John L. Humes

Macrophages synthesise and release prostaglandins in response to inflammatory stimuli

WHEN macrophages encounter inflammatory stimuli either in vivo or in vitro, they respond by releasing a number of products which may account for the central role that this cell has in chronic inflammatory diseases1. These products include hydrolytic enzymes active at neutral2 or acidic pH (ref. 1), components of both the classical3 and alternate pathway4 of complement, factors modulating responses of lymphocytes to antigens and mitogens5, and factor(s) influencing the proliferation6 and synthesis of collagen7 by fibroblasts. We now show that macrophages whose phospholipid components were labelled with 3H-arachidonic acid also synthesise and release 3H-prostaglandins (PGs) in response to inflammatory stimuli. These observations are consistent with the findings that human macrophages on intrauterine devices8 and guinea pig macrophages responding to lymphokines9 release PGs.

Physiological and pharmacological regulation of prostaglandin and leukotriene production by macrophages

The synthesis and secretion of prostaglandins and leukotrienes by mouse peritioneal macrophages is under several regulatory controls. Arachidonic acid must first be released from phospholipid stores by the action of phospholipases. Macrophages have the capacity to deacylate arachidonic acid directly from the SN2 position of phospholipids via the action of a phospholipase A2. In addition, these cells contain a phospholipase C capable of removing inositolphosphate from phosphatidylinositol generating diacylglycerol. Another enzyme, diacylglycerol lipase is present to then generate arachidonic acid. The free arachidonic acid then enters the cyclooxygenase pathway to generate prostaglandins, the lipoxygenase pathway to generate leukotrienes or both pathways. The nature of the inflammatory stimulus added to these cells determines which of the above pathways become operative. Zymosan and the Ca+ + ionophore, A23187 stimulate the synthesis of both prostaglandins and leukotrienes whereas phorbol myristate acetate and lipopolysaccharide induce only the synthesis of prostaglandins. In addition, the synthesis of these two products by macrophages can be regulated by certain antiinflammatory compounds. Indomethacin, aspirin, ibuprofen and benoxaprofen are only inhibitors of the prostaglandin pathway, whereas BW755C, 5,8,11 ETYA, NDGA and sulindac sulfide (high doses) are inhibitors of the synthesis of both prostaglandins and leukotrienes. Dapsone, an effective drug for leprosy, also inhibits the synthesis of both of these products.

Antigen-antibody complexes stimulate the synthesis and release of prostaglandins by mouse peritoneal macrophages.

Antigen-antibody complexes (Ag/Ab) formed at equivalence stimulate the release of arachidonic acid and synthesis of prostaglandin E2 and 6-keto-prostaglandin F1 alpha by resident mouse peritoneal macrophages. Prostaglandin synthesis and secretion is stimulated by submicrogram quantities of Ag/Ab which increases in a dose-dependent manner. This release is time-dependent and occurs in the absence of any loss of cell viability as indicated by increased cellular levels of lactate dehydrogenase without concomitant loss of this activity to the media and the continued secretion of a constitutive cellular product, lysozyme. The stimulated synthesis of prostaglandins by Ag/Ab is inhibited by indomethacin and physiological levels of antiinflammatory glucocorticoids.

rTNFα facilitates human polymorphonuclear leukocyte adherence to fibrinogen matrices with mobilization of specific and tertiary but not azurophilic granule markers

rTNFα facilitates highly reproducible adherence of polymorphonuclear leukocyte (PMN) to fibrinogen‐coated surfaces in a concentration‐ and time‐dependent manner. The adhesion was maximal with 1.0 nM rTNFα within 40–50 min at 37°C. A monoclonal antibody (1B4) directed toward the β2‐chain of the integrin receptor for fibrinogen (CD11b, CD18) completely inhibited the rTNFα induced adhesion. TNFα caused a time‐dependent secretion of the granule markers gelatinase and lactoferrin but no liberation of myeloperoxidase and minimal production of Aα(1–21), a specific cleavage product of fibrinogen generated by elastase, as markers for the azurophilic granule. PMN adhered to fibrinogen in the presence of rTNFα could be further stimulated with cytochalasin B and N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) to release azurophilic granule markers as measured by increasing MPO activity and Aα(1–21) production over time. Thus the rTNFα‐facilitated adherence of PMN to a fibrinogen matrix provides a system for partial activation of PMN resulting in release of markers of specific and tertiary but not azurophilic granules. Moreover, these conditions should provide an opportunity to define more clearly the signal transduction processes involved in azurophilic granule release.

A new assay for measuring adenyl cyclase activity in intact cells

Abstract A radiometric assay has been described for the measurement of newly synthesized cyclic AMP in isolated fat cells. With this technique, isolated fat cells or tissue slices are preincubated with adenine-8-C 14 in the presence of glucose which allows the intracellular build-up of labeled ATP. Subsequent treatment with lipolytic hormones causes an increased conversion of labeled ATP to cyclic AMP, the latter being measured by scintillation counting after separation from other nucleotides. Since only newly synthesized nucleotide is measured, this method is particularly useful for determining the role of agents affecting the rate of formation and breakdwon of cyclic AMP.

Pharmacological effects of non-steroidal antiinflammatory agents on prostaglandin and leukotriene synthesis in mouse peritoneal macrophages

Resident mouse peritoneal macrophages, exposed to zymosan, synthesized and released products of both the cyclooxygenase and lipoxygenase pathways. The effects of various non-steroidal antiinflammatory agents were evaluated for their abilities to inhibit zymosan-stimulated prostaglandin E2 (PGE2) and leukotriene C4 (LTC4) synthesis. The order of potencies to inhibit PGE2 synthesis and release was: indomethacin greater than or equal to sulindac sulfide greater than ibuprofen greater than or equal to aspirin greater than 3-amino-1-[3-(trifluoromethyl)-phenyl]-2-pyrazoline (BW755C) greater than benoxaprofen greater than or equal to nordihydroguaiaretic acid (NDGA) greater than 5,8,11-eicosatriynoic acid (ETYA). BW755C and ETYA also inhibited zymosan-stimulated LTC4 production. None of the compounds tested showed selective inhibition of lipoxygenase products.

Effect of RNA and protein synthesis inhibitors on the release of inflammatory mediators by macrophages responding to phorbol myristate acetate

The interaction of phorbol myristate acetate with resident populations of mouse peritoneal macrophages causes an increased release of arachidonic acid followed by increased synthesis and secretion of prostaglandin E2 and 6-keto-prostaglandin F1 alpha. In addition, phorbol myristate acetate causes the selective release of lysosomal acid hydrolases from resident and elicited macrophages. These effects of phorbol myristate acetate on macrophages do not cause lactate dehydrogenase to leak into the culture media. The phorbol myristate acetate-induced release of arachidonic acid and increased synthesis and secretion of prostaglandins by macrophages can be inhibited by RNA and protein synthesis inhibitors, whereas the release of lysosomal hydrolases is unaffected. 0.1 microgram/ml actinomycin D blocked the increased prostaglandin production due to this inflammatory agent by more than 80%, and 3 microgram/ml cycloheximide blocked prostaglandin production by 78%. Similar results with these metabolic inhibitors were found with another stimulator of prostaglandin production, zymosan. However, these inhibitors do not interfere with lysosomal hydrolase releases caused by zymosan or phorbol myristate acetate. It appears that one of the results of the interaction of macrophages with inflammatory stimuli is the synthesis of a rapidly turning-over protein which regulates the production of prostaglandins. It is also clear that the secretion of prostaglandins and lysosomal hydrolases are independently regulated.

Role of leukocyte factors and cationic polyelectrolytes in phagocytosis of group A streptococci and Candida albicans by neutrophils, macrophages, fibroblasts and epithelial cells: modulation by anionic polyelectrolytes in relation to pathogenesis of chronic inflammation.

A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.A variety of cationic polyelectrolytes opsonized group A streptococci andCandida albicans to phagocytosis by human polymorphonuclear leukocytes and by mouse peritoneal macrophages. The most potent opsonins for streptococci were specific antibodies supplemented with complement, nuclear histone, polylysine, polyarginine, ribonuclease, leukocyte lysates, leukocyte cationic protein and, to a lesser extent, lysozyme and myeloperoxidase. Histone, RNAse, leukocyte extracts, and platelet extracts also functioned as opsonins for phagocytosis of streptococci in the peritoneal cavity, where phagocytic indices, higher than those obtained for the in vitro phagocytosis, were obtained. Fresh serum, polylysine, polyarginine, and nuclear histone acted as good opsonins forCandida, but none of the other factors tested were active. In order for the cationic proteins and leukocyte extracts to function as opsonins, they must be present on the particle surface. These agents were poor opsonins when applied on the macrophages. Nuclear histone, polylysine, polyarginine, and fresh human serum also functioned as good opsonins for the uptake ofCandida by mouse fibroblasts. On the other hand, none of the other substances which opsonized streptococci were effective withCandida. The phagocytic capabilities of fibroblast polykaryons were much higher than those of ordinary spindle-shaped mouse fibroblasts. Histone also functioned as a good opsonic agent for the uptake ofCandida by human fibroblasts, HeLa cells, epithelial cells, monkey kidney cells, and rat heart cells. On the other hand, neither leukocyte extracts nor ribonuclease LCP or MPO functioned as opsonins for these mammalian cells.Candida, taken up by fibroblasts, were present within tight phagosomes, but no fusion of lysosomes with the phagosome occurred. A small proportion of the internalized yeast cells underwent partial plasmolysis, but little damage to the rigid cell walls was observed within 24–48 h of internalization. Phagocytosis of streptococci andCandida by macrophages and the uptake ofCandida by fibroblasts were both strongly inhibited by liquoid (polyanethole sulfonic acid sodium salt). This anionic polyelectrolyte also markedly inhibited the release ofN-acetylglucosaminidase from macrophages without affecting cell viability (LDH release). Hyaluronic acid, DNA, and dextran sulfate markedly inhibited the uptake of histone-coated particles by macrophages. On the other hand, hyaluronic acid and DNA enhanced the uptake ofCandida by fibroblasts. The effect of these anionic polyelectrolytes on phagocytosis of serum-opsonized particles by macrophages was not consistent. While in some experiments it blocked phagocytosis, in others it either had no effect or even enhanced the uptake of the particles. Phagocytosis of microorganisms by “nonprofessional” phagocytes like fibroblasts and the paucity in these cells of hydrolases capable of breaking down microbial cell wall components may contribute to the persistence of non-biodegradable components of bacteria in tissues and to the perpetuation of chronic inflammatory sequellae. Cationic polyelectrolytes may also prove important as “helper” opsonins and as agents capable of enhancing the penetration into cells of both viable and nonviable particles, genetic material, and drugs.

The fibrinogen cleavage product A alpha-Val(360), a specific marker of neutrophil elastase activity in vivo

Background Alpha-1-antitrypsin (A1AT) deficiency is the only recognised genetic risk factor for chronic obstructive pulmonary disease (COPD), a leading cause of morbidity and mortality worldwide. Since A1AT is the major inhibitor of neutrophil elastase (NE), this enzyme has become widely implicated in the pathogenesis of COPD in general; however, there is currently no specific biomarker for its pre-inhibition activity. Such a biomarker should be a measure of elastase-specific COPD disease activity with the potential to assess early targeted therapeutic intervention, in contrast to traditional and non-specific disease severity markers such as forced expiratory volume in 1 s. Methods In pilot studies, plasma Aα-Val360 and markers of neutrophil activation were measured in 95 subjects with a range of A1AT concentrations. Aα-Val360 and sputum elastase activity were also measured in a further seven PiZ A1AT-deficient subjects over the course of an acute exacerbation. Finally, Aα-Val360 was measured in plasma from subjects randomised to receive A1AT replacement or placebo in the EXACTLE trial. Results The plasma concentrations of Aα-Val360 and A1AT related exponentially, consistent with previous theoretical and in vitro experimental data. L-233 (an intracellular NE inhibitor) blocked generation of Aα-Val360 and subsequent A1AT/NE complex formation. Aα-Val360 was related to the spirometric severity of lung disease in A1AT deficiency, to sputum elastase activity in acute exacerbations and was decreased in subjects receiving A1AT replacement therapy (while remaining constant in those receiving placebo). Conclusions Aα-Val360 represents the first specific footprint of pre-inhibition NE activity and is a potential biomarker of disease activity and progression in subjects with elastase-dependent COPD. Trial registration The EXACTLE study was registered in ClinicalTrials.gov as ‘Antitrypsin (AAT) to Treat Emphysema in AAT-Deficient Patients’; ClinicalTrials.gov Identifier: NCT00263887.

Prostaglandin antagonists: Studies on the mode of action of polyphloretin phosphate

Summary Studies with polyphloretin phosphate (PPP), a substance that has been reported to block the action of prostaglandins (PGE1 and PGF2α) in certain in vivo and in vitro situations, reveal it has no effect upon PGE1 stimulated cyclic AMP formation in mouse ovaries or rabbit myometrium. Instead, PPP is shown to act at a site subsequent to cyclic AMP formation, tentatively identified as the cyclic AMP dependent protein kinase step. Thus, PPP is not a true prostaglandin antagonist in these tissues, since it does not block the primary PGE1 event. The data presented, combined with the in vivo antiprostaglandin effect of PPP, support our earlier proposal that cyclic AMP is an obligatory intermediate in the action of prostaglandins.