Identification of a second neutrophil-chemoattractant cytokine generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to melanoma-growth-stimulatory activity.

The intraperitoneal injection of zymosan in the rabbit results in the generation of an inflammatory exudate containing oedema-forming and chemoattractant activities. Previous studies demonstrated the early appearance of the complement fragment C5a, followed by the generation of two mediators related to the cytokine interleukin-8 that were separable by cation-exchange h.p.l.c. N-Terminal amino acid sequencing identified one of these mediators as rabbit interleukin-8. This paper describes the purification of the second cytokine by cation-exchange, gel-filtration and reversed-phase h.p.l.c. The purified material had both oedema-forming and chemoattractant activity when assayed in rabbit skin in vivo. On SDS/PAGE a single 6-8 kDa band was observed and N-terminal amino acid sequencing of the reduced and alkylated protein positively identified 36 amino acids. This sequence revealed the rabbit homologue of melanoma-growth-stimulatory activity. The identification of these two cytokines in vivo will provide an opportunity to investigate the importance of their co-release in the inflammatory process.

A novel neutrophil chemoattractant generated during an inflammatory reaction in the rabbit peritoneal cavity in vivo. Purification, partial amino acid sequence and structural relationship to interleukin 8.

An inflammatory reaction was induced in vivo by injection of zymosan into the peritoneal cavity of the rabbit. The inflammatory exudate was found to contain oedema-inducing and neutrophil chemoattractant activity when assayed in rabbit skin in vivo, using 125I-albumin and 111In-neutrophils. This activity was additional to that of complement fragment C5a, which was removed by an affinity gel. Two chemoattractants were isolated by cation-exchange, gel-filtration and reversed-phase h.p.l.c. One of these, which ran as a single band of 6-8 kDa on SDS/PAGE, was subjected to N-terminal sequence analysis without reduction and alkylation of cysteine residues. Positive identification of 28 of the first 31 amino acids revealed a rabbit homologue of interleukin-8 (75% sequence identity with human interleukin-8). The demonstration of interleukin-8 as a major neutrophil chemoattractant in an inflammatory reaction in vivo provides the basis for further investigations into the role of this cytokine in the inflammatory process.

Modulation of beta-endorphin secretion from mouse pituitary tumor cells by calmodulin inhibitor W7.

The involvement of calmodulin in the secretion of beta-endorphin from the mouse anterior pituitary tumor cell line, AtT-20, was investigated. The calmodulin inhibitor W7 potentiated secretion produced by 8-BrcAMP, and induced a secretory response to arginine vasopressin, which did not elevate beta-endorphin levels when added alone. Release of hormone in response to CRF was not affected. Calmodulin phosphodiesterase inhibitor 8-MeOMeMIX produced a dose-dependent increase in 8-BrcAMP stimulation, suggesting that inhibition of cAMP degradation is the mechanism of enhancement of 8-BrcAMP-induced secretion in the presence of W7.

Leukotriene biosynthesis and metabolism detected by the combined use of HPLC and radioimmunoassay.

A radioimmunoassay for leukotriene D4 (LTD4) has been developed which exhibits sufficiently high sensitivity to be useful in conjunction with RP-HPLC in the detection of LTC4, LTD4 and LTE4 in physiological samples. The detection limit of the assay was approximately 240 amoles, using antiserum TG1 at a dilution of 6 X 10(3), with 50% displacement at 70 fmoles. Antiserum NW1, also at a dilution of 6 X 10(3), displayed a detection limit of 9 fmoles with 50% displacement at 100 fmoles. The two antisera have similiar crossreactivities, both manifesting useful affinities for LTE4 and LTC4, and low or negligible affinities for other arachidonic acid metabolites, or their derivatives. The radioimmunoassay was used to detect 1) LTC4, LTD4 and LTE4 released from perfused rat lung in response to platelet-activating factor (PAF) stimulation, 2) conversion of exogenous LTD4 to LTE4 in human blood, and 3) endogenous leukotrienes in human blood samples.