蜂毒肽的溶血作用与红细胞膜上两种酶活性变化的关系

从蜂毒肽作用于红细胞膜上的Na^＋-K^＋-ATPase和葡萄糖-6-磷酸脱氢酶(G-6-PD)活性变化的角度,利用分光光度法测定酶活性,研究蜂毒肽与红细胞及膜作用过程中可能的靶点,讨论了蜂毒肽溶血过程与RBC膜上2种酶活性的变化．结果发现,蜂毒肽抑制RBC膜上酶活性的主要模式为附着/插入质膜与游离态并存模式,附着/插入质膜中的作用大于游离态的作用．Na^＋-K^＋-ATPase的K⁺结合位点是蜂毒肽的1个作用靶点．蜂毒肽插膜过程与其对此酶的作用随时间延长同步发生．蜂毒肽通过作用于葡萄糖-6-磷酸和NADP使G-6-PD的催化受到缓慢抑制,蜂毒肽形成四聚体的程度与酶活性密切相关．EDTA抑制蜂毒肽聚集,干扰蜂毒肽作用于G-6-P,蜂毒肽作用于底物G-6-P及辅酶NADP的生化机理相似,蜂毒肽抑制作用与G-6-PD的结构无关.     

Phe-D-Leu-Phe-D-Leu-Phe derivatives as formylpeptide receptor antagonists in human neutrophils: cellular and conformational aspects.

We synthesized several Phe-d-Leu-Phe-d-Leu-Phe analogues in which tert-butyloxycarbonyl and four different ureido substituents were included at the N-terminal of the peptides, obtained as free acid and methyl-ester derivatives. Their biological action was analysed on human neutrophil responses induced by N-formyl-Met-Leu-Phe (fMLF). Several in vitro assays were carried out: receptor binding, measurement of Ca2+ intracellular concentration, chemotaxis, superoxide anion production and enzyme release. A conformational investigation, using infrared absorption and circular dichroism, was also performed. Our results demonstrate that the compounds examined prefer an ordered conformation (beta-turn) in amphipathic environment, and are able to antagonize the neutrophil functions evoked by fMLF. Moreover, the extent of inhibition of Ca2+ intracellular enhancement, as well as of superoxide anion production and granule enzyme release, appears related to their affinity toward the formylpeptide receptor. The free acid peptide derivatives appear to be more active antagonists than the methyl-ester ones.     

Bimodal regulatory effect of melittin and phospholipase A2-activating protein on human type II secretory phospholipase A2

Melittin and phospholipase A2-activating protein (PLAP) are known as efficient activators of secretory phospholipase A2(sPLA2) types I, II, and III when phospholipid liposomes are used as substrate. The present study demonstrates that both peptides can either inhibit or activate sPLA2 depending on the peptide/phospholipid ratio when erythrocyte membranes serve as a biologically relevant substrate. Low concentrations of melittin and PLAP were observed to inhibit sPLA2-triggered release of fatty acids from erythrocyte membranes. The inhibition was reversed at melittin concentrations above 1 microM. PLAP-induced inhibition of sPLA2 persisted steadily throughout the used concentration range (0-150 nM). The two peptides induced a dose-dependent activation of sPLA2 at low concentrations, followed by inhibition when model membranes were used as substrate. This opposite modulatory effect on biological membranes and model membranes is discussed with respect to different mechanisms the interaction of the regulatory peptides with the enzyme molecules and the substrate vesicles.     

Melittin exerts multiple effects on the release of free fatty acids from L1210 cells: lack of selective activation of phospholipase A2 by melittin

Melittin is known as a phospholipase A2 (PLA2) activator, but the selectivity of its effect on PLA2 is uncertain. We examined the selectivity of melittin effect on the release of free fatty acids (FFAs) from L1210 cells using various inhibitors. A systemic lipid analysis by HPLC and GLC revealed that melittin induced release of various FFAs including saturated, monounsaturated, and polyunsaturated FFAs. Various PLA2 inhibitors examined exerted only minimal effects on the melittin-induced arachidonic acid (AA) and palmitic acid (PAL) releases. Specific inhibitors of phosphatidylinositol-phospholipase C (U73122) and diacylglycerol lipase (RHC80267) exerted significant inhibitory effects on both AA and PAL releases. These results suggest that melittin-induced FFA release is most likely due to multiple participations of various types of lipases. Since BAPTA/AM, an intracellular Ca2+ chelator, did not influence the FFA release, the Ca2+ influxed by melittin appeared not to be a key factor for the FFA release. The mimicking of the melittin-induced FFA release by digitonin, a membrane-permeabilizing agent, implies that the membrane-perturbing action of melittin is likely the cause of the FFA release. Melittin also induced release of multiple FFAs from other cell lines including P388D1 and HL60. The rapid melittin-stimulated phospholipase D (PLD) observed in L1210 cells appeared not directly related to the steady release of FFA, as indicated by the fact that the PLD was not blocked by RHC80267. In view of melittin's multiple effects on the composition of cellular lipids, we conclude that melittin does neither exclusively release any single FFA nor selectively activate PLA2 in L1210 cells. The problem of using melittin as a PLA2 activator is discussed.     

Viscous macromolecules inhibit erythrocyte hemolysis induced by snake venom phospholipase A2

The effect of medium viscosity on lysis of red blood cells (RBC) induced by snake venom phospholipase A2 (PLA2) was examined. The medium viscosity was modified by the addition of various macromolecules which differ in their chemical nature and in their capacity to increase fluid viscosity. PLA2 and Ca++ were applied to cells suspended in viscous medium to induce hemolysis. It was found that the hemolysis is inhibited in direct proportion to increasing viscosity of the extracellular fluid. This phenomenon was observed with aggregated as well as disaggregated RBC. To examine whether the viscosity interferes with the accessibility of the enzyme to the cell, the medium viscosity was modified after binding of the enzyme to the cells; PLA2 was added to a RBC suspension in the presence of Ba++ which binds the enzyme to the cell membrane but does not activate it. The cell-enzyme complex was separated by gel filtration and suspended in viscous medium in the presence of Ca++ which activates the reaction. Also in this case RBC lysis was inhibited as the medium viscosity was increased. It is proposed that the action of PLA2 on RBC membrane is regulated by the viscosity of the cell surface aqueous environment.     

Monosodium urate crystals stimulate phospholipase A2 enzyme activities and the synthesis of a phospholipase A2-activating protein

Eicosanoids are important mediators of the inflammatory response to monosodium urate crystals (MSUC) that results in gout. Phospholipase enzymes cleave fatty acids from membrane phospholipids, and this is thought to be the rate-limiting step in eicosanoid production. To understand better the mechanism of eicosanoid production in this disease, we stimulated human peripheral blood neutrophils and monocytes with MSUC and measured phospholipase enzyme activities. MSUC stimulated both intracellular and secretory phospholipase A2 enzyme activities in a time and concentration-dependent manner. Specificity was observed, as phospholipase C activities were not affected. Pretreatment with colchicine, but not aspirin, indomethacin, allopurinol, or islet activating protein, abrogated the enhanced phospholipase A2 activities. We have recently isolated and characterized a phospholipase A2 activating protein termed PLAP from synovial fluid from patients with rheumatoid arthritis, and from murine and bovine cell lines. PLAP was detected in gouty synovial fluid by immunodot blotting and ELISA assays and expressed the same characteristics as PLAP identified from other sources. To examine the role of PLAP in MSUC-induced phospholipase A2 stimulation, we treated cells with MSUC and observed an increase in immunoreactive PLAP. This response also could be blunted by colchicine, but not other drugs. Both phospholipase A2 and PLAP induced production by human monocytes of PGE2 and leukotriene B4 by neutrophils. These findings suggest that phospholipase A2 activation in response to MSUC requires an intact microtubule structure, and that phospholipase A2 and PLAP may be important modulators of at least a portion of the gouty inflammatory response.     

Alternagin-C, a nonRGD-disintegrin, induces neutrophil migration via integrin signaling.

Recently, a new protein containing a disintegrin domain, alternagin-C (Alt-C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt-C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt-C on human neutrophil chemotaxis in vitro and the activation of integrin-mediated pathways. Alt-C showed a potent chemotactic effect for human neutrophils when compared to N-formyl-methionyl-leucyl-phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt-C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt-C and fMLP. A significant increase of F-actin content was observed in cells treated with Alt-C, showing that the chemotactic activity of Alt-C on neutrophils is driven by actin cytoskeleton dynamic changes. Furthermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3-kinase. Alt-C was also able to induce a significant increase in extracellular signal-regulated kinase 2 nuclear translocation. The chemotactic activity of Alt-C was partially inhibited by LY294002, a specific phosphatidylinositol 3-kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt-C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin-activated pathways and that these effects could be related to the ECD mogif present in disintegrin-like domain.     

Tissue plasminogen activator and plasmin independently decrease human neutrophil activation

Tissue-plasminogen activator (t-PA) and plasmin both decrease platelet aggregation, which may contribute to thrombolysis and tissue salvage. Since neutrophils may contribute to reperfusion injury, we examined the effects of t-PA and plasmin on human neutrophil function. t-PA (1 to 100 micrograms/ml) decreased f-MLP-induced chemotaxis and ionophore A23187-induced superoxide and LTB4 release in isolated neutrophils, and these effects were not blocked by the plasmin-inhibitor epsilon-aminocaproic acid (epsilon-ACA). On the other hand, plasmin (0.05 to 0.5 units/ml) also decreased these neutrophil functions but its effects were blocked in the presence of epsilon-ACA. Thus, while both t-PA and plasmin decrease neutrophil functions, effects of t-PA are independent of plasmin generation. Cumulative effects of t-PA and plasmin on neutrophil functions may relate to the overall efficacy of t-PA in thrombotic disorders.     

MASP-1 Induces a Unique Cytokine Pattern in Endothelial Cells: A Novel Link between Complement System and Neutrophil Granulocytes

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca²⁺-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms.     

NATO advanced study institute on “drugs affecting leukotrienes and other eicosanoid pathways”

Several vasodilator compounds (e.g. acetylcholine) relax isolated arteries by a mechanism dependent on an intact endotheliu. The inhibitors of phospholipase A₂, mepacrine and p-bromophenacylbromide, reversed the acetylcholine-induced relaxations of rabbit aortic strips. We therefore tested the effect of melittin, a compound which stimulates phospholipase activity. Melittin induced concentration-dependent rapid relaxations of strips of rabbit aorta, preconstricted with noradrenaline, if the endothelium was intact. Strips with noradrenaline, if the endothelium aorta, preconstricted with removed endothelium were not relaxed but rather slightly contracted by the compound. Similar to acetylcholine-induced relaxations melittin relaxations could be completely prevented by nordihydroguaiaretic acid, an inhibitor of lipoxygenases, but not by even high concentrations of indomethacin, an inhibitor of cyclooxygenase. Also the dual inhibitors of cyclooxygenase and lipoxygenases 5,8,11,14-eicosatetraynoic acid (ETYA) and 3-amino-1-(3-trifluoromethylphenly)-2-pyrazoline (BW 755C) inhibited relaxations induced by both melittin and acetylcholine.We therefore conclude that melittin induces the release and/or formation of a substance (an eicosanoid?) by endothelial cells which in turn relaxes the smooth muscle of the blood vessel. This compound is probably a lipoxygenase product of arachidonic acid. The mechanism of relaxation by melittin resembles that induced by acetylcholine.     

蜂毒溶血肽对鸡红细胞及膜的生化作用

本文采用荧光分光光度、薄层层析、原子吸收、荧光显微图像等多种生化技术,系统研究了蜂毒肽作用于鸡红细胞及膜的生化机理。结果表明：蜂毒肽影响红细胞膜上及胞内两种酶的功能。它抑制膜Na⁺-K⁺-ATPase活性,导致胞内外离子转运异常,K⁺浓度失衡;它也抑制细胞内葡萄糖-6-磷酸脱氢酶活性,其正电区域干扰胞内带负电小分子的作用,影响红细胞正常代谢。蜂毒肽干扰膜中阴离子通道的转运功能,使细胞渗透压改变,引起膨胀而溶血。蜂毒肽对有核红细胞核内DNA没有作用,与其他抗微生物多肽作用的靶向不同。据此认为,抗菌蛋白类抗生素对细菌作用的生化机理与传统抗生素不同,这是细菌对其不易产生耐药性的重要原因。     

Evaluation of synthetic sphingosine, lysosphingolipids and glycosphingolipids as inhibitors of functional responses of human neutrophils.

Human neutrophils, when exposed to soluble stimuli, aggregate, release oxygenated products of arachidonic acid and generate active oxygen species. Sphingolipid-derived products such as sphingosine and lysosphingolipids have been shown to exert selective actions on a variety of cell types, including neutrophils. Therefore, to determine the structural basis for selective inhibition of neutrophil responses by naturally occurring sphingolipids, seven compounds were prepared by total organic synthesis, and their impact on neutrophils in suspension has been studied. The compounds synthesized included sphingosine, psychosine, lactosyl lysosphingolipid, globotriaosyl (Gb3) lysosphingolipid, galactosyl cerebroside, lactosyl ceramide and Gb3 ceramide. The neutrophil responses studied were aggregation, leukotriene generation and superoxide anion production. When exposed to non-cytotoxic levels of the synthetic compounds, as monitored by exclusion of Trypan Blue, none of the synthetic sphingolipids inhibited A23187-induced aggregation of neutrophils. Only lactosyl lysosphingolipid, at a concentration of 1 microM, significantly inhibited aggregation induced by fMetLeuPhe; the other compounds in this series including sphingosine were without effect at equal molar concentrations (1 microM). Aggregation induced by phorbol 12-myristate 13-acetate (PMA) (0.1 microM) was significantly blocked by only two of the synthetic sphingolipids (1 microM). At concentrations below 1 microM, these inhibitory actions were not evident, nor was it possible to assign a structure-activity relationship for this series of compounds. None of the synthetic sphingolipids effectively inhibited the generation of superoxide anions induced by PMA. In addition, neither synthetic sphingosine nor psychosine affected either the formation or metabolism of leukotriene B4. Taken together, the results provide further evidence that sphingolipids, when added to intact cells, are not potent selective inhibitors of functional responses of human neutrophils.     

The evaluation and structure-activity relationships of 2-benzoylaminobenzoic esters and their analogues as anti-inflammatory and anti-platelet aggregation agents

Forty-seven 2-benzoylaminobenzoic esters were synthesized and evaluated in anti-platelet aggregation, inhibition of superoxide anion generation, and inhibition of neutrophil elastase release assays. Most 2-benzoylamino-4-chlorobenzoic acid derivatives showed selective inhibitory effects on arachidonic acid (AA)-induced platelet aggregation. Among them, compounds 6b and 7b exhibited more potent inhibitory effects (ca. 200-fold) than aspirin. Additionally, compounds 1a and 5a showed strong inhibitory effects on neutrophil superoxide generation with IC(50) values of 0.65 and 0.17 microM, respectively. However, compounds 6d and 6e exhibited dual inhibitory effects on platelet aggregation and neutrophil elastase (NE) release; therefore, these two compounds may be new leads for development as anti-inflammatory and anti-platelet aggregatory agents.     

Presynaptic facilitation of glutamate release from isolated hippocampal mossy fiber nerve endings by arachidonic acid

Hippocampal mossy fiber synaptosomes were used to investigate the role of arachidonic acid in the release of endogenous glutamate and the long-lasting facilitation of glutamate release associated with long-term potentiation. Exogenous arachidonate induced a dose-dependent efflux of glutamate from the hippocampal mossy fiber synaptosomes and this effect was mimicked by melittin. Neither treatment induced the release of occluded lactate dehydrogenase at the concentrations used in these experiments. In each case, removal of the biochemical stimulus allowed for glutamate efflux to return to spontaneous levels. However, there was a persistent effect of exposure to either arachidonate or melittin, since these compounds facilitated the glutamate release induced by the subsequent addition of 35 mM KCl. This facilitation of glutamate release resulted from an enhancement of both the magnitude and duration of the response to depolarization. Although exogenous prostanoids were also able to stimulate the release of glutamate, they appeared to play no direct role in secretion processes, since inhibition of eicosanoid synthesis potentiated the glutamate efflux in response to membrane depolarization or exogenous arachidonic acid. We suggest that the calcium-dependent accumulation of arachidonic acid in presynaptic membranes plays a central role in the release of endogenous glutamate and that the persistent effects of arachidonic acid may be related to the maintenance of long-term potentiation in the hippocampal mossy fiber-CA3 synapse.     

Modulation of neutrophil phospholipase C activity and cyclic AMP levels by fMLP-OMe analogues

The N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-OMe (1) analogues for-Thp-Leu-Ain-OMe (2), for-Thp-Leu-Phe-OMe (3), for-Met-Leu-Ain-OMe (4), for-Met-Delta(z)Leu-Phe-OMe (5), for-Met-Lys-Phe-For-Met-Lys-Phe (6), for-Met-Leu-Pheol-COMe (7), and for-Nle-Leu-Phe-OMe (8) have been studied. Some of these have been found selective towards the activation of different biological responses of human neutrophils. In particular, peptides 2 and 3, which evoke only chemotaxis, are ineffective in enhancing inositol phosphate, as well as cyclic AMP (cAMP) levels. On the contrary, analogues 5 and 7, which induce superoxide anion production and degranulation, but not chemotaxis, significantly increase the levels of the two intracellular messengers, as is the case of the full agonists 1 and 6. The Ca(2+) ionophore A23187 also activates phospholipase C (PLC) and increases the nucleotide levels; when tested in combination with peptide 1 or 5, a supra-additive enhancement of cAMP concentration is obtained. The PLC blocker, U-73122, inhibits the formylpeptide-induced inositol phosphate formation, as well as cAMP increase. Moreover, this drug drastically reduces superoxide anion release triggered by 1 or 5, whereas it inhibits to a much lesser extent neutrophil chemotaxis induced by 1 or 2. Our results suggest that: (i) PLC stimulation is involved in cAMP enhancement by formylpeptides; (ii) the activation of PLC by formylpeptides, in conditions of increased Ca(2+) influx, induces a supra-additive enhancement of the nucleotide; (iii) the inability of pure chemoattractants to significantly alter the PLC activity or cAMP level, differently from full agonists or peptides specific in inducing superoxide anion release, appears as a general property. Thus, the activation of neutrophil PLC seems essential for superoxide anion release, but less involved in the chemotactic response.     

Melittin induces both time-dependent aggregation and inhibition of Na,K-ATPase from duck salt glands however these two processes appear to occur independently

Using cupric phenanthroline as a cross-linking agent, we have shown that melittin induced time-dependent aggregations of Na,K-ATPase in microsomal fractions and in preparations of purified Na,K-ATPase from duck salt glands. Incubation of melittin with these preparations also led to the progressive loss of Na,K-ATPase activity. At melittin/protein molar ratio of 5:1, we did not observe inhibition of Na,K-ATPase in the microsomal fraction but the process of enzyme aggregation occurred. At higher melittin/protein molar ratios (10:1 and 30:1), the inhibition of the enzyme and its aggregation proceeded simultaneously but the rates of these processes and maximal values achieved were different. At a melittin/protein ratio of 30:1, Na,K-ATPase inhibition may be described as a biexponential curve with the values for pseudo-first order rate constants being 2.7 and 0.15 min(-1). However, the aggregation may be presented by a monoexponential curve with a pseudo-first order rate constant of 0.15 min(-1). In purified preparations of Na,K-ATPase, the maximal aggregation (about 90%) was achieved at a melittin/protein molar ratio of 2:1, and a further increase in the melittin/protein ratio increased the rate of aggregation but did not affect the value of maximal aggregation. The results show that melittin induced both aggregation and inhibition of Na,K-ATPase but these two processes proceeded independently.     

Eicosanoid Release Is Increased by Membrane Destabilization and CFTR Inhibition in Calu-3 Cells

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2α) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2α. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-α. This was concomitant with increased IL-8 synthesis and cPLA2α activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-β-cyclodextrin induced further cPLA2α activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-α-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2α and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-α-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.     

Physiological concept for a blood based CFTR test.

We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) could be involved in the volume regulation of human red blood cells (RBC). Experiments were based on two gadolinium (Gd(3+)) sensitive mechanisms, i.e. inhibition of ATP release (thetaATP(i)) and membrane destabilization. RBC of either cystic fibrosis (CF) patients or healthy donors (non-CF) were exposed to KCl buffer containing Gd(3+). A significantly larger quantity of non-CF RBC (2.55 %) hemolyzed as compared to CF RBC (0.89 %). It was found that both of the Gd(3+) mechanisms simultaneously are needed to achieve hemolysis, since either overriding thetaATP(i) by exogenous ATP addition prevented Gd(3+) induced hemolysis, or mimicking thetaATP(i) by apyrase in absence of Gd(3+) could not trigger hemolysis. Additionally, ion driven volume uptake was found to be a prerequisite for Gd3+ induced hemolysis as chloride and potassium channel blockers reduced the Gd(3+) response. The results show that in non-CF RBC Gd(3+) exerts its dual effect leading to hemolysis. On the contrary, in CF RBC, lacking CFTR dependent ATP release, the sole Gd(3+) effect of membrane destabilization is not sufficient to induce hemolysis similar to non-CF. This concept could form the basis of a novel method suitable for testing CFTR function in a blood sample.     

Purification and partial biochemical characterization of a human monocyte-derived, neutrophil-activating peptide that lacks interleukin 1 activity

A novel monocyte-derived neutrophil-activating peptide (MONAP) produced by lipopolysaccharide- and phorbol myristate acetate-stimulated human peripheral blood monocytes was purified by sequential ion exchange-high performance liquid chromatography (HPLC), size exclusion HPLC, and reversed phase HPLC. Biologic activities of the purified cytokine were monitored by either an enzyme release assay or a chemotaxis assay, using peripheral human neutrophils. Purified MONAP was found to be homogeneous, giving a single peak on size-exclusion HPLC, reversed-phase HPLC, as well as a single 10-kDa band on silver-stained polyacrylamide gels. Purified MONAP stimulate human neutrophil chemotaxis at an estimated molarity of 5 x 10(-11) M. Half-maximal enzyme release of cytochalasin B pretreated neutrophils occurred at 2 to 3 x 10(-10) M, whereas superoxide anion production elicited by various concentrations of MONAP was found to be low. Isolated human peripheral monocytes, as well as human eosinophils, showed no chemotactic response to MONAP, indicating neutrophil specificity. MONAP activity was separated from thymocyte-stimulating activity by reversed-phase HPLC, indicating nonidentity with interleukin (IL)-1. This was further supported by heat resistance of MONAP, which is in contrast to the heat sensitivity of IL-1. In addition, IL-1 obtained as a by-product during isolation of MONAP did not stimulate human neutrophil chemotaxis.     

Selective inhibition of human neutrophil chemotaxis to N-formyl-methionyl-leucyl-phenylalanine by sulfones

The therapeutic efficacy of the sulfones, dapsone, and sulfoxone in neutrophilic dermatoses may be related to the effects of these drugs on neutrophil function. Therefore we determined whether neutrophil chemotactic migration to various chemoattractants could be inhibited by sulfones in vitro. The chemotactic responses of human neutrophils from healthy donors were tested by using N-formyl-methionyl-leucyl-phenylalanine (F-met-leu-phe), purified human C5a, and leukocyte-derived chemotactic factor (LDCF). Therapeutic concentrations of sulfones selectively inhibited neutrophil chemotaxis to F-met-leu-phe, but did not affect neutrophil chemotaxis to LDCF or C5a. Inhibition of neutrophil chemotaxis to F-met-leu-phe was induced by both dapsone and sulfoxone at a concentration of 10 micrograms/ml without affecting random migration, and the inhibition was reversed by washing the neutrophils. When dapsone- and sulfoxone-treated neutrophils (100 micrograms/ml) were stimulated with F-met-leu-phe, neutrophil superoxide generation was not inhibited. Sulfapyridine (10 micrograms/ml) also selectively inhibited neutrophil chemotaxis to F-met-leu-phe; however, sulfamethoxazole and sulfisoxazole did not affect chemotaxis. The inhibitory effects of dapsone, sulfoxone, and sulfapyridine could not be demonstrated with granulocytes from rabbits or guinea pigs nor with human monocytes. Experiments with radiolabeled dapsone showed rapid, nonspecific, and reversible binding of dapsone to human neutrophils. These data suggest that a mechanism of action of sulfones in neutrophilic dermatoses may be a selective inhibition of neutrophil migration to as yet undefined chemoattractants in the skin.