William Galbraith

Interleukin-1β stimulates phospholipase A2 mRNA synthesis in rabbit articular chondrocytes

A cDNA that codes for human rheumatoid synovial fluid phospholipase A2 (PLA2) hybridized with a RNA of the same size (900 base pairs) isolated from rabbit articular chondrocytes (RAC). Treatment of RAC with 100 ng/ml recombinant human interleukin-1 beta (IL-1) for 24 hours caused a 13-fold increase in mRNA for this PLA2. Timecourse studies demonstrated that maximal induction of PLA2 mRNA occurred by 16 hours post addition of IL-1 (100 ng/ml). Augmentation of RAC PLA2 mRNA levels was dose-dependent; half-maximal induction was estimated to require 0.15 ng/ml IL-1. Actinomycin D inhibited IL-1 effects on PLA2 mRNA levels. Coordinate effects of IL-1 on RAC PLA2 activity were observed with respect to time and dose dependence as well as actinomycin D sensitivity.

Antiinflammatory phospholipase-A2 inhibitors. I

Abstract A novel series of dehydroabietylamine derivatives has been synthesized and shown to be inhibitors of phospholipase-A 2 (PLA 2 ). These compounds exhibit in vivo antiinflammatory activity in the rat carrageenan paw edema assay and support the concept of PLA 2 inhibition as an approach to the discovery of novel antiinflammatories.

In vivo inhibition of tumor growth of B16 melanoma by recombinant interleukin 1β: II. Mechanism of inhibition: The role of polymorphonuclear leukocytes

Recombinant human interleukin 1 beta (IL 1 beta) inhibits growth of B16 melanoma in syngeneic C57BL/6 mice in a dose-dependent manner when given intratumorally, intradermally, or intramuscularly over a period of 5 to 7 days. Inhibition of tumor growth was rapid and measurable within 3 days after the initial injection and occurred regardless of the route of injection. However, only intratumoral (ITU) injections of IL 1 beta resulted in greater than 90% inhibition in tumor growth. This enhanced inhibition of tumor growth was not dependent on T or NK cells since inhibition of tumor growth occurred in nude and Beige mice. Also, a profound lymphopenia occurred in mice receiving IL 1 beta. Inhibition of tumor growth did correlate with an increase in the number of polymorphonuclear leukocytes (PMNs) in the circulation. However, only ITU injections of IL 1 beta increased the number of PMNs within the tumors. IM injections of IL 1 beta, while increasing the number of PMNs in the circulation, did not increase the influx of PMNs into the tumors. Furthermore, the transfer of PMNs directly into B16 tumors caused a 49% reduction in tumor growth without the presence of IL 1 beta. These results suggest that in vivo, PMNs may effectively control the growth of tumors and that IL 1 beta may increase this effectiveness by increasing the number of PMNs in the circulation and by locally stimulating the production of chemotactic factors for PMNs within the tumor.

Effect of antiinflammatory drugs on human interleukin-1-induced cartilage degradation

Human monocyte IL-1 stimulated the release of proteoglycans from cartilage in organ culture in a concentration-related manner. This stimulation required protein synthesis as shown by inhibition with cycloheximide. The metal chelator, 1,10-phenanthroline, inhibited breakdown, suggesting the involvement of a metalloproteinase. Various nonsteroidal anti-inflammatory drugs (100 μM), and the corticosteroids, dexamethasone and hydrocortisone (1–10 μM), were not effective in blocking proteoglycan release. Of the disease modifying agents tested, levamisole was ineffective while the antimalarials, chloroquine (100 μM) and hydroxychloroquine (100 μM), inhibited the action of IL-1. The free- radical inhibitor SOD (5000 U/ml but not 1000 U/ml) was effective while catalase (8000 U/ml) was not. The protective effects of SOD and the antimalarials suggest that oxygen reactive species may play a role, while lack of inhibition with NSAIDs and corticosteroids indicate that arachidonic acid metabolites may not be important in this degradative process.

Novel Synthesis of Potent Site-Specific Phospholipase A2 Inhibitors

Phospholipase A2 (PLA2) is an esterase responsible for the liberation of phospholipid-bound arachidonic acid, a biosynthetic precursor of putative inflammatory mediators. Arachidonic acid is metabolized by cyclooxygenase and lipoxygenase to the corresponding prostaglandins and leukotrienes (Fig. 1). Traditional antiinflammatory therapy has relied on cyclooxygenase and more recently on lipoxygenase blockade (Shen, 1981), but direct control of arachidonic acid pools has remained relatively unexplored. Recent evidence (Hirata et al., 1980; Blackwell et al., 1980; Rothhut et al., 1983) demonstrates that antiinflammatory steroids control polyunsaturated fatty acid release at both cyclooxygense and lipoxygenase pathways by enhancing the production of PLA2 inhibitory proteins (lipomodulin, macrocortin, renocortin). Direct phospholipase A2 site-specific inhibition, therefore, offers new opportunities in antiinflammatory treatment.

Topical anti-inflammatory activity of DuP 654, a 2-substituted 1-naphthol

Recent work suggests that one of the common biochemical characteristics of skin inflammatory diseases such as psoriasis is altered arachidonic acid metabolism with elevated levels of prostaglandins and leukotrienes. DuP 654, a 2-substituted 1-naphthol, is an exceptionally potent inhibitor of 5-lipoxygenase. DuP 654 was tested in various models of skin inflammation and was found to be potent at inhibiting edema induced by the topical application of arachidonic acid, tetradecanoyl phorbol acetate or the calcium ionophore A23187. DuP 654 was also effective in a murine model of contact sensitivity. DuP 654 was effective at reducing the numbers of infiltrating polymorphonuclear leukocytes in AA and TPA induced edema. These data, taken together, suggest that DuP 654 may be effective in treating human skin diseases.

Phospholipase A2 (PLA2) activity in undifferentiated U937 cells

PLA2 activity has been described in U937 cells. The present study characterized PLA2 activity in these undifferentiated cells. Cells were grown in suspension culture, harvested by centrifugation, and washed and homogenized in a neutral buffer containing standard proteinase inhibitors. A low speed supernatant was fractionated either by acid extraction or by sucrose density gradient centrifugation. PLA2 activity was measured using either L-α-1-palmitoyl-2-arachidonoyl [1-14C]-phosphatidylcholine or heat-inactivated [3H]oleic acid-labeledE. coli as substrates. Substrate-specific PLA2 activity was found in the acid-extracted and in the 25% sucrose fractions. Standard inhibitors were investigated with these PLA2 activities. Our results suggest undifferentiated U937 cells contain three distinct PLA2 activities. This is the first indication that more than one PLA2 activity is present in undifferentiated U937 cells.

Eicosanoid production and cell accumulation induced by intrapleural injection of sodium arachidonate in the rat. Characterization of the model

Intrapleural injection of sodium arachidonate to rats produced both a cellular response and the generation of hydroxy fatty acid metabolites of arachidonic acid without edema. The cellular influx was inhibited by mixed 5-lipoxygenase/cyclooxygenase inhibitors and by a glucocorticoid but not by cyclooxygenase inhibitors. These results suggest that a model using intrapleural injection of sodium arachidonate may have utility for detecting systemically active 5-lipoxygenase inhibitors

Effects of IL-1 muteins on cartilage degradation and as inducers of acute inflammation

IL-1 peptides with N-terminal amino acid mutations were cloned and expressed to help characterize structural requirements for activity. Addition of Thr-Asn to the N-terminus (DP516) or substitution of the first two residues (Ala-Pro) of mature, native IL-1β with Thr-Met (DuP118) had no effect on the potency of the muteins in stimulating proteoglycan breakdown and inhibiting proteoglycan synthesisin vitro, or inducing mouse paw swellingin vivo. When Arg in position 4 of DuP118 was replaced by Glu (Glu-4), proteoglycan-synthesis-inhibitory activity was reduced to 20% and proteoglycan-degrading activity to 2% of that induced by native IL-1β. Glu-4 was much less active in inducing mouse paw swelling and gave maximal swelling about 40% that of native IL-1β. The data suggest that the presence of the positively charged side chain of Arg in position 4 may be important for the activity of IL-1 and may be useful in designing specific IL-1 receptor agonists/antagonists.