Tumour necrosis factor-alpha expression and cell recruitment in Sephadex particle-induced lung inflammation: effects of dexamethasone and cyclosporin A.

1. Tumour necrosis factor-alpha (TNF-alpha) is a cytokine with diverse properties consistent with a possible role in inflammatory disease. We investigated whether TNF-alpha is induced during the progression of lung inflammation elicited by a particulate non-antigenic stimulus, and whether pharmacological control of TNF-alpha expression influences recruitment of specific inflammatory cell types. 2. A single intravenous injection of Sephadex particles into rats led to extensive granulomatous inflammation in lung alveolar and bronchial tissue that peaked in intensity after 24-72 h. Mononuclear cells were the principal component of granulomas, but neutrophils and eosinophils were also abundant. Numbers of mononuclear cells, neutrophils and eosinophils recovered by bronchoalveolar lavage (BAL) peaked at 72 h, 48 h and 72 h, respectively. 3. Messenger RNA encoding TNF-alpha was induced in lung epithelial cells, lung granulomas and BAL cells 6 h after Sephadex administration and remained elevated for 72 h before declining to baseline by 7 days. In BAL cell populations TNF-alpha protein was localized to mononuclear cells at all times points pre- and post-Sephadex administration. 4. Treatment of rats with dexamethasone significantly reduced the Sephadex-induced recruitment of mononuclear cells, neutrophils and eosinophils into the bronchoalveolar cavity, and significantly reduced TNF-alpha mRNA expression by BAL cells. 5. Treatment of rats with cyclosporin A was without effect on Sephadex-induced elevations of mononuclear cell numbers and expression of TNF-alpha, but did reduce significantly recruitment of neutrophils and eosinophils to BAL cell populations. 6. These results show that a sequential asthma-like recruitment of neutrophils, eosinophils and mononuclear cells into lung tissue can be induced by single exposure to a non-antigenic stimulus. Pharmacological and histological studies reveal that mononuclear cell mobilization relates closely to induced TNF-alpha expression, whereas mobilization of neutrophils and eosinophils appears secondary to expression of the cytokine.

Dispersion and characterisation of mast cells from human skin.

A technique is described for the enzymatic dispersion of mast cells in high yield from human infant foreskin. Dispersed mast cells exhibit high viability as assessed by light microscopy, low spontaneous histamine release, and survival in culture. Dispersed mast cells release histamine in response to immunological stimulation and synthetic secretagogues including ionophore A23187, compound 48/80 and poly-L-lysine. Reactivity to these stimuli indicates that cutaneous mast cells differ in their properties from human pulmonary mast cells.

Histamine secretion from human skin slices induced by anti-IgE and artificial secretagogues and the effects of sodium cromoglycate and salbutamol.

Human cutaneous mast cells show functional differences from their counterparts in other tissues. Following passive sensitization with 1% atopic serum for 30 min at 37 degrees C human skin slices released histamine after challenge with anti-human IgE in a concentration dependent manner. Maximum release of 14 +/- 2% was achieved with a 1/10 dilution of anti-IgE. Passive sensitization with 10% atopic serum increased the secretory response to anti-IgE but histamine release was only concentration related over the entire 1/1000 to 1/10 dilution range in half of the specimens studied, the remainder showing high dose tolerance to anti-IgE. Negligible histamine release occurred with anti-IgE challenge of slices which had not been passively sensitized. The histamine releasing ability of A23187 in human skin slices was similar to that observed in lung and adenoidal mast cells being concentration dependent over the range 0.1-3 microM with a maximum release of 25 +/- 3%. In contrast to human lung and adenoidal mast cells, poly-L-lysine and compound 48/80 induced histamine release from skin slices. Poly-L-lysine induced a concentration-dependent release of histamine over the range 0.01-10 microM with a maximum of 27 +/- 3%. The response to compound 48/80 was variable, releasing in some but not all specimens. Histamine release caused by anti-IgE, A23187 and poly-L-lysine was shown to be dependent upon extracellular calcium while release stimulated by compound 48/80 was calcium independent. The chemotactic peptide, formyl-methionyl-leucyl-phenylalanine, over the range 0.01-10 microM failed to release histamine from skin slices.(ABSTRACT TRUNCATED AT 250 WORDS)

Scatchard plots with a positive slope: dependence upon ligand concentration.

The inverse dependence of binding constants (nK) upon albumin concentration for 2-(4'-hydroxybenzeneazo) benzoic acid (HABA) has been investigated with bovine and human albumin by equilibrium dialysis. Data obtained by the use of a range of concentrations of either bovine or human albumin, together with a single ligand concentration, gave positive Scatchard plots indicative of binding constants inversely dependent upon protein concentration. The slope of each plot was influenced by the particular ligand concentration used and an increase in the ligand concentration in the case of both bovine and human albumin resulted in a decrease in the slope of the positive Scatchard plot. Higher concentrations of ligand produced a transformation from a positive to a negative plot with both bovine and human albumin.

Drug binding defect of uraemic plasma: unlikely involvement of carbamoylated albumin.

Carbamoylation of bovine and human albumin in vitro decreased the binding of methyl red and salicylic acid. Charcoal extraction of the carbamoylated albumin under acid conditions produced some decrease in the degree of carbamoylation, but did not substantially improve the binding of methyl red and salicylate. Albumin from rats with glycerol-induced renal failure showed no significant degree of carbamoylation compared to controls. Carbamoylation is not responsible for the binding defect of uraemic rat plasma, nor is likely to be involved in the case of human uraemic plasma.