Ethanol upregulates glucocorticoid-induced leucine zipper expression and modulates cellular inflammatory responses in lung epithelial cells.

Alcohol abuse is associated with immunosuppressive and infectious sequelae. Particularly, alcoholics are more susceptible to pulmonary infections. In this report, gene transcriptional profiles of primary human airway epithelial cells exposed to varying doses of alcohol (0, 50, and 100 mM) were obtained. Comparison of gene transcription levels in 0 mM alcohol treatments with those in 50 mM alcohol treatments resulted in 2 genes being upregulated and 16 genes downregulated by at least 2-fold. Moreover, 0 mM and 100 mM alcohol exposure led to the upregulation of 14 genes and downregulation of 157 genes. Among the upregulated genes, glucocorticoid-induced leucine zipper (GILZ) responded to alcohol in a dose-dependent manner. Moreover, GILZ protein levels also correlated with this transcriptional pattern. Lentiviral expression of GILZ small interfering RNA in human airway epithelial cells diminished the alcohol-induced upregulation, confirming that GILZ is indeed an alcohol-responsive gene. Gene silencing of GILZ in A549 cells resulted in secretion of significantly higher amounts of inflammatory cytokines in response to IL-1beta stimulation. The GILZ-silenced cells were more resistant to alcohol-mediated suppression of cytokine secretion. Further data demonstrated that the glucocorticoid receptor is involved in the regulation of GILZ by alcohol. Because GILZ is a key glucocorticoid-responsive factor mediating the anti-inflammatory and immunosuppressive actions of steroids, we propose that similar signaling pathways may play a role in the anti-inflammatory and immunosuppressive effects of alcohol.

Evaluation of novel dry eye model: preganglionic parasympathetic denervation in rabbit.

PURPOSE: To evaluate ocular surface status after interruption of preganglionic, parasympathetic neural control after surgical removal of the greater superficial petrosal nerve (GSPN). METHODS: New Zealand White rabbits underwent unilateral section and removal of a 5-mm portion of the GSPN by a route through the inner ear; no ocular or orbital tissue was involved. Before and 7 days after surgery, all animals underwent preliminary examination, including fluorescein staining, rose bengal instillation, blink rate, tear breakup time (BUT), tear flow, and impression cytology. Total tarsorrhaphy was carried out in four additional rabbits, and another four animals underwent unilateral sham procedures. The GSPN, pterygopalatine ganglion, lacrimal gland, and conjunctiva were evaluated by light and transmission electron microscopy (TEM). RESULTS: GSPN sectioning resulted in significant changes of the ocular surface after 7 days: intense rose bengal staining of the conjunctiva, fluorescein staining of the cornea, increased blink rate (P < 0.05), decreased BUT (P < 0.005), decreased tear flow by 26% (P < 0.005), and decreased goblet cell density (P < 0.01). TEM revealed massive accumulation of secretory granules in lacrimal acinar cells. The changes were also seen after tarsorrhaphy. Neither the contralateral control nor the sham eyes were affected. CONCLUSIONS: The effects of GSPN nerve section led to the rapid onset of a dry eye condition in the rabbits that continued for at least 1 week. The authors suggest that continuous neural drive of the pterygopalatine ganglion is necessary to maintain adequate tear flow and mucin secretion. It is likely the trigeminal system is the afferent origin of this continuous neural tone.

Elevated ankyrin G in a plexiform neurofibroma and neuromas associated with pain.

UNLABELLED: Ankyrin G has recently been shown to be responsible for activation of sodium channels in the developing and regenerating axonal membrane. Via this sodium channel mechanism, elevated ankyrin G levels have been linked with spontaneous ectopic hyperexcitability and thus with pain phenomena in nervous tissue. Ankyrin G, a transmembrane, structural protein of the axon, was examined in four conditions: (a) painful plexiform neurofibroma; (b) painful neuroma; (c) non-painful neuromas; (d) normal nerve. Neurofibroma tissue was obtained from an 18-year old male patient who developed an intensely painful, plexiform neurofibroma of the posterior femoral cutaneous nerve and subsequently underwent surgery. Sample proteins were separated by PAGE and labeled with anti-ankyrin G antibodies in a Western blot procedure. RESULTS: The ankyrin G band density (mug) of protein for the painful neurofibroma was 6014 and was 3557 for the painful neuroma as compared to 3041, 1988 and 606 (mean+/-SD=1878+/-1221) for the three non-painful neuromas. Ankyrin G expression in normal nerves (8 specimens from 7 patients) was comparatively less (mean+/-SD=411+/-339). CONCLUSION: Our results may represent the first evidence for abnormally increased levels of ankyrin G protein with painful neurofibromas. Due to ankyrin G's multifunctional role in the development and remodeling of excitable membranes, it can be hypothesized that the significant increase contributes to the development of hyperexcitable axonal membranes in neurofibromas and potentially other peripheral pain conditions.

Ankyrin G and voltage gated sodium channels colocalize in human neuroma--key proteins of membrane remodeling after axonal injury.

We tested if ankyrin G could be detected in human neuroma, if it colocalized with site-specific peripheral nerve sodium channels that accumulate at axon tips of injured nerve, and if there are differences in the distribution of these proteins in non-painful neuroma and painful neuroma tissue vs. normal nerve. Frozen sections from one painful, six non-painful, and three normal nerves were immunocytochemically examined. A double labeling technique with highly specific antibodies against peripheral nerve type 1 (Na(v)1.7), and peripheral nerve type 3 (Na(v)1.8) sodium channels and anti-ankyrin G antibodies detected sodium channels and ankyrin G on the same section, using confocal laser scanning microscopy. Ankyrin G colocalized with both types of sodium channels. Neuroma specimens exhibited considerably larger immunofluorescence for both sodium channels and ankyrin G compared with normal nerve. The painful neuroma presented an even more pronounced immunolabeling in clusters. Findings support results from animal models that link ankyrin G with clustering of sodium channels at axon tips of unmyelinated, sprouting fibers. A common (repair-) mechanism that exists throughout the human nervous system for clustering sodium channels at a high density is assumed. A dysregulation in this membrane remodeling mechanism might be an initial step in a cascade that leads to a painful rather than a non-painful neuroma.

Painful neuromas: a potential role for a structural transmembrane protein, ankyrin G.

OBJECT: Severe nerve injury induces the formation of a neuroma. Some neuromas cause excruciating pain. Overexpression of Na+ channels leads to hyperexcitability and painful phenomena. Ankyrin G, a multifunctional transmembrane protein of the axolemma, might be a key protein in neuroma formation because it binds Na+ channels in the initial segments of a regenerating axon and links with neuronal cell adhesion molecules. The authors wanted to determine if ankyrin G could be detected in neuroma, and if present, whether there would be differences in distribution between nonpainful neuromas, painful neuromas, and normal nerve. METHODS: First, frozen sections of nine nerve specimens obtained from six patients (six nonpainful neuromas, one painful neuroma, and two normal nerves) were immunocytochemically screened for ankyrin G by using confocal laser scanning microscopy. Second, specimens from 29 patients (seven painful neuromas, 15 nonpainful neuromas, and seven normal nerves) were examined using immunoblot analysis for their ankyrin G content. Western blot analysis detected ankyrin G, which was visualized by applying the enhanced chemiluminescence technique. Computerized densitometry was used to quantitate ankyrin G expression by comparing band intensities. Normal nerve served as control. Neurofilament was used as a marker for nerve tissue content. Ankyrin G could be detected and was found to be increased in neuromas. The mean band intensity values were 1838 for painful neuromas, 1166 for nonpainful neuromas, and 411 for normal nerves. In two cases the authors were able to compare specimens of painful neuroma and normal nerve from the same patient. The painful neuromas exhibited considerably higher levels of ankyrin G. Painful neuroma and normal nerve densitometry values were 499 and 165, respectively, for one patient, and 4254 and 821, respectively, for the other patient. Painful neuromas were also found to have higher neurofilament values than nonpainful neuromas. CONCLUSIONS: Altered regulation of ankyrin G after nerve injury may lead to hyperexcitability and painful phenomena via clustering of Na+ channels. A propensity to overexpress ankyrin G after peripheral nerve trauma may turn out to be a factor in the development of painful neuromas and neuropathic pain. The relevant literature regarding the importance of ankyrin G for nerve regeneration and nerve membrane remodeling is reviewed.