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Background: IL-8 is an important activator and chemoattractant for neutrophils that is produced by normal human bronchial epithelial (NHBE) cells through mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 pathways. Dapsone, a synthetic sulfone, is widely used to treat chronic neutrophil dermatoses. We investigated the effects of dapsone on polarized IL-8 secretion from lipopolysaccharide (LPS)-stimulated NHBE cells and further evaluated its ability to decrease LPS-induced inflammation in the ferret airway., Methods: NHBE cells were grown at air-liquid interface (ALI) to ciliated differentiation. Baseline and endotoxin (LPS)-stimulated IL-8 secretion was measured by enzyme-linked immunosorbent assay at air and basal sides with and without dapsone. Western blotting was used to determine signaling pathways. In vivo, ferrets were exposed to intratracheal LPS over a period of 5 days. Once inflammation was established, oral or nebulized dapsone was administered for 5 days. Intraepithelial neutrophil accumulation was analyzed histologically, and mucociliary transport was measured on the excised trachea., Results: Dapsone, 1 μg/mL, did not influence unstimulated (basal) IL-8 secretion. Apical LPS stimulation induced both apical and basolateral IL-8, but basolateral LPS increased only basolateral IL-8. Dapsone inhibited polarized IL-8 secretion from ALI-conditioned cells. Dapsone also decreased LPS-induced IL-8 mRNA level. LPS led to phosphorylation of extracellular signal-regulated kinase 1/2, but not p38 MAPK or c-Jun NH(2)-terminal kinase. LPS also induced NF-κB p65 phosphorylation, an effect that was inhibited by dapsone. Both oral and aerosol dapsone decreased LPS-induced intraepithelial neutrophil accumulation, but only treatment with aerosol dapsone restored mucociliary transport to normal., Conclusions: Dapsone, given either systemically or as an aerosol, may be useful in treating neutrophilic airway inflammation.

As proteolipid of the myelin sheath and its parent glial membrane may possible interact with bacterial pyrogen (lipopolysaccharide, LPS) during penetration into the brain, we investigated the interaction of LPS with proteolipid derived from the cerebrum of rabbits, rats and chickens. Intravenous administration of LPS (1 microgram/kg) produced a febrile response in rabbits, but not in rats and chickens. Marked hyperthermia was observed in these three species after intracisternal administration of LPS (0.01-0.1 microgram/kg). Dinitrophenol (30 mg/kg s.c.) induced a high fever in these three species tested, particularly in the chickens. The pyrogenicity of LPS given intravenously to rabbits was inactivated by incubation of LPS with proteolipid in vitro. Inactivation effects of proteolipid extracted from the three species was in the order of: chickens, rats and rabbits. In rats, the inactivation effects of proteolipid from the adult animal were more potent than in the case of newborn animals. The febrile response induced by dinitrophenol and leucocytic pyrogen in rabbits, however, was not suppressed by incubation with proteolipid extracted from the rabbit brain. These results suggest that proteolipids do play an important role in the mechanism of penetration of LPS into the brain.

To elucidate the mechanisms of fever response by bacterial pyrogen (lipopolysaccharide, LPS), we investigated both pyrogenicity and Limulus amoebocyte lysate (LAL) gelation activity of cerebrospinal fluid (CSF) withdrawn from febrile rabbits induced by i.v. injection of LPS. One ml of CSF was withdrawn from donor animals 2 hr after i.v. injection of E. coli LPS at graded doses of O, 1, 10 and 50 micrograms/kg, and its pyrogenicity was checked by administration into cisterna magna of recipient animals. Pyrogenicity was revealed only in the CSF withdrawn from the group injected with 50 micrograms/kg of LPS. Differences of both protein content and concentrations of ions (Na, K and Ca) were not obtained among the CSF withdrawn from control and LPS-injected groups. All of the above CSF had no LAL gelation activity, but the activity could be detected in the CSF withdrawn from animals injected with a higher dose of LPS (500 micrograms/kg, i.v.). LAL gelation activity of LPS dissolved in normal CSF was 2 orders less potent in comparison with that of LPS dissolved in saline, which suggested the presence of inhibitor(s) in CSF for LAL assay. Pyrogenicity was not revealed in the CSF withdrawn from hyperthermic rabbits induced by administration of reserpine after pretreatment with a monoamine oxidase inhibitor. These findings suggest that the central action of LPS is involved in pyrogen fever and that monoamines are not closely related to pyrogen fever.

We reported previously that the acute and fetal toxicities of aspirin (ASA) were enhanced by bacterial endotoxin (LPS). In order to clarify the mechanism of the enhancement by LPS, the effects of LPS on the toxicities of salicylic acid (SA), the main metabolite of ASA, were investigated in rats. The following results were obtained: 1) The acute toxicity of SA was significantly potentiated by LPS in male rats. The LD50 of SA with LPS was about one third of that of SA alone. 2) The increase of maternal body weight was inhibited significantly after administration of SA (383 mg/kg, p.o.) with LPS (20 micrograms/kg, i.v.), but not after administration of SA alone. 3) The fetal toxicity of SA including fetal death, resorption, growth retardation and skeletal variations was slightly observed in the dam receiving a single dose of SA on the 15th day of pregnancy, but it was markedly increased by LPS (20 micrograms/kg, i.v.). 4) The half-life period of SA in plasma was increased significantly by the co-administration of LPS in male rats after administration of ASA or SA. All of these phenomena in the rats given SA closely resembled the phenomena previously reported in the rats given ASA. These results suggest that SA might play a main role in the acute and fetal toxicities of ASA, and one of the mechanism of the enhancement effect by LPS on ASA-induced fetal toxicity might be related to the increase of SA concentration in the fetus.

The specificity of endotoxin (lipopolysaccharide, LPS) in the carbocyanine dye reaction was investigated, and then a stoichiometric study of the dye-LPS interaction was conducted with attention to the relationship of biological activities of LPS to the reactivity with the dye. Absorption maxima of some bacterial components in the dye reaction were as follows; LPS from both Escherichia coli and Pseudomonas aeruginosa and lipid A from E. coli LPS, 465 nm; Shigella flexneri LPS, 460 nm; Salmonella minnesota R595 glycolipid, 470 nm; polysaccharide from E. coli LPS, 650 nm; yeast RNA, 620 nm; streptococcal M protein and pyrogenic exotoxin, 610 nm; and free fatty acids, 445-450 nm. The absorbance at 465 nm was increased approximately threefold by sonicating LPS for 1-3 min, which roughly paralleled the decrease in turbidity of the LPS aqueous solution. The Limulus amoebocyte lysate (LAL) gelation activity of LPS increased 10-fold when LPS was sonicated for 0.5-5 min, but it decreased to the control level after further treatment. This decrease, however, was overcome by sonication in the presence of 5 mmol of L-ascorbic acid used as an antioxidant. The LAL gelation activity of LPS was inactivated in parallel with an increase in the ratio (w/w) of dye to LPS from 1.73 to 6.90 in the dye-LPS mixture. Pyrogenicity of LPS was also clearly inactivated when the ratio was over 1.73. The ratios of the height of the beta band at 465 nm (dye-LPS complex) to that of the alpha band at 510 nm (free dye) were increased by sonicating LPS, indicating that the binding character, or stacking tendency, was increased by sonicating LPS.

In order to elucidate the dependency of pyrogenicity of lipopolysaccharide (LPS) on the lipid A structure, we investigated the pyrogenicity of both LPS and lipid A prepared from Mima polymorpha var. oxidans which is deficient in 3-hydroxymyristic acids linked to the 3-hydroxy group of other fatty acids. LPS and lipid A were also prepared as reference compounds from Escherichia coli UKT-B. Furthermore, the establishment of reliable indices for pyrogenicity was undertaken. The following results were obtained. The correlation in linearity was demonstrated between maximal increase in body temperature (delta Tmax) and dose of LPS or lipid A complexed with bovine serum albumin (BSA). The dose-response curves based on delta Tmax were more reliable statistically than the Fever Index-4h representing the area under fever curves for 4 h. The minimum pyrogenic dose (MPD) of E. coli LPS was 1.6 X 10(-3) micrograms/kg i.v. In contrast, the MPD of M. polymorpha LPS was 7.0 X 10(-3) micrograms/kg i.v. By intracisternal injection, the MPD of E. coli LPS was 2.5 X 10(-6) micrograms/kg and that of M. polymorpha LPS 1.0 X 10(-4) micrograms/kg. The end points of Limulus amoebocyte lysate gelation were 10(-5) micrograms/ml in E. coli LPS and 10(-3) micrograms/ml in M. polymorpha LPS. The MPDs of lipid A/BSA complexes by i.v. injection were 0.15 micrograms/kg in E. coli and 2.5 micrograms/kg in M. polymorpha. The rabbits immunized with E. coli lipid A/BSA complex acquired pyrogenic tolerance to the parent LPS but the cross tolerance to M. polymorpha LPS was not observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Our previous finding that the cerebral proteolipid could inactivate the pyrogenicity of lipopolysaccharide (LPS) in vitro was also studied by Sephadex LH-20 column chromatography and the following results were obtained. When rabbit cerebral proteolipid was chromatographed, two main protein peaks were obtained. One appeared in the chloroform (C)/methanol (M) 6:1 and the other C/M 4:1 effluent, designated as fraction IV and fraction V, respectively. When the incubation mixture of proteolipid and LPS was chromatographed, a new protein peak appeared in the C effluent. The new protein peak was suggested to be a complex of proteolipid protein and LPS, because pyrogenicity could be detected in the protein fractions only after treatment with 2% SDS. Fraction V but not fraction IV inactivated the pyrogenicity of LPS in vitro. By re-chromatography of the incubation mixture of fraction V and LPS, a complex of protein and LPS was also eluted in the C effluent. On the other hand, by rechromatography of the incubation mixture of fraction IV and LPS, such a complex was not detected in the C effluent. The present results suggest that the proteolipid apoprotein eluted in the C/M 4:1 effluent on a Sephadex LH-20 column plays an important role in the inactivation of the pyrogenicity of LPS.

The development of pyrogenic tolerance was studied in rabbits treated with varying doses of E. coli lipopolysaccharide (LPS). The following results were obtained. 1) Development of pyrogenic tolerance seemed to proceed in two steps: that is, the first in which tolerance appeared rapidly and the second in which tolerance proceeded more gradually or steadily in response to repeated injections of a constant dose. 2) Tolerance induced by the latter method was not absolute; the rabbits were still sensitive to increased doses of LPS. 3) Rabbits immunized with E. coli vaccine lost their pyrogenic sensitivity of parent LPS to some extent. 4) Following intracisternal injection of LPS into tolerant rabbit, pyrogenic response was not decreased but rather enhanced in comparison with control. 5) The contents of nor-epinephrine and serotonin in the brain did not differ between normal and tolerant rabbits. 6) The mechanisms of endotoxin tolerance remain to be further studied.