1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and in-aminohexanoate (in-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.

From rabbit heterophile leukocytes, granules were prepared which had a high specific activity of acid hydrolases. When lysates of such granules were injected into the skin and joints of other rabbits, acute inflammation was regularly produced. Repeated intraarticular injections led to hypertrophy and byperplasia of synovial lining cells, round cell infiltra-

Prostaglandin E1 (PGE1) and oils enriched in its precursor fatty acids suppress inflammation and joint tissue injury in several animal models. Since synovial cell proliferation is a hallmark of rheumatoid arthritis, we studied the effect of dihomo‐γ‐linolenic acid (DGLA), an immediate precursor of PGE1, on the growth of human adherent synovial cells (ASC) in tissue culture. When stimulated by appropriate concentrations of recombinant interleukin‐1β (rIL‐1β), ASC proliferate and produce PGE. DGLA‐enriched medium suppressed both baseline and rIL‐1β–stimulated ASC growth fivefold, compared with medium supplemented with arachidonic acid. Indomethacin reduced the effect of the DGLA. Synovial cells incorporated the DGLA, and rIL‐1β–stimulated cells that were incubated with DGLA exhibited a 14‐fold increase in PGE1 (to 25.2 ± 6.0 ng/ml, mean ± SD) and a 70% decrease in PGE2 (to 25.2 ± 4.2 ng/ml) compared with cells in control medium. At equivalent concentrations (5 × 10−7M), PGE1 increased the level of cellular cAMP to a greater extent than did PGE2 (16.8 ± 2.0 pmoles versus 4.3 ± 1.9 pmoles, mean ± SEM). Exogenous PGE1 was also a more effective inhibitor of cell growth. Similarly, cAMP concentrations in cells exposed to DGLA for 6 hours were greater than concentrations in arachidonic acidenriched cultures (17.8 ± 3.3 pmoles versus 2.1 ± 2.0 pmoles). These observations suggest that DGLA can restrain ASC growth, an effect which may be due to its capacity to increase PGE1 production and subsequent cellular cAMP concentration.

No abstract

Immunosuppressive effects of E-series prostaglandins have been demonstrated in many in vitro assays of immune responsiveness as well as in autoimmune diseases. To explore the mechanisms underlying prostaglandin El (PGE1)-associated immunosuppression in autoimmunity, we treated SJL mice immunized to produce immune-mediated interstitial nephritis with PGE1, PGFu, or vehicle alone. Mice receiving PGE1 treatment do not develop interstitial nephritis, nor do they display delayed-type hypersensitivity (DTH) to the immunizing renal tubular antigen preparation. The observed immunosuppression is critically dependent on PGE1 administration during the period of effector T cell induction. We therefore investigated the effect of PGE1 on the in vitro induction of DTH effector T cells reactive to renal tubular antigens (SRTA). PGE1 inhibits effector T cell induction in a dose-dependent, reversible manner, but has no inhibitory effect on fully differentiated DTH effector cells or SRTA-reactive cell lines. The PGE1 effect is indirect and mediated via nonspecific suppressor lymphokines. This suppression can be overcome by recombinant interleukin 1 (IL-i), which suggests a mechanism related to either diminished IL-1 secretion or target cell sensitivity to IL-1.