Induction of interleukin-8 release by lung epithelium with cystic fibrosis epithelial lining fluid is marginally affected by inhibitors of interleukin-1beta.

Interleukin-1beta (IL-1beta) and neutrophil elastase (NE) are present in the epithelial lining fluid (ELF) of patients with cystic fibrosis (CF). Both factors activate surrounding cells including lung epithelial cells, causing release of IL-8, a potent chemoattractant for neutrophils. Previous studies showed up-regulation of IL-8 release by lung epithelial cells as a function of NE in CF; however, few studies addressed the relationship between IL-1beta and activation of lung epithelial cells in CF lungs. Confluent layers of A549 cells, a type II-like human lung epithelial cell line, were incubated overnight with IL-1beta (0-5 ng/ml) or NE (100 nM), and supernatants were analyzed for IL-8 by enzyme-linked immunosorbent assay (ELISA). Both IL-1beta and NE led to a significant increase in IL-8: 12.8 +/- 2.8 ng/ml and 0.8 +/- 0.3 ng/ml, respectively. Next, bronchoalveolar lavage (BAL) samples were obtained from one healthy adult volunteer and six patients with CF and measured for IL-8 and IL-1beta concentrations by ELISA. Both IL-8 (range 169.00 +/- 56.57 to 1742.04 +/- 338.98 pg/ml) and IL-1beta (range 0-24.26 +/- 0.52 pg/ml) were detected in CF specimens, whereas neither was detected in the volunteer's specimen. Normal and CF BALs then were incubated overnight at a 1:10 dilution with confluent A549 cells. Analysis by ELISA of cell-free supernatants revealed increased IL-8 production from cells stimulated with CF BALs only. Similar experiments were performed with BAL supernatants that had been incubated with soluble IL-1 type II receptor, soluble IL-1 receptor antagonist, or a peptide inhibitor of NE. Addition of IL-1 inhibitors had a marginal effect on the amount of IL-8 release after incubation with CF BAL samples, whereas inhibition of NE had no effect. Our results indicate that other factors present in ELF in CF account for IL-8 release from lung epithelial cells.

Extracellular regulation of interleukin (IL)-1beta through lung epithelial cells and defective IL-1 type II receptor expression.

Interleukin (IL)-1beta is produced primarily by activated mononuclear phagocytic cells in the lung airway and functions as a potent proinflammatory cytokine. Release of IL-1beta in the airway microenvironment induces the production of proinflammatory factors from parenchymal airway cells, including IL-8. To study the regulation of lung epithelial cell responsiveness to IL-1beta, the human type II-like airway epithelial cell line A549 and primary normal human bronchial epithelial (NHBE) cells were assayed for IL-1-specific response modifiers. Specifically, the IL-1 type I receptor (IL-1RI), IL-1 type II receptor (IL-1RII), IL-1 receptor accessory protein (IL-1RAcP), and IL-1 receptor antagonist (IL-1Ra) were analyzed. Constitutive expression of IL-1RI, IL-1RAcP, and IL-1Ra was detected in both immortalized and primary human airway epithelial cells. Interestingly, a complete absence of IL-1RII expression was demonstrated under all study conditions in both A549 and NHBE cells. Both cell types were responsive to IL-1beta at concentrations as low as 50 to 500 pg/ml when measured by IL-8 release into cell supernatants. IL-1beta-induced chemokine production and release were inhibited by a 10- to 1,000-fold molar excess of recombinant IL-1RII or IL-1Ra, whereas IL-1RI was a less effective inhibitor. On the basis of our results, we propose that human lung epithelial cells lack the ability to downregulate IL-1beta activity extracellularly because of an inability to express IL-1RII. Release of extracellular IL-1 inhibitors, including soluble IL-1Ra and soluble IL-1RII, by other inflammatory cells present in the airway may be critical for regulation of IL-1beta activity in the airway microenvironment.

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa.

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.

Alpha 1-antitrypsin and protease complexation is induced by lipopolysaccharide, interleukin-1beta, and tumor necrosis factor-alpha in monocytes.

Local regulation of alpha1-antitrypsin (alpha1-AT) may have importance in maintenance of the protease-antiprotease balance in the microenvironment of inflammatory cells. We therefore studied whether lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), and tumor necrosis factor-alpha (TNFalpha) affect the pericellular concentration of alpha1-AT in human peripheral blood mononuclear cells (PBMC). PBMC taken from normal healthy volunteers were treated with LPS, IL-1beta, and TNFalpha, and the concentration of human alpha1-AT in conditioned supernatants was measured. When compared with unstimulated control supernatants (147 +/- 19 ng/ml), LPS (439 +/- 66 ng/ml; p < or = 0.001), IL-1beta (263 +/- 37 ng/ml; p < or = 0.01), and TNFalpha (316 +/- 59 ng/ml; p < or = 0.05) induced a 2- to 3-fold increase of alpha1-AT. Up-regulation of alpha1-AT protein correlated with an increase in alpha1-AT mRNA, suggesting a simultaneous increase in alpha1-AT synthesis. Despite the increase in alpha1-AT concentration, functional antiprotease activity could not be detected. Furthermore, protease activity was present in all samples, with the amount of activity being inversely related to the amount of alpha1-AT measured in supernatants. These findings suggest that local inflammatory conditions up-regulate alpha1-AT production by monocytes which complex with a protease derived from the PBMC population.

A nondiabetic neurotrophic ulcer of the heel.

This case deals with the etiology and treatment of a pure neurotrophic ulcer of the foot, an uncommon lesion. It is primarily caused by increased collagenolytic activity of anesthetic skin over a bony prominence. This type of ulcer in the heel is best treated by a skin flap from an adjacent innervated area of the plantar aspect of the foot. A split thickness skin graft covers the donor site.