Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3.

Two important cytokines mediating inflammation are tumor necrosis factor alpha (TNFalpha) and IL-1beta, both of which require conversion to soluble forms by converting enzymes. The importance of TNFalpha-converting enzyme and IL-1beta-converting enzyme in the production of circulating TNFalpha and IL-1beta in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFalpha and/or IL-1beta by neutrophil-derived proteinases, immunoreactive TNFalpha and IL-1beta release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFalpha and IL-1beta release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.

In vitro studies of a bradykinin B1/B2 antagonist linked to a human neutrophil elastase inhibitor; a heterodimer for the treatment of inflammatory disorders.

Inflammatory disorders typically have a complex etiology and involve a multitude of inflammatory mediators, and hence, a polytherapeutic approach to these diseases would seem appropriate. In certain chronic inflammatory conditions, we believe that bradykinin (BK) and human neutrophil elastase (HNE) are cooperatively involved. We have previously synthesized compounds with inhibitory activity toward both the BK B2 receptor and HNE. The present study describes single compounds designed to incorporate HNE inhibitory activity and BK B1 and B2 antagonist activity. A proprietary HNE inhibitor (HNEI, CP-955) was directly linked via amide bond formation to a peptide-based combined BK B1/B2 antagonist (B-9430). Three compounds were made using different linking positions in the antagonist peptide. For all compounds, B1 and B2 receptor binding in human cloned receptors was at least 10-fold less than that of B-9430, whereas in the in vitro guinea pig ileum B2 receptor functional assay, the compounds had potencies equivalent to B-9430. Compound I was found to have a fourfold increase in HNEI activity compared with CP-955, whereas compounds II and III were inactive. These data clearly demonstrate that it is possible to retain BK B1/B2 receptor antagonist and HNE activity in a heterodimer.

Purification of a novel glycosylated ferritin from horse heart.

We have previously shown that mRNA coding for ferritin L subunit is present on both cytosolic ribosomes and endoplasmic reticulum-bound ribosomes in rat heart tissue [Campbell et al. (1989) Arch Biochem Biophys 273:89-98]; from this we infer that heart tissue is capable of making a secreted ferritin. We now report the purification from horse heart, of a ferritin that specifically binds to Concanavalin A-Sepharose and is immunologically cross-reactive with antibodies raised against both horse cellular ferritin and horse serum ferritin. Where cellular ferritin is 10 nm in diameter and contains primarily 21-kDa subunits (as determined by gel exclusion chromatography and electron microscopy), the glycosylated heart ferritin is smaller with diameters of 3-5 nm. Antisera raised against serum ferritin cross-reacted with the glycosylated heart ferritin did but did not show significant cross-reactivity with cellular ferritin thus indicating that serum ferritin and glycosylated heart ferritin have antigenic determinants which may not be present on cellular ferritin. The glycosylated ferritin also differs from cellular ferritin in subunit composition, with subunits of 66, 60.5, 53.5, 43.5, and 29.5 kDa, as shown by SDS-PAGE and Western blot analysis. Interestingly, ferritin purified from horse serum contains subunits of similar size.