exposure to methylmercury (MeHg) causes persistent changes in learning and motivational behavior in mice. In this study, we report that the depression-like behavior in MeHg-exposed male mice is reversed by chronic treatment with the antidepressant fluoxetine. Behavioral alterations are associated with a decrease in brain-derived neurotrophic factor (BDNF) mRNA in the hippocampal dentate gyrus and fluoxetine treatment restores BDNF mRNA expression. We also show that MeHg-exposure induces long-lasting repressive state of the chromatin structure at the BDNF promoter region, in particular DNA hypermethylation, an increase in histone H3-K27 tri-methylation and a decrease in H3 acetylation at the promoter IV. While fluoxetine treatment does not alter hypermethylation of H3-K27, it significantly up-regulates H3 acetylation at the BDNF promoter IV in MeHg-exposed mice. Our study shows that developmental exposure to low levels of MeHg predisposes mice to depression and induces epigenetic suppression of BDNF gene expression in the hippocampus.     

Enhanced synthesis of brain-derived neurotrophic factor in the lesioned peripheral nerve: different mechanisms are responsible for the regulation of BDNF and NGF mRNA

Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are molecules which regulate the development and maintenance of specific functions in different populations of peripheral and central neurons, amongst them sensory neurons of neural crest and placode origin. Under physiological conditions NGF is synthesized by peripheral target tissues, whereas BDNF synthesis is highest in the CNS. This situation changes dramatically after lesion of peripheral nerves. As previously shown, there is a marked rapid increase in NGF mRNA in the nonneuronal cells of the damaged nerve. The prolonged elevation of NGF mRNA levels is related to the immigration of activated macrophages, interleukin-1 being the most essential mediator of this effect. Here we show that transsection of the rat sciatic nerve also leads to a very marked increase in BDNF mRNA, the final levels being even ten times higher than those of NGF mRNA. However, the time-course and spatial pattern of BDNF mRNA expression are distinctly different. There is a continuous slow increase of BDNF mRNA starting after day 3 post-lesion and reaching maximal levels 3-4 wk later. These distinct differences suggest different mechanisms of regulation of NGF and BDNF synthesis in non-neuronal cells of the nerve. This was substantiated by the demonstration of differential regulation of these mRNAs in organ culture of rat sciatic nerve and Schwann cell culture. Furthermore, using bioassays and specific antibodies we showed that cultured Schwann cells are a rich source of BDNF- and ciliary neurotrophic factor (CNTF)-like neurotrophic activity in addition to NGF. Antisera raised against a BDNF-peptide demonstrated BDNF-immunoreactivity in pure cultured Schwann cells, but not in fibroblasts derived from sciatic nerve.     

Differential Regulation of Hippocampal Neurotrophins During Aging in Rats

Abstract: Neurotrophins are a family of neurotrophic factors with considerable structural homology. We used sensitive and specific two-site enzyme immunoassays to assess age-associated changes in levels of three neurotrophins—nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3)—in the hippocampus of Fischer 344 rats. Expressions of these proteins and their mRNAs were compared in the same animals. More than 200 ng of BDNF per gram of tissue was detected in the hippocampus of 2-month-old rats. This amount was two and 100 times greater than that of NT-3 and NGF, respectively. The levels of BDNF and NT-3 increased further 2–6 months after birth, whereas NGF content declined during this period, and the altered protein levels of all three neurotrophins were maintained 6–18 months postnatally. In contrast to the patterns of protein expression, BDNF mRNA levels increased during both of these periods, and the NT-3 mRNA levels appeared to decline. Changes in the expression of BDNF mRNA and NGF protein were opposite to those reported to occur in Alzheimer's disease. These results suggest that, during normal aging in rats, neurotrophin expression is regulated independently at both the mRNA and posttranslational levels. Any deficiency in their regulation might contribute to neurodegenerative disorders.     

Expression peculiarities of EGR1, neurotrophins and their receptor genes in human lung cancer and in normal lung tissue

Expression of certain neurotrophin genes and their receptors, as well as NGF-induced gene EGRI was studied in human normal lung, squamous cell lung cancer, and adenocarcinoma tissues. Differential expression pattern of NGF, BDNF, and NT-3 mRNA was established by RT-PCR in normal human lung. NGF expression level varying from minor to significant was demonstrated in double specimens (histologically diagnosed human lung cancer and appropriate adjacent tissue). Interestingly, a half of the double specimens studied demonstrated the differential expression pattern in both cancer and adjacent tissues, whereas in other cases no difference in the NGF expression between these pair of tissues was observed. In the majority of the double specimens, we detected low levels of NT3 and BDNF expression for both cancer and adjacent tissue. No expression of TrkA, TrkB, p75 was found in double specimens and normal tissues. Differential expression patterns of TrkC were observed in normal tissues as well as in certain double specimens. High levels of EGR1 expression were detected in normal tissues. No EGRI expression was observed in cancer tissue compared to its high expression level in adjacent tissue in the majority of double specimens.     

Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons

Regulation of calbindin and calretinin expression by brain-derived neurotrophic factor (BDNF) was examined in primary cultures of cortical neurons using immunocytochemistry and northern blot analysis. Here we report that regulation of calretinin expression by BDNF is in marked contrast to that of calbindin. Indeed, chronic exposure of cultured cortical neurons for 5 days to increasing concentrations of BDNF (0.1-10 ng/ml) resulted in a concentration-dependent decrease in the number of calretinin-positive neurons and a concentration-dependent increase in the number of calbindin-immunoreactive neurons. Consistent with the immunocytochemical analysis, BDNF reduced calretinin mRNA levels and up-regulated calbindin mRNA expression, providing evidence that modifications in gene expression accounted for the changes in the number of calretinin- and calbindin-containing neurons. Among other members of the neurotrophin family, neurotrophin-4 (NT-4), which also acts by activating tyrosine kinase TrkB receptors, exerted effects comparable to those of BDNF, whereas nerve growth factor (NGF) was ineffective. As for BDNF and NT-4, incubation of cortical neurons with neurotrophin-3 (NT-3) also led to a decrease in calretinin expression. However, in contrast to BDNF and NT-4, NT-3 did not affect calbindin expression. Double-labeling experiments evidenced that calretinin- and calbindin-containing neurons belong to distinct neuronal subpopulations, suggesting that BDNF and NT-4 exert opposite effects according to the neurochemical phenotype of the target cell.     

BDNF-overexpression regulates the reactivity of small pulmonary arteries to neurokinin A

Brain-derived neurotrophic factor (BDNF) is a key modulator during the development of jugular and nodose ganglia neurons, which represent the origin of a large proportion of the sensory innervation of the lung. It belongs to the family of neurotrophins, which have been shown to induce the expression of tachykinins. To assess the interactions of BDNF and the tachykinin neurokinin A (NKA) in small pulmonary vessels, BDNF-transgenic mice were examined for tachykinin contents in the airways, heart, trigeminal ganglion and jugular and nodose ganglion complex (JNC) using reverse phase HPLC (rpHPLC) and radioimmunoassay. BDNF-overexpression led to increased NKA levels in the heart and the JNC, whereas only slightly enhanced levels in the trigeminal ganglion were detected. Lower NKA levels were found in the lung. To assess vasoreactivity in small arteries, precision cut lung slices were subjected to videomorphometry and the response to NKA was examined, which showed significantly stronger effects in the BDNF-transgenic mice, while NK-2 receptor mRNA expression, assayed by real-time RT-PCR, was reduced. In conclusion, BDNF-overexpression results in decreased levels of NKA in the lung with subsequently increased NKA-sensitivity of small arteries. These findings point to a modulatory role of neurotrophins in small respiratory vessel tone regulation.     

Exacerbation of cigarette smoke-induced pulmonary inflammation by Staphylococcus aureus Enterotoxin B in mice

Background

Cigarette smoke (CS) is a major risk factor for the development of COPD. CS exposure is associated with an increased risk of bacterial colonization and respiratory tract infection, because of suppressed antibacterial activities of the immune system and delayed clearance of microbial agents from the lungs. Colonization with Staphylococcus aureus results in release of virulent enterotoxins, with superantigen activity which causes T cell activation.

Objective

To study the effect of Staphylococcus aureus enterotoxin B (SEB) on CS-induced inflammation, in a mouse model of COPD.

Methods

C57/Bl6 mice were exposed to CS or air for 4 weeks (5 cigarettes/exposure, 4x/day, 5 days/week). Endonasal SEB (10 μg/ml) or saline was concomitantly applied starting from week 3, on alternate days. 24 h after the last CS and SEB exposure, mice were sacrificed and bronchoalveolar lavage (BAL) fluid and lung tissue were collected.

Results

Combined exposure to CS and SEB resulted in a raised number of lymphocytes and neutrophils in BAL, as well as increased numbers of CD8⁺ T lymphocytes and granulocytes in lung tissue, compared to sole CS or SEB exposure. Moreover, concomitant CS/SEB exposure induced both IL-13 mRNA expression in lungs and goblet cell hyperplasia in the airway wall. In addition, combined CS/SEB exposure stimulated the formation of dense, organized aggregates of B- and T- lymphocytes in lungs, as well as significant higher CXCL-13 (protein, mRNA) and CCL19 (mRNA) levels in lungs.

Conclusions

Combined CS and SEB exposure aggravates CS-induced inflammation in mice, suggesting that Staphylococcus aureus could influence the pathogenesis of COPD.     

Regional distribution of brain-derived neurotrophic factor mRNA in the adult mouse brain.

Brain-derived neurotrophic factor (BDNF) is a protein that allows the survival of specific neuronal populations. This study reports on the distribution of the BDNF mRNA in the adult mouse brain, where the BDNF gene is strongly expressed, using quantitative Northern blot analysis and in situ hybridization. All brain regions examined were found to contain substantial amounts of BDNF mRNA, the highest levels being found in the hippocampus followed by the cerebral cortex. In the hippocampus, which is also the site of highest nerve growth factor (NGF) gene expression in the central nervous system (CNS), there is approximately 50-fold more BDNF mRNA than NGF mRNA. In other brain regions, such as the granule cell layer of the cerebellum, the differences between the levels of BDNF and NGF mRNAs are even more pronounced. The BDNF mRNA was localized by in situ hybridization in hippocampal neurons (pyramidal and granule cells). These data suggest that BDNF may play an important role in the CNS for a wide variety of adult neurons.     

Increased level of serum vascular endothelial growth factor by long-term exposure to hypergravity.

We have previously demonstrated that short-term exposure to hypergravity at 2G for 4 h induces expression of cyclooxygenase-2 (COX-2) in the mouse heart. Moreover, expression of vascular endothelial growth factor (VEGF) is also induced in the heart in a COX-2-dependent manner. Here, we demonstrate that long-term exposure of mice to 2G for 24 h resulted in a significant increase of serum VEGF level, although expression of COX-2 and VEGF in the heart decreased to the 1G-control level. Moreover, increase of serum VEGF was not suppressed by treatment with COX-2 inhibitor, indicating that VEGF was induced in a COX-2-independent manner. These results suggest that gravitational force contributes to maintenance of the serum VEGF level.     

Coordinated involvement of mast cells and T cells in allergic mucosal inflammation: critical role of the CC chemokine ligand 1:CCR8 axis

CCL1 is the predominant chemokine secreted from IgE-activated human and mouse mast cells in vitro, colocalizes to mast cells in lung biopsies, and is elevated in asthmatic airways. CCR8, the receptor for CCL1, is expressed by approximately 70% of CD4(+) T lymphocytes recruited to the asthmatic airways, and the number of CCR8-expressing cells is increased 3-fold in the airways of asthmatic subjects compared with normal volunteers. In vivo, CCL1 expression in the lung is reduced in mast cell-deficient mice after aeroallergen provocation. Neutralization of CCL1 or CCR8 deficiency results in reduced mucosal lung inflammation, airway hyperresponsiveness, and mucus hypersecretion to a similar degree as detected in mast cell-deficient mice. Adenoviral delivery of CCL1 to the lungs of mast cell-deficient mice restores airway hyperresponsiveness, lung inflammation, and mucus hypersecretion to the degree observed in wild-type mice. The consequences of CCR8 deficiency, including a marked reduction in Th2 cytokine levels, are comparable with those observed by depletion of CD4(+) T lymphocytes. Thus, mast cell-derived CCL1- and CCR8-expressing CD4(+) effector T lymphocytes play an essential role in orchestrating lung mucosal inflammatory responses.     

Mast cells and macrophages in normal C57/BL/6 mice

Mast cells and macrophages have an important role in immunity and inflammation. Because mice are used extensively for experimental studies investigating immunological and inflammatory responses, we examined mast cell and macrophage distribution in normal murine tissues. Mast cells were abundant in the murine dermis, tongue, and skeletal muscle but were rarely found in the heart, lung, spleen, kidney, liver, and the bowel mucosa. In contrast, dogs exhibited large numbers of mast cells in the lung parenchyma, liver, and bowel. Some murine dermal mast cells had long cytoplasmic projections filled with granular content. Mouse mast cells demonstrated intense histamine immunoreactivity and were identified with histochemical enzymatic techniques for tryptase and chymase. Macrophages, identified using the monoclonal antibody F4/80, were abundant in the spleen, lung, liver, kidney, and bowel but relatively rare in the heart, tongue, and dermis. Using a nuclease protection assay we investigated mRNA expression of stem cell factor (SCF), a crucial survival factor for mast cells, and the macrophage growth factors macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating factor (GM-CSF). Stem cell factor mRNA was highly expressed in the murine lung. Relatively low levels of SCF mRNA expression were found in the tongue and earlobe, which are tissues containing a high number of mast cells. Macrophage CSF and GM-CSF mRNA was highly expressed in the lung and spleen. The murine heart, an organ with a low macrophage content, expressed high levels of M-CSF but negligible levels of GM-CSF mRNA. Constitutive growth factor mRNA expression in murine tissues without significant populations of mast cells and macrophages may suggest an alternative role for these factors in tissue homeostasis.     

Brain-Derived Neurotrophic Factor Is More Highly Conserved in Structure and Function than Nerve Growth Factor During Vertebrate Evolution

Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fish Xiphophorus, with the mammalian homologues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purified recombinant BDNF. However, the activity of recombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng/ml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF had reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates from a common ancestral gene about 600 million years ago. By RNA gel blot analysis NGF mRNA was detected during late embryonic development; BDNF was present in adult brain.     

Increased levels of messenger RNAs for neurotrophic factors in the brain during kindling epileptogenesis.

Kindling, induced by repeated subconvulsive electrical or chemical stimulations leads to progressive and permanent amplification of seizure activity, culminating in generalized seizures. We report that kindling induced by electrical stimulation in the ventral hippocampus leads to a marked and transient increase in mRNA for NGF and BDNF in the dentate gyrus, the parietal cortex, and the piriform cortex. BDNF mRNA increased also in the pyramidal layer of hippocampus and in the amygdaloid complex. No change was seen in the level of HDNF/NT-3 mRNA. The increased expression of NGF and BDNF mRNAs was not influenced by pretreatment with the NMDA receptor antagonist MK801, but was partially blocked by the quisqualate, AMPA receptor antagonist NBQX. The presumed subsequent increase of the trophic factors themselves may be important for kindling-associated plasticity in specific neuronal systems in the hippocampus, which could promote hyperexcitability and contribute to the development of epileptic syndromes.     

Expression profiles of BDNF splice variants in cultured DRG neurons stimulated with NGF

Expression of brain-derived neurotrophic factor (BDNF) mRNA is increased in the dorsal root ganglion (DRG) in response to peripheral inflammation. Nerve growth factor (NGF) from inflammatory tissue is thought to induce expression of BDNF. Recently, it was reported that the BDNF gene has eight non-coding exons that are transcribed independently into several splice variants. Expression of these splice variants in DRG neurons stimulated with NGF has not been studied. We examined changes in expression of BDNF splice variants in a rat model of peripheral inflammation and in cultured DRG neurons exposed to NGF. Total BDNF mRNA was increased by inflammation in vivo and by NGF in vitro. Among all splice variants, exon 1-9 showed the greatest increase in expression in both experiments. Our results indicate that exon 1-9 contributes to changes in total BDNF levels and may play an important role in the acute response of DRG to NGF.     

Regulation and function of CCSP during pulmonary Pseudomonas aeruginosa infection in vivo

Clara cell secretory protein (CCSP) is a 16-kDa homodimeric polypeptide secreted by respiratory epithelial cells in the conducting airways of the lung. To assess the role of CCSP in bacterial inflammation and to discern whether CCSP expression is influenced by bacterial infection, CCSP-deficient [(-/-)] gene-targeted mice and wild-type mice were given Pseudomonas aeruginosa intratracheally. Infiltration by polymorphonuclear cells was significantly increased in the lungs of CCSP(-/-) mice 6 and 24 h after the administration of the bacteria. The number of viable bacteria isolated from the lungs in CCSP(-/-) mice was decreased compared with that in wild-type mice. Concentrations of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha were modestly increased after 6 and 24 h, respectively, in CCSP(-/-) mice. The concentration of CCSP protein in lung homogenates decreased for 1-5 days after infection and recovered by 14 days after infection. Likewise, CCSP mRNA and immunostaining for CCSP markedly decreased in respiratory epithelial cells after infection. CCSP deficiency was associated with enhanced pulmonary inflammation and improved killing of bacteria after acute pulmonary infection with P. aeruginosa. The finding that Pseudomonas infection inhibited CCSP expression provides further support for the concept that CCSP plays a role in the modulation of pulmonary inflammation during infection and recovery.     

NGF, BDNF, leptin, and mast cells in human coronary atherosclerosis and metabolic syndrome

While multiple growth factor, cytokines, and immune cells are identified in atherosclerotic lesions, as well as an essential nonneuronal function of neurotrophins implicated in cardiovascular tissue development and in lipid and glucose metabolism, the role of the neurotrophins NGF and BDNF and also the adipokine leptin in human coronary atherosclerosis and related disorders, such as metabolic syndrome, remains unclear. Here we report that (i) both the amount and the immunoreactivity of NGF was reduced and the expression of p75NGF receptor and the number of mast cell increased in human atherosclerotic coronary arteries (n = 12) compared with control specimens (n = 9) obtained from autopsy cases, and (ii) NGF and BDNF plasma levels were reduced in patients with metabolic syndrome (n = 23) compared with control subjects (n = 10). Also, in metabolic syndrome patients, a positive correlation between the plasma leptin levels and the number of adipose tissue mast cells was found, suggesting that leptin may be a novel adipoimmune mediator. Altogether, the results provide the first correlative evidence for the potential involvement of NGF, BDNF, leptin, and mast cells in human coronary atherosclerosis and metabolic syndrome, implying neuroimmune and adipoimmune pathways in the pathobiology of these cardiovascular disorders.