Dysregulation of arachidonic acid release and metabolism by atopic mononuclear cells

We studied the ability of monocytes to metabolize [3H]arachidonic acid (AA) provided exogenously by activated T cells, and the extent to which dexamethasone suppressed eicosanoid production by normal and atopic cells. [3H]AA metabolites were identified using a reverse-phase high pressure liquid chromatography system (HPLC). Unstimulated and PHA-stimulated T cells from normal and atopic subjects exhibited a similar uptake and time-dependent release of radiolabel, 90% of which was identified as free AA. The addition of autologous normal and atopic monocytes to these cultures enhanced the release of radiolabel, even in the absence of stimulation with mitogen. Atopic T cell/monocyte cultures released significantly (P = 0.046) more radiolabel than normal cells when stimulated with PHA. Furthermore, the monocytes from both normal and atopic subjects metabolized T cell derived [3H]AA into cyclo-oxygenase (CO) and lipoxygenase (LO) products. Under unstimulated conditions, atopic cells produced significantly (P = 0.04) less CO products than normal cells. In contrast, under PHA and calcium ionophore-stimulated conditions, the atopic cells produced significantly (P = 0.048) more prostaglandins than normal donor cells. Furthermore, although the total release of radioactivity was comparable in both groups, significantly less (P = 0.02) free AA remained in ionophore-stimulated culture supernatants from atopic cells. In order to study the regulation of AA release by normal and atopic T cells, dexamethasone (1 microM) was added to T cell cultures. Dexamethasone inhibited the release of [3H]AA from normal T cells to a significantly (P = 0.003) greater extent than it did to atopic cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human mononuclear phagocytes from different anatomical sites differ in their capacity to metabolize arachidonic acid.

Human mononuclear phagocytes have the capacity to metabolize arachidonic acid (AA) into prostaglandins (PG) endowed with potent activities in immune responses and inflammatory processes. We have evaluated AA metabolism in human mononuclear phagocytes harvested from different anatomical sites (blood monocytes, macrophages from milk, peritoneal cavity and alveolar spaces). At thin layer radiochromatography, the AA metabolites mainly present were PGE2, TxB2 and, only in bronchoalveolar macrophages, a peak comigrating with PGD2. No appreciable levels of 6-keto-PGF1 alpha were observed. These data were confirmed using specific radioimmunoassays for TxB2, PGE2 and 6-keto-PGF1 alpha. Blood monocytes and bronchoalveolar macrophages were the poorest producers of PG, with the possible exception of PGD2 in bronchoalveolar macrophages. The high amounts of TxB2 and PGE2 produced by milk macrophages could contribute to the levels of PG in human milk. Thus, human mononuclear phagocytes obtained from diverse anatomical sites are considerably heterogeneous in terms of AA metabolism.     

Mast cell histamine release induced by intermediate products of arachidonic acid metabolism

This study was performed to evaluate the role of intermediate products of arachidonic acid metabolism on histamine release from rat serosal mast cells. Arachidonic acid in concentrations ranging from 10(-9) to 10(-4) M caused no histamine release from purified rat peritoneal mast cells. High concentrations (10(-6)-10(-6) M) of the terminal products of the arachidonic acid metabolism were also devoid of any significant histamine-releasing properties. The metabolic activation of arachidonic acid with prostaglandin-H-(PGH)-synthase isolated from calf seminal vesicles, evoked a significant release of histamine from rat serosal mast cells. The liberation of histamine was not accompanied by a significant leakage of lactic dehydrogenase (LDH) and the electron microscopical features were consistent with an exocytotic release. The phenomenon was blocked by reduced glutathione (GSSH) and by D-mannitol, a hydroxyl free-radical scavenger. These results suggest that free radical derivatives of arachidonic acid are generated during the catalysis which triggers mast cell histamine release.     

The effect of inflammatory stimuli on the stroma of neoplasms: the involvement of mononuclear phagocytes

The phlogistic response of the stroma supporting a transplantable rat fibrosarcoma and the ability of animals bearing a neoplasm to mount an inflammatory reaction to non-specific stimuli were investigated. In the unstimulated neoplasm the concentration of macrophages in the stroma was similar to that of normal connective tissue. The mononuclear exudate induced by cotton pellets or glass coverslips implanted in the neoplastic tissue, however, was reduced when compared with that in normal connective tissues at both 7 and 14 days after implantation of these foreign bodies. Since the emigration of radioactively labelled monocytes in both inflamed normal connective tissue and inflamed stromal tissue in the neoplasm proved to be similar, the assumption has been made that neoplastic cells inhibit the local accumulation of the newly emigrated monocytes. This macrophage dispersing effect only occurs in the neoplasm and there is no systemic action. The only systemic effect detected is an anti-inflammatory action which was significant only between the 1st and the 2nd wk after transplantation of the neoplasm into neonatal recipients.     

Histamine release from serosal mast cells by intermediate products of arachidonic acid metabolism

In the present paper we report the results of experiments carried out to measure the release of histamine from isolated rat mast cells during the metabolic activation of arachidonic acid. Arachidonic acid (10^–8–10^–4 M) and the terminal products (10^–6 M) of the arachidonic acid pathways were devoid of any significant histamine releasing properties. A substantial amount of histamine was released from rat mast cells by low concentrations of arachidonic acid during incubation with prostanoid generating systems, such as guinea-pig lung microsomes, rat serosal macrophages and polymorphonuclear cells and prostaglandin-H-synthase from calf seminal vesicles. The release of histamine was not accompanied by a leakage of lactate dehydrogenase and was blocked byd-mannitol and by lipoxygenase and cyclo-oxygenase pathway inhibitors. The data are consistent with the hypothesis that free radical derivatives of arachidonic acid, originating from hydroperoxy fatty acids, are generated during catalysis, causing mast cell histamine release.     

Hemodialysis-related induction of beta 2-microglobulin synthesis and release by mononuclear phagocytes

We have investigated beta 2-microglobulin (beta 2M) synthesis and release by blood leukocytes and isolated mononuclear phagocytes. Since beta 2M was discovered to form amyloid fibrils in patients undergoing long term, chronic hemodialysis and monomeric beta 2M levels in plasma of these patients are highly elevated, and since hemodialysis-related factors that increase beta 2M production are unknown, we evaluated beta 2M production by mononuclear phagocytes under a variety of conditions. We utilized a novel enzyme-lined immunoabsorbant assay to quantitate beta 2M release. Adherence of macrophages onto polystyrene or Cuprophan membranes does not induce beta 2M synthesis or release. In contrast, interaction of macrophages with lipopolysaccharide, gamma-interferon, tumor necrosis factor, or interleukin 1 induces synthesis or release. In contrast, interaction of macrophages with lipopolysaccharide, gamma-interferon, tumor necrosis factor, or interleukin 1 induces synthesis and release of beta 2M, indicating that beta 2M synthesis is increased during macrophage activation. Exposing leukocytes or macrophages to changes in osmotic or oncotic pressure induces a rapid release of beta 2M and interleukin 1 into the cellular medium. These results suggest that during hemodialysis, beta 2M production is more likely to result from endotoxin contamination, or osmotic and oncotic changes, rather than direct interaction of mononuclear phagocytes with cellulosic membranes.     

Inhibition of arachidonic acid release from human peripheral mononuclear cells by heat shock treatment and geldanamycin

The objective of this study was to investigate the effects of heat shock (HS) treatment and geldanamycin (GA) on the release of arachidonic acid (AA) from human peripheral blood mononuclear cells (PBMC), monocytes and lymphocytes. Mononuclear cells prepared from blood of healthy subjects were preincubated with (3)H-AA. The release of (3)H-AA incorporated into the membrane was studied after pretreatment of cells by HS (43 degrees C, 1 h) and GA. The activation of AA producing enzymes was achieved by the addition of phorbol 12-myristate 13-acetate (PMA) or by the combination of PMA+calcium ionophore A-23187. Treatment of cells by HS inhibited the release of AA. Furthermore, the release of AA by PBMC was dose dependently inhibited by GA. The combination of treatments by HS and GA augmented the inhibition of AA release. The HS response involves a diminished release of AA from PBMC. The inhibitory effect of GA on the AA release is a new element in the antiinflammatory pharmacological ability of this drug.     

Chronic inflammatory bowel disease--increased plasminogen activator secretion by mononuclear phagocytes.

The secretion of the neutral protease plasminogen activator (PA) by cultured macrophages (M phi) was studied in hospitalized patients suffering from chronic inflammatory bowel disease (IBD). There was markedly enhanced secretion of PA by M phi derived from circulating monocytes of the IBD population (18) compared to an age-matched population (16) which was not afflicted by intestinal disease (P less than 0 . 001). Mean M phi PA activity was greater in the population of 11 Crohn's disease patients (P less than 0 . 01) than in a group of seven ulcerative colitis sufferers (P less than 0 . 05) when compared to the control population. While both the treated and untreated hospitalized IBD populations showed increased M phi PA specific activity, results for the nine untreated patients (5 . 56 +/- 1.14 units/micrograms M phi DNA) were substantially higher than those found in the treated IBD population (2 . 91 +/- 0 . 62 units/micrograms M phi DNA) (P less than 0 . 01). These findings reflect the activity of M phi in IBD and suggest a means by which tissue injury is mediated in these conditions.     

The response of human peripheral blood mononuclear phagocytes to rheumatoid arthritis

The maturity of peripheral blood mononuclear phagocytes (B-MPs) from patients with rheumatoid arthritis (RA), osteoarthritis (OA), and reference ("normal") subjects was compared. Mononuclear cell isolates from peripheral blood were separated on discontinuous gradients of Percoll into low density (more mature) and high density (less mature) subpopulations. Contrary to expectations, the proportion of immature B-MPs in RA patients was found to be significantly lower than that in reference subjects. In RA patients with synovial effusions the proportion of immature B-MPs approached but did not exceed those found in reference subjects, despite the fact that 31% of these patients displayed a peripheral blood monocytosis. It was concluded that the bone marrow precursor population had adapted to the long-term demand for B-MPs in the course of this chronic inflammatory disease.     

Differential release of mediators from human basophils: differences in arachidonic acid metabolism following activation by unrelated stimuli

We have examined the release of histamine and LTC4 from purified human basophils challenged with several different stimuli, both physiological and nonphysiological. Basophils (n = 16) challenged with 0.1 micrograms/ml anti-IgE released 38 +/- 4% of their available histamine and 39 +/- 12 ng LTC4/10(6) basophils within 15-30 min. F-Met peptide (n = 8) caused the release of 54 +/- 8% histamine and 42 +/- 25 ng LTC4/10(6) basophils within a period of 2-5 min. C5a caused the release of 22 +/- 3% histamine from selected donors but failed to initiate any LTC4 release unless combined with D2O or 5 mM extracellular calcium. The two nonphysiological stimuli A23187 and TPA caused extensive histamine release, 67 +/- 8 and 82 +/- 11%, respectively, and while A23187 initiated a large and rapid release of leukotriene, TPA failed to release any LTC4 even when combined with D2O or 2-5 mM extracellular calcium. Increased concentrations of extracellular calcium enhanced anti-IgE and f-Met peptide induced release of LTC4 but inhibited the A23187 induced release of leukotriene. A single peak of immunoreactive leukotriene C4 that comigrated with the authentic standard was identified using HPLC followed by radioimmunoassay. No LTD4 or LTE4 could be detected. Purified human basophils incubated with 0.2 microM [3H]AA incorporated 290 pmol/10(6) cells, or 32 +/- 5% of the available label within 60 min. The [3H]AA was taken principally into the phospholipids (73 +/- 5%), with 20 +/- 3% as neutral lipid, and only 5 +/- 2% remaining as the free acid. Three phospholipid subclasses, phosphatidylcholine, PC (24 +/- 2%), phosphatidylinositol, PI (22 +/- 1%), and phosphatidylethanolamine, PE (15 +/- 3%), accounted for the majority of the incorporated [3H]AA while the remainder of the phospholipids accounted for less than 5% of the total cpm. HPLC analysis of the lipid mediators released during stimulation with 0.1 micrograms/ml anti-IgE revealed [3H]LTC4 (2.4 +/- 1.0%), [3H]5HETE (1.0 +/- 0.1%), unmetabolized [3H]AA (91 +/- 2%), and an unidentified peak (3.4 +/- 1.4%). The unknown metabolite eluted with the prostaglandins, was inhibited by indomethacin, and appeared to have a relatively high specific activity. It may thus represent an artifact of the labeling procedure rather than a novel basophil-derived prostaglandin.     

Killing of Listeria monocytogenes by inflammatory neutrophils and mononuclear phagocytes from immune and nonimmune mice

Acquired resistance to the facultative intracellular bacterium Listeria monocytogenes is thought to require immunologically activated macrophages. Using peritoneal exudate cells from nonimmunized mice in a suspension bactericidal assay, however, we found that peritoneal neutrophils obtained early during the inflammatory process (4 hr after elicitation) and macrophages obtained later during inflammation (maximal listericidal activity at 48 hr after elicitation) were able to kill Listeria in vitro. The kinetics of expression of bactericidal activity by inflammatory neutrophils and macrophages against both L monocytogenes and E coli were similar. Although intraperitoneal immunization or intravenous hyperimmunization markedly enhanced resistance of mice to Listeria in vivo, immunization did not increase the ability of inflammatory peritoneal phagocytes to kill Listeria in vitro. However, in response to intraperitoneal injection of proteose-peptone or dead Listeria, immunized mice mobilized more neutrophils and monocytes into the inflamed peritoneum. These data suggest that, rather than systemic activation of mononuclear phagocyte bactericidal activity, increased mobilization of neutrophils and mononuclear phagocytes into sites of infection may be of prime importance in resistance to listeriosis.     

Myoblasts produce IL-6 in response to inflammatory stimuli

Muscle fibers are the target of T cell-mediated cytotoxic reactions in polymyositis and inclusion body myositis, while the success of myoblast transplantation depends on the absence of an immune rejection against the myofibers. In order to study the behaviour of muscle cells in an inflammatory milieu, we investigated the production of IL-6 and its modulation, including the second messenger pathways controlling it, in in vitro highly purified human myoblast cultures. We found that IL- 1beta, tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS) stimulated myoblast IL-6 secretion in a dose- and time-dependent manner, whereas forskolin and cholera toxin did not. HA1004 at 10 microM did not significantly affect the IL-1beta- and TNF-alpha-induced IL-6 secretion, suggesting that cAMP and protein kinase A are not sufficient to stimulate this process. To investigate the role of protein kinase C (PKC) in this signal transduction, we employed the inhibitor calphostin C, and the activators phorbol-12-myristate-13- acetate (PMA) and calcium ionophore A23187. Calphostin C blocked IL-6 secretion, PMA had a small stimulatory effect and A23187 had no effect; moreover, PKC down-regulation by PMA did not inhibit IL-1beta stimulation, while it reduced TNF-alpha stimulation. These data indicate that different PKC isoforms may be involved in TNF-alpha and IL-1beta signal transduction. Such a difference can distinguish the action of two traditionally 'overlapping' inflammatory cytokines. Our data suggest that muscle cells, like myoblasts, satellite cells and in vivo regenerating myofibers, may discriminate between different stimuli and produce IL-6 when activated in response to muscle injury.      

Selective induction of mononuclear phagocytes to produce neopterin by interferons

Interferon-gamma (IFN-gamma) has been shown to be a potent inducer of neopterin secretion by human peripheral blood monocytes/macrophages (1). In this paper, it is shown that other known stimuli of monocytes (e.g., to secrete proteases or to migrate) such as zymosan-activated human serum, lipopolysaccharide, human C3/iC3 and zymosan coated with complement were unable to trigger monocytes/macrophages to release neopterin. Monocytes/macrophages could be stimulated solely by IFN-gamma (25 U/ml) and IFN-alpha at very high concentrations (10,000 U/ml). In the case of human peripheral blood mononuclear cells (PBMNC), basically the same pattern was observed. If however, in the buffer controls PBMNC showed some neopterin release, all stimuli triggered an increase of neopterin secretion: 10,000 U/ml IFN-alpha induced the same amount of secreted neopterin as did 25 U/ml of IFN-gamma. Both caused higher levels of neopterin secretion than ZAS, LPS and C3/iC3. Amongst the supernatants from PBMNC, only those which were obtained from cells activated with IFN-gamma or -alpha stimulated monocytes/macrophages to produce neopterin. Supernatants from lymphocytes activated with zymosan, lipopolysaccharide and interferon did not contain neopterin, nor did the latter induce monocytes/macrophages to generate and secrete neopterin. Antibodies against IFN-gamma inhibited the triggering effect of the supernatants except when generated by IFN-alpha at 10,000 U/ml. These results demonstrate that both interferons, IFN-gamma and IFN-alpha, the latter only at a 400-fold higher concentration, can trigger monocytes/macrophages directly to secrete neopterin. ZAS, LPS and C3/iC3 are weakly effective only on a mixture of lymphocytes and monocytes/macrophages, provided this cell mixture shows already a basic spontaneous neopterin release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conversion of arachidonic acid in human maternal and neonatal mononuclear leukocytes

The metabolism of exogenous [1-¹⁴C]arachidonic acid in neonatal and maternal peripheral mononuclear leukocytes was studied. Both neonatal and maternal leukocytes converted arachidonic acid to hydroxy acids and to prostaglandin E₂, but small amounts of PGF_2α and thromboxane B₂ were also found. In addition a polar arachidonic acid metabolite with conjugated double bonds was identified in the supernatant from both maternal and neonatal leukocytes. This might be a leukotriene, but further attempts at biochemical characterization are necessary in order to confirm this.