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PubMed1985

The role of the polymorphonuclear leukocyte in hyperalgesia.

The results of recent studies of the mechanism of leukotriene B4-induced hyperalgesia suggest a dependence on polymorphonuclear leukocytes (PMNLs). In this study, we addressed the contribution of PMNLs to hyperalgesia evoked by the peptide chemotactic factors N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the anaphylatoxin fragment of the fifth component of the complement pathway (C5a). Local injection of glycogen, which attracts but does not activate PMNLs, produced a marked shift to the left (toward lower concentrations) in the concentration dependence curve of fMLP-induced hyperalgesia. In addition, PMNL repletion by transfusion with syngeneic PMNLs reestablished fMLP-induced hyperalgesia in PMNL-depleted rats. Finally, supernatants from rat and human PMNLs, that had been stimulated with fMLP in vitro, produced hyperalgesia in PMNL-depleted rats. Preliminary characterization of the hyperalgesia-inducing activity released by stimulated PMNLs indicated that it is lipid in nature. The nonsteroidal anti-inflammatory indomethacin did not attenuate C5a and fMLP-induced hyperalgesia. Thus, the hyperalgesia produced by fMLP and C5a is similar to that produced by leukotriene B4 in that it is dependent on PMNLs and independent of the cyclo-oxygenation of arachidonic acid. Taken together, these data suggest that structurally diverse PMNL-chemotactic factors produce hyperalgesia by a novel mechanism, involving PMNL-derived factors.

Animals↗

PubMed1987

Human GM-CSF primes neutrophils for enhanced oxidative metabolism in response to the major physiological chemoattractants.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a T cell-derived lymphokine which induces hematopoietic precursor cells to proliferate in vitro and differentiate to neutrophils and macrophages. GM-CSF also inhibits the motility of mature neutrophils (NIF-T activity), and primes neutrophils to enhance oxidative metabolism in response to the bacterial chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (f-MLP). The present study was designed to determine whether this lymphokine also enhances neutrophil oxidative metabolism in response to the other major physiological chemoattractants which include complement-derived C5a, and the 5-lipoxygenation product of arachidonic acid, leukotriene B4 (LTB4). Superoxide anion production was measured as superoxide dismutase-inhibitable cytochrome C reduction. Purified biosynthetic GM-CSF enhanced superoxide anion production by neutrophils in response to f-MLP, C5a desArg, and LTB4. In contrast to several other factors which prime neutrophils, GM-CSF did not prime for an enhanced oxidative response to phorbol myristate acetate (PMA). These results suggest that GM-CSF may be an endogenous regulator of neutrophil inflammatory responses induced by the major physiological chemoattractants.

Chemotactic Factors↗

PubMed1998

Effects of some anti-asthma drugs on human eosinophil superoxide anions release and degranulation.

BACKGROUND: Eosinophil infiltration of bronchial tissues and subsequent release of inflammatory mediators by them are the hallmarks of bronchial asthma but it has not yet been clarified whether anti-asthma drugs affect these cells directly. In this study, we investigated the direct effects of 8 clinically used anti-asthma drugs [salbutamol, salmeterol, theophylline, denbufylline, disodium cromoglycate (DSCG), azelastine, ketotifen and dexamethasone] on superoxide anions (O2-) and eosinophil peroxidase (EPO) release from human blood eosinophils in vitro. METHODS: Highly purified eosinophils were stimulated for O2- release with platelet-activating factor (PAF) or interleukin-5 (IL-5), while for EPO release complement fragment (C5a) or N-formyl-methionyl-leucyl-phenylalanine (FMLP) was employed. Generated products were assayed by standard techniques. RESULTS: All the drugs, except ketotifen and dexamethasone, inhibited PAF-induced O2- release in a dose-dependent manner. The IC50 values were 0.7, 5.8, 330, 3,500, 4,200 and 6,250 nM for DSCG, denbufylline, salmeterol, azelastine, salbutamol and theophylline, respectively. On IL-5-induced release, the effects were similar except that salbutamol completely failed to inhibit the release induced by this stimulus. In contrast, EPO release was generally poorly inhibited, especially when the release was induced by C5a. Only theophylline and azelastine (both at 10(-4) M or more) were able to inhibit EPO release by both C5a and FMLP. Salbutamol and, to a lesser extent, salmeterol inhibited FMLP-, but not C5a-induced EPO release, while all the other drugs tested were inactive. CONCLUSIONS: The results show that some of the anti-asthma drugs, but not all, do exert direct effects on human blood eosinophils but these effects may be stimulus-dependent and by far more pronounced against O2- release than against degranulation.

Anti-Asthmatic Agents↗

PubMed2003

Anti-inflammatory properties of piperlactam S: modulation of complement 5a-induced chemotaxis and inflammatory cytokines production in macrophages.

Macrophages infiltrate tissues in response to chemoattractants including complement 5a (C5a). Infiltrating macrophages clear microorganisms but also can cause tissue damage. We hypothesized that prevention of macrophages from excessive recruitment into infected sites may underlie the anti-inflammatory effects of piperlactam S, an alkaloid isolated from Piper kadsura (Choisy) Ohwi. To test this hypothesis, chemotactic migration of RAW264.7 macrophages was induced by C5a and the effects of piperlactam S were studied. The results showed that piperlactam S (1-30 microM) concentration-dependently suppressed C5a-induced migration across a fibrinogen-coated barrier with an IC50 of 4.5 +/- 0.3 microM. At 30 microM, piperlactam S inhibited chemotaxis by more than 95 % and also decreased phagocytosis by 25 % without reducing macrophage viability and adherent capacity. Furthermore, piperlactam S treated cells adhered but failed to spread and elongate as in control cells. Finally, piperlactam S inhibited the C5a-stimulated release of tumor necrosis factor-alpha and interleukin-1beta. We conclude that retardation of macrophage recruitment by interfering with the migration process and suppression of cytokines production might underlie the potential usefulness of piperlactam S as an anti-inflammatory agent.

Animals↗

PubMed1982

Release of leukotrienes from guinea pig lung stimulated by C5ades Arg anaphylatoxin.

The complement anaphylatoxins C5a and C5Ades Arg contract guinea pig peripheral airway preparations and trachea by a mechanism largely independent of histamine release. In trachea the contractions are inhibited by FPL 55712, a relatively specific inhibitor of slow-reacting substance of anaphylaxis (SRS-A). SRS-A is now known to be a mixture of leukotrienes C4, D4, and E4 (LTC4, LTD4, LTE4). These data suggest that C5-derived anaphylatoxins stimulate production and release of leukotrienes in pulmonary tissues. To define these observations more precisely, fragments of guinea pig lung were incubated with porcine C5ades Arg, and the supernatant fluids were analyzed for leukotrienes by using both pharmacologic and chemical methods. In addition to histamine, a smooth muscle contracting activity characteristic of SRS-A was released from C5a-treated lung preparations. The contractile substance was identified as a leukotriene based on: 1) the characteristic contraction of guinea pig ileum, 2) inhibition of the contractile activity by FPL 55712, 3) enhanced release of activity in the presence of indomethacin or L-cysteine, 4) chromatographic behavior of ethanol-extracted active material on Amberlite XAD-7 resin, and 5) cochromatography of the active material on reverse-phase, high performance liquid chromatography with standard LTD4. We therefore concluded the humoral factor C5ades Arg induces a leukotriene release reaction in guinea pig lung tissue. This particular response of pulmonary tissue to anaphylatoxin has not been appreciated previously as an immediate effect of complement activation on the pathophysiology of the lung.

Anaphylatoxins↗

PubMed1991

Neutrophil chemotactic factors.

Polymorphonuclear leukocytes (neutrophils) are recruited to inflammatory sites by a variety of soluble mediators (chemoattractants) that stimulate neutrophil directed migration (chemotaxis). Many neutrophil chemoattractants such as neutrophil activating proteins, leukotriene B4 (LTB4), platelet activating factor, and complement-derived C5a, are generated endogenously by host cells or enzymatic cleavage of host proteins. Other chemoattractants such as N-formyl peptides are generated exogenously by bacteria that invade the host. Oxidative modification of methionine residues or changes in the amino acid sequence of peptide chemoattractants dramatically alter their chemoattractive properties. Many of the well-defined neutrophil chemotactic factors and studies of their structure-function relationships will be reviewed.

Animals↗

PubMed1993

Increased lower airways responsiveness associated with sinusitis in a rabbit model.

The association between sinusitis and asthma has been clinically recognized, but its precise nature is controversial. We studied the relationship between sinusitis and lower airways function in a rabbit model of sterile maxillary sinusitis induced by the chemotactic complement fragment C5a des arg. Animals kept in a head-up position following experimental sinusitis demonstrated a significant increase in airways responsiveness (AWR) to histamine. In contrast, animals of which the sinuses were injected with a saline diluent and in which no sinus inflammation developed had no increase in AWR. Experiments were subsequently carried out to investigate some of the potential mechanisms for these observations. A group of rabbits in which a distal site of inflammation was induced did not demonstrate increased AWR. Likewise, when sinus inflammation was established but passage of fluid to the lower airways was prevented, either by intubation or by head-down positioning, there was no change in AWR. A fourth experiment was designed to decrease complement factor activity before placing the animals in a head-up position. This led to the same degree of increased AWR. Neither the histology nor the bronchoalveolar lavage data demonstrated significant lower airways inflammation in any group. We conclude that sterile sinusitis can increase lower airways responsiveness to histamine in a rabbit model. The most likely mechanism for this effect is the postnasal dripping of cells or cell products into the lower airway. The role of a nasobronchial reflex or inflammatory mediator absorption could not be demonstrated in this model.

Animals↗

PubMed1987

Antigenic identification of chemotactic factor inactivator in normal human serum and bronchoalveolar lavage fluid.

Several proteins have been described that can modulate the activity of the complement component C5a, a potent chemoattractant for neutrophils. One of these inhibitors has been termed chemotactic factor inactivator (CFI). We hypothesized that CFI was antigenically present in normal human serum and that antigenic levels would correlate with the ability of serum to inhibit C5a. To test this hypothesis, CFI was purified from normal human serum, antibodies to CFI were developed in rabbits, and these reagents were used to develop an enzyme-linked immunoadsorbent assay to measure CFI. Sera from 32 normal volunteers were assayed for CFI and found to contain 77 +/- 29 micrograms/ml (range 17 to 137 micrograms/ml). Partially purified CFI from normal human sera was found to inhibit 61% +/- 9% (range 45% to 75%) of the ability of C5a to attract human neutrophils. Importantly, the ability of CFI to inhibit C5a-induced neutrophil chemotaxis correlated with the antigenic amounts of CFI (r = 0.68, P less than 0.05), suggesting that CFI is a major inhibitor of C5a. This was confirmed by the finding that (1) all C5a inhibitory activity coprecipitated with CFI by ammonium sulfate precipitation (45% to 65% saturation), and (2) depletion of this ammonium sulfate fraction of CFI resulted in a major loss in its ability to inhibit the chemotactic activity of C5a (57% vs. 8% inhibition, P less than 0.01). To determine whether CFI could play a role in the modulation of inflammation at tissue sites, normal bronchoalveolar lavage fluid was evaluated for the presence of CFI. CFI was identified in all fluids (mean 0.50 +/- 0.09 micrograms/mg albumin, range 0.14 to 1.43 micrograms/mg albumin.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1999

Inhibition of a membrane complement regulatory protein by a monoclonal antibody induces acute lethal shock in rats primed with lipopolysaccharide.

Rats pretreated with traces of LPS developed acute fatal shock syndrome after i.v. administration of a mAb that inhibits the function of a membrane complement regulatory molecule. Such a shock was not observed after the administration of large amounts of LPS instead of the mAb following LPS pretreatment. The lethal response did not occur in rats depleted of either leukocytes or complement, and a C5a receptor antagonist was found to inhibit the reaction. Furthermore, LPS-treated rats did not suffer fatal shock following the injection of cobra venom factor, which activates complement in the fluid phase so extensively as to exhaust complement capacity. Therefore, complement activation on cell membranes is a requirement for this type of acute reaction.

Acute Disease↗

PubMed1996

Effects of theophylline and rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of human eosinophils from normal and atopic subjects.

1. The effects of the non-selective phosphodiesterase (PDE) inhibitor theophylline and the selective PDE4 inhibitor rolipram on leukotriene C4 (LTC4) synthesis and chemotaxis of complement 5a (C5a)- and platelet-activating factor (PAF)-stimulated human eosinophils obtained from normal and atopic donors were investigated. 2. Eosinophils were purified from peripheral venous blood of normal and atopic subjects by an immunomagnetic procedure to a purity > 99%. Eosinophils were stimulated with PAF (0.1 microM) or C5a 0.1 microM for 15 min and LTC4 was measured by radioimmunoassay (RIA). Eosinophil chemotaxis in response to PAF and C5a was assessed with 48-well microchambers (Boyden). 3. Under these conditions substantial amounts of LTC4 (about 300-1000 pg per 10(6) cells) were only detectable in the presence of indomethacin (0.1-10 microM). To explain this finding it was hypothesized that indomethacin reversed the inhibition of LTC4 synthesis by endogenously synthesized prostaglandins, in particular prostaglandin E2 (PGE2). In fact, eosinophils release 23 pg PGE2 per 10(6) cells following PAF stimulation; this PGE2 synthesis was completely inhibited by indomethacin and readdition of PGE2 inhibited eosinophil LTC4 synthesis (IC50 = 3 nM). The following experiments were performed in the presence of 10 microM indomethacin. 4. Theophylline (IC50 approximately 50 microM) and rolipram (IC50 approximately 0.03-0.2 microM) suppressed PAF- and C5a-stimulated LTC4 synthesis. This PDE inhibitor-induced suppression of LTC4 generation is mediated by activation of protein kinase A, since it was reversed by the protein kinase A inhibitor Rp-8-Br-cyclic AMPS. In addition, exogenous arachidonic acid concentration-dependently (0.3 microM-3 microM) reversed the inhibition of LTC4 synthesis by the PDE inhibitors, indicating that theophylline and rolipram suppress the mobilization of arachidonic acid. The beta 2-adrenoceptor agonist salbutamol inhibited eosinophil LTC4 synthesis (IC50 = 0.08 microM). The combination of salbutamol with theophylline (10 microM) or rolipram (3 nM) appeared to be additive. 5. Theophylline (IC50 approximately 40 microM), rolipram (IC50 approximately 0.02 microM [C5a], approximately 0.6 microM [PAF]) and PGE2 (IC50 approximately 3 nM) inhibited C5a- and PAF-stimulated eosinophil chemotaxis. The combination of PGE2 with theophylline resulted in an additive effect. 6. Both C5a- and PAF-stimulated eosinophil chemotaxis and LTC4 generation were significantly elevated in eosinophils from atopic individuals compared to normal subjects. However, eosinophils from normal and atopic individuals were not different with respect to their total cyclic AMP-PDE and PDE4 isoenzyme activities as well as the potencies of theophylline and rolipram to suppress LTC4 generation and chemotaxis.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

Chemotaxis of blood neutrophils from patients with primary ciliary dyskinesia.

Primary ciliary dyskinesia is characterized by chronic upper and lower respiratory infections which are caused by the grossly impaired ciliary transport. Since the cilia and neutrophils both utilize microtubular system for their movement, it has been speculated that neutrophil motility such as chemotaxis might be impaired in patients with primary ciliary dyskinesia. Neutrophils were purified from whole blood from 16 patients with primary ciliary dyskinesia and from 15 healthy controls. Chemotactic responses of neutrophils to leukotriene B(4) (LTB(4)), complement 5a (C5a), and formylmethionylleucylphenylalanine (fMLP) were examined using the under agarose method. The chemotactic differentials in response to LTB4, C5a, and fMLP in neutrophils from the patient group were significantly lower than the corresponding values in neutrophils from the control group (p<0.05 for all comparisons). The difference in chemotactic index between the two groups was statistically significant for LTB4 and fMLP (p<0.05 for both comparisons), but not for C5a (p=0.20). Neutrophils from patients with primary ciliary dyskinesia showed a decreased chemotactic response as compared with those from normal subjects. It is concluded that the increased frequency of respiratory tract infection in patients with primary ciliary dyskinesia is possibly due to the defective directional migration of neutrophils, as well as to the defective mucociliary clearance of the airways.

Adolescent↗

PubMed1982

Cellular changes in bone marrow of malaria-infected mice. III. Chemotaxis of granulocytes.

The number of bone marrow cells and their chemotactic activity was studied during malaria infection. Two days after infection of Balb/c mice with Plasmodium berghei, an increase in granulocyte number was observed in the blood. A modified Boyden chamber chemotaxis assay was employed to investigate the mechanism of granulocyte accumulation in the blood. Bone marrow cells from normal mice, from mice during a primary lethal infection and from immune mice after challenge were compared. The complement factor C5a showed chemotactic activity for bone marrow cells; a significant decrease of chemotaxis was only observed after 6 days of primary infection. Extracts of spleen, liver and infected erythrocytes lacked chemotactic activity, or caused inhibition of cell migration. Serum from mice with a 2-day primary infection contained chemotactic activity. The active component was heat labile, protease sensitive and had an estimated molecular weight of 250,000.

Animals↗

PubMed2005

Requisite role for complement C5 and the C5a receptor in granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate.

The development of pulmonary granulomatous lesions during mycobacterial infection is a complex phenomenon, in part caused by responses elicited towards the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor). The molecular mechanisms underlying granuloma formation following challenge with TDM are not yet completely understood. The present study defines pathologic differences in acute response to Mycobacterium tuberculosis TDM in C57BL/6 mice and mice lacking the C5a receptor (C5aR-/-). Mice were intravenously injected with TDM prepared in water-in-oil-in-water emulsion and examined for histologic response and changes in proinflammatory cytokines and chemokines in lung tissue. Control C5a receptor-sufficient mice demonstrated a granulomatous response that peaked between days 4 and 7. Increased production of macrophage inflammatory protein-1 alpha (MIP-1alpha), interleukin-1beta (IL-1beta) and CXC chemokine KC (CXCL1) correlated with development of granulomas, along with modest change in tumor necrosis factor-alpha (TNF-alpha). In contrast, the C5aR-/- mice revealed markedly exacerbated inflammatory response. The receptor-deficient mice also demonstrated a lack of coherent granulomatous response, with severe oedema present and instances of lymphocytic cuffing around pulmonary vessels. Lung weight index was increased in the C5aR-/- mice, correlating with increased MIP-1alpha, KC, IL-1beta and TNF-alpha over that identified in the congenic C5aR-sufficient controls. Correlate experiments performed in C5-deficient (B10.D2-H2d H2-T18c Hco/oSnJ) mice revealed similar results, leading to the conclusion that C5 plays a significant role in mediation of chemotactic and activation events that are the basis for maturation of granulomatous responses to TDM.

Animals↗

PubMed2002

Apoptosis of bovine neutrophils following diapedesis through a monolayer of endothelial and mammary epithelial cells.

In a two-chamber system, isolated blood polymorphonuclear neutrophil leukocytes (PMN) were allowed to migrate (5 h, 37degrees C) in response to bovine complement component C5a across calfskin and rat-tail type I collagen-coated micropore membranes, arterial endothelial, or mammary epithelial cell monolayer on calfskin and rat-tail collagen-coated membranes, respectively. Migration through calfskin collagen-coated membranes resulted in 14.5% +/- 3.4% apoptotic PMN, which was significantly higher than 6.6% +/- 1.2% apoptotic nonmigrated C5a-treated PMN. The addition of an endothelial or epithelial cell monolayer to collagen-coated membranes prevented apoptosis of migrated PMN. After removing the membranes, nonmigrated (untreated and C5a treated) and migrated PMN were incubated for an additional 20 h. At this time point, 69.1% +/- 4.5% and 47% +/- 4.5% of PMN that have migrated through a calfskin-coated membrane and an endothelial monolayer, respectively, were apoptotic, compared with 28.2% +/- 3.0% and 21.1% +/- 4.5% apoptotic untreated and C5a-treated PMN, respectively; 46.9% +/- 4.8% of PMN that have migrated through rat-tail-coated membranes were apoptotic compared with 14.7% +/- 2.3% and 9.3% +/- 1.2% apoptotic untreated and C5a-treated PMN, respectively. Migration across rat-tail collagen-coated membranes with a monolayer of epithelial cells did not affect apoptosis of migrated PMN, even after 20 h of incubation. In conclusion, migration of PMN across collagen-coated membranes (either calfskin or rat-tail collagen) induced an apoptotic response, which was downregulated by a monolayer of endothelial cells and was negated by an epithelial cell monolayer.

Animals↗

PubMed1984

Inhibition of human polymorphonuclear leukocyte cell responses by ibuprofen.

The stimulation of cell swelling, cell aggregation, polymorphonuclear leukocyte locomotion, and lysosomal enzyme release in response to chemoattractant were all inhibited by ibuprofen, a nonsteroidal anti-inflammatory agent. The dosages needed to induce 50% inhibition (ID50) were 5.9, 7.6, 60, and 95 micrograms/mL, respectively. Aside from the differences in ID50, there was also a difference in the degree of maximum inhibition (Imax) of the complement C5a-stimulated responses observed, so that at achievable serum drug concentrations of 20-50 micrograms/mL, inhibition of 67-78% for cell swelling, 69-82% for cell aggregation, 20-35% for migration response, and 17-38% for lysosomal enzyme release were demonstrated. Also observed were a minor stimulatory effect on nitroblue tetrazolium reduction and an inhibitory effect on the ability to kill Staphylococcus aureus, but only at very high concentrations (approximately 2 mg/mL).

Blood Bactericidal Activity↗

PubMed1998

Dialysis neutropenia: the role of the cytoskeleton.

Dialysis neutropenia is the result of pulmonary sequestration of neutrophils after complement activation by the dialyzer membrane. Increased expression of neutrophil adhesion receptors, such as CD11b/CD18, suggests that neutrophil adhesion to the capillary endothelium is a possible mechanism. An alternative hypothesis is that the complement fragment C5a modulates neutrophil mechanical properties via the cytoskeleton-largely filamentous actin (F-actin)-stiffening them and thereby slowing their passage through the pulmonary capillaries. To investigate this hypothesis, we developed an assay to measure the F-actin content of neutrophils in whole blood using flow cytometry and the stain NBD-phallacidin. We measured neutrophil F-actin content during hemodialysis of patients with polysulfone (N = 6), Hemophan (N = 6), and Cuprophan membranes sterilized with either ethylene oxide (N = 5) or steam (N = 6). Cell counts, neutrophil and monocyte CD11b expression and plasma C5a concentrations were also measured. The results confirm the strong relationship between the degree of neutropenia, increases in CD11b expression and plasma C5a levels reported by previous researchers. Modulation of the F-actin content of neutrophils was also strongly related to C5a levels, indicating that the neutrophil cytoskeleton is active during dialysis. Modeling of cell counts suggests that with Cuprophan a substantial fraction of neutrophils and monocytes are sequestered before they even pass through the dialyzer, suggesting some form of systemic activation of these cells. Evidence for systemic activation was also seen in measurements of F-actin content, but not CD11b expression, a finding that strengthens the case for the involvement of the cytoskeleton in dialysis neutropenia.

Actins↗

PubMed2004

Chemotaxis inhibitory protein of Staphylococcus aureus, a bacterial antiinflammatory agent.

Leukocyte migration is a key event both in host defense against invading pathogens as well as in inflammation. Bacteria generate chemoattractants primarily by excretion (formylated peptides), complement activation (C5a), and subsequently through activation of leukocytes (e.g., leukotriene B4, platelet-activating factor, and interleukin 8). Here we describe a new protein secreted by Staphylococcus aureus that specifically impairs the response of neutrophils and monocytes to formylated peptides and C5a. This chemotaxis inhibitory protein of S. aureus (CHIPS) is a 14.1-kD protein encoded on a bacteriophage and is found in >60% of clinical isolates. CHIPS reduces the neutrophil recruitment toward C5a in a mouse peritonitis model, even though its activity is much more potent on human than on mouse cells. These findings suggest a new immune escape mechanism of S. aureus and put forward CHIPS as a potential new antiinflammatory therapeutic compound.

Amino Acid Sequence↗

PubMed1994

Priming for the synthesis of 5-lipoxygenase products in human blood ex vivo by human granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha.

BACKGROUND: Previous studies reported the priming effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF alpha) on leukotriene synthesis by isolated polymorphonuclear leukocytes; however, little is known as of yet of these biologic effects of the two cytokines in a physiologic environment. EXPERIMENTAL DESIGN: In this study, we investigate the effects of GM-CSF and TNF alpha on the synthesis of 5-lipoxygenase (5-LO) products in heparinized blood stimulated ex vivo, using reverse phase high performance liquid chromatography analysis of deproteinized plasma samples. RESULTS: Stimulation of blood with f-Met-Leu-Phe resulted in the accumulation of up to 30 pmol of 5-LO products/ml of plasma. Preincubation of blood with 100 pM GM-CSF or 1.2 nM (200 units/ml) TNF alpha for 30 minutes at 37 degrees C before stimulation with f-Met-Leu-Phe resulted in a marked enhancement (> 5-fold) of the synthesis of leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid, which were formed in equivalent amounts. GM-CSF and TNF alpha priming activities were detectable at concentrations as low as 3 pM and 6 pM (1 unit/ml), respectively. The preincubation times required for optimal priming by GM-CSF and TNF alpha were different (40 and 10 minutes, respectively), and the effects of the two cytokines on leukotriene B4 and 5(S)-hydroxyeicosatetraenoic acid synthesis were additive, suggesting different priming mechanisms. The synthesis of 5-LO products in primed blood was also induced by platelet-activating factor, the complement fragment C5a, the particulate stimulus zymosan, and the ionophore A23187, but not by interleukin-8. Polymorphonuclear leukocytes and mononuclear cells accounted for 80% and 20% of the synthesis of 5-LO products, respectively. CONCLUSIONS: These data demonstrate that GM-CSF and TNF alpha exert very potent priming effects on the biosynthesis of 5-LO products in whole blood stimulated by various stimuli and strongly support that these cytokines could be important modulators of lipid mediator synthesis in physiologic and pathophysiologic conditions.

Arachidonate 5-Lipoxygenase↗