Bo Ringertz

Leukotriene-induced neutrophil aggregation in vitro

1. INTRODUCTION Neutrophilic polymorphonuclear (PMN) leuko- cytes react with increased adherence, oxidative metabolism, chemotaxis, degranulation and bacte- rial killing after contact with substances generated in an inflammatory or infected site, e.g., the C5a- fragment and bacterial products, such as fMLP. One of the earliest recognized manifestations of an acute inflammatory response is granulocyte adhe- sion to the vascular endothelium, adjacent to the inflammatory focus. This increased adhesiveness may also lead to an increment in granulocyte ag- gregation, which can be measured in vitro in an aggregometer. A novel group of mediators of inflammation are called leukotrienes [l]. These are formed within PMNs after stimulation with fMLP or the calcium ionophore A23 187 by a lipoxygenase-dependent oxygenation of arachidonic acid. The generated epoxide leukotriene 4 (LTA4) is unstable and converted to leukotriene B4 (LTB4) by enzymatic hydrolysis. Two other 5,12-dihydroxy isomers (compounds IJI) can also be formed by a non- enzymatic hydrolysis of LTA,+ LTB4 can be further metabolized to a w-hydrox- ylated compound (20-OH-LTB4) and the corre- sponding dicarboxylic acid, (20-COOH-LTB4) [2].

The effects of lipoxin A and lipoxin B on functional responses of human granulocytes.

Lipoxin A and lipoxin B (LXA and LXB) are formed from arachidonic acid by leukocyte 5- and 15-lipoxygenases. We have assessed the effects of synthetic lipoxins on functional responses of human granulocytes. LXA stimulated migration at 1 nM. The effect was highly stereospecific, since e.g. 6S-LXA and LXB were less active than LXA. Neither synthetic LXA nor several of its stereoisomers provoked degranulation or aggregation. LXB and its isomers did not induce any of these functional responses. These results indicate that migratory granulocyte responses to LXA are highly stereospecific.

Rat neutrophil function, and leukotriene generation in essential fatty acid deficiency

Since the essential fatty acid linoleic acid is the precursor of arachidonic acid and thus of leukotriene B4 (LTB4), essential fatty acid deficiency (EFAD) may result in decreased synthesis of this stimulator of neutrophil granulocyte functions. Peritoneal and blood neutrophils from rats fed a diet with only 0.3% of energy requirements as linoleic acid and exhibiting biochemical evidence of EFAD showed substantial functional impairments compared to neutrophils from rats maintained on a diet with 3% of the energy requirement as linoleic acid. Oxidative burst activation (assessed by chemiluminescence), chemotaxis and aggregation were impaired upon stimulation with formylpeptides or the ionophore A23187. In contrast, these functions were intact on stimulation with exogenous LTB4. Chemiluminescence was slightly but not significantly enhanced in EFAD rat neutrophils compared to controls when stimulated with phorbol myristate acetate (PMA). There were no differences between EFAD and control peritoneal neutrophils in the number of f-metleu-phe (fMLP) receptors, or in their affinity for the ligand, assessed with fML(3H)P. The fraction of responding cells also were similar, assessed with dichlorofluorescein diacetate fluorescence. Moreover, the endogenous LTB4 production in response to A23187 or fMLP was decreased by 57.7% and 63.5%, respectively, in EFAD peritoneal neutrophils. Thus, EFAD was associated with reductions of LTB4 production and neutrophil responsiveness to A23187 and formylpeptides but not to LTB4 or PMA, which supports the hypothesis that endogenous LTB4 may contribute to the activation of neutrophil functions involved in inflammation and host defense.

Leukotriene B4 triggers highly characteristic and specific functional responses in neutrophils: studies of stimulus specific mechanisms.

By using human neutrophils we studied the on-off phenomenon for leukotriene B4 (LTB4) -induced functional responses compared with fMetLeuPhe (fMLP). LTB4 induced rapidly appearing and disappearing neutrophil chemiluminescent (CL), superoxide anion formation, aggregatory and membrane depolarizing responses, whereas fMLP responses were slower both in onset and termination. Increases of intracellular calcium concentrations (as reflected by quin2 and fura-2 fluorescence) were of similar magnitude for both stimuli; however, LTB4 responses were more rapidly terminated and fMLP responses were biphasic. When intracellular calcium fluxes, calmodulin or protein kinase C activities were inhibited by quin2, trifluoperazine, verapamil or 3,4,5-trimethoxybenzoic acid 8-diethylamino)octyl ester (TMB-8), profound changes were noted for chemiluminescent and aggregation kinetics induced by fMLP, whereas kinetics of LTB4 responses were less affected. When drugs were used to modulate cAMP levels, or to inhibit cyclo- and lipoxygenase metabolites of arachidonic acid, no effects on response kinetics were observed. Cytochalasin B both amplified and delayed responses although chemiluminescent responses to fMLP were amplified more than those to LTB4. Despite those effects cytochalasin B did not enhance peak fura-2 or quin2 responses to either fMLP or LTB4. Thus, LTB4 rapidly initiates functional responses in neutrophils, and stimulus-specific response patterns are already discernable during the mobilization of calcium, and can be modulated by interference with calcium-dependent reactions.

Lipoxin A4 Induces Neutrophil-Dependent Cytotoxicity for Human Endothelial Cells

The authors have assessed the capacity of neutrophil granulocytes (PMN) to kill cultured human umbilical–vein endothelial cells (HUVEC) in vitro (as release of 51Cr) in response to the recently described double dioxygenation product of arachidonic acid, lipoxin A4 (LXA4). LXA4 conferred a marked cytotoxicity, whereas formyl–methionyl–leucyl–phenylalanine (fMLP) was less potent. The LXA4 and fMLP effects were dose dependent, with a maximum at 100 nM (which caused 2.7–and 2.3–fold increases of 51 Cr release, respectively, relative to buffer–treated controls). The LXA4 and fMLP responses increased with the PMN concentration, depended on the fetal calf serum concentration, incubation temperature and duration and the presence of calcium and magnesium ions.

Effects of auranofin on leukotriene production and leukotriene stimulated neutrophil function

Arachidonic acid is metabolized in neutrophils by lipoxygenase to leukotrienes, which are suggested to play a central role in inflammation. The antirheumatic drug auranofin (4 μg/ml) was found not to inhibit neutrophil production of the lipoxygenase products 5-HETE, 15-HETE and LTB4,in vitro when stimulated with the calcium ionophore A23187. Auranofin, however, modulated neutrophil aggregation, enzyme release and chemotaxis induced by LTB4. The results suggest that auranofin may exert some of its antirheumatic effects through affecting neutrophil responses to leukotrienes.

Essential fatty acid deficiency in rats: Effects on arachidonate metabolism, generation of cyclooxygenase products and functional responses in neutrophils

Linoleic and arachidonic acid concentrations in neutrophils from rats maintained on a diet with only 0.3% of the energy content as essential fatty acid (EFA, EFAD group) were reduced by 70 +/- 2.2% and 34.8 +/- 5.2%, respectively, compared with controls fed a diet with a normal 3% EFA content. Neutrophil chemiluminescence and aggregation induced by f-Met-Leu-Phe was substantially reduced in the EFAD group. Production of 6-keto-prostaglandin F1 alpha and thromboxane B2 were significantly lower in the EFAD neutrophils when stimulated by the ionophore A23187, whereas there was no difference when leukotriene B4 was used as stimulus.

Effects of Lipoxins A and B on Functional Responses of Human Granulocytes

The effects of synthetic lipoxin A and B (LXA and LXB) as well as several of their isomers were assessed as inducers of functional responses of human granulocytes. LXA stimulated migration at 1 nM and exhibited a variable effect as inducer of chemiluminescence at concentrations greater than or equal to 1 microM. Both migration and chemiluminescence evoked by LXA proved to be highly stereospecific, since e.g. 6S-LXA was less active than LXA. Neither synthetic LXA nor several of its stereoisomers provoked degranulation, aggregation, membrane potential changes or intracellular calcium fluxes. In addition, LXB and its isomers did not stimulate aggregation, degranulation or chemiluminescence. Pretreatment of granulocytes with LXA did not modify subsequent challenges with leukotriene B4. Together, these results indicate that granulocyte responses to LXA are highly stereospecific and are mediated by other mechanisms than those evoked by leukotriene B4.

Effects of an essential fatty acid-supplemented diet on leukotriene B4-induced rat neutrophil functions

This study evaluated the effect of dietary supplementation with the essential fatty acid linoleic acid to 10% of the energy content of a diet on the stimulus-response coupling of rat peritoneal neutrophils. When stimulated with leukotriene B4 neutrophils from essential fatty acid supplemented rats responded with a significantly more pronounced oxidative metabolism (assessed as luminol augmented chemiluminescence) relative to control cells from rats on a normal 3% of total energy essential fatty acid diet. Chemiluminescence response to the formylpeptide N-formyl-norleucyl-leucyl-phenylalanine-norleucyl-thyrosyl-leucine was similarly enhanced. In contrast, responses elicited by the lectin concanavalin A did not differ between the two dietary groups. In response to leukotriene B4 a dose-related inhibition of neutrophil aggregation was observed, whereas chemotaxis did not differ between the two groups. Thus, linoleate supplementation is associated with a stimulus-specific modulation of neutrophil oxidative and aggregatory responses suggesting an effect on early, conceivably receptor-linked, steps of the stimulus-response coupling.

The Effect of 20‐Trifluoromethyl Leukotriene B4 on Neutrophil Functional Responses

20‐triHuoromelhyl‐lcLikotriene B4 (20CF3LTB4) is a stable derivative of leukotriene B4 (LTB4) that is not subjected to co‐oxidation to less active metabolites. 2OCK3LTB4 was as potent as LTB4 as a chemotactic, adhesion‐promoting and aggregatory agent for human neutrophils, but bad only 11 ± 3% of the ability to induce an Oxidative response. Nonetheless, both compounds were equally efficient in order to confer a rapid and monophasic increment of the concentration of cytosolic calcium. The kinetics of the calcium, aggregatory and chemiluminescent responses to 2OCF3‐LTB4 were similar to that of LTB4. These findings suggest that the insertion of the trifluoromethyl group into the LTB4 molecule causes a shift of the biological activity profile, suggesting that 20CF3LTB4 binds mainly to high affinity LTB4 receptors. Moreover, the similarity of the response kinetics of LTB4 and 20CF3‐ LTB4 suggests that the mechanism for the rapid and transient responses of LTB4 is not due to its w‐oxidation.