In vitro effects of platelet factor 4 on normal human neutrophil functions

Platelet factor (PF4) prepared from human outdated platelets by heparinagarose affinity chromatography was confirmed to be chemotactic for human neutrophils and in a concentration-dependent fashion caused significant release of lysosomal enzymes (myeloperoxidase, lysozyme, beta-glucuronidase) from human neutrophils treated with cytochalasin B. Lysosomal enzyme release from PF4-stimulated neutrophils was rapid and reached a plateau by 1-3 min. PF4 did not cause release of the cytoplasmic enzyme lactate dehydrogenase which indicates that exocytosis of granule-containing lysosomal enzymes did not result from cytolysis. In contrast, superoxide anion generation from human neutrophils stimulated with PF4 was undetectable even at the highest PF4 concentration tested (2 X 10(-5) M). Pretreatment of neutrophils with PF4 caused significant increased adherence of neutrophils to plastic surfaces and cultured pulmonary artery endothelial cells. The concentration of PF4 that elicited neutrophil chemotaxis, lysosomal enzyme release and increased adherence is slightly higher than those concentrations found in normal human sera. However, the results suggest that PF4 may be an important mediator in neutrophil-platelet interactions and the induction of acute inflammation especially at sites of platelet microthrombi where the concentration of PF4 would be elevated.     

Inhibition of human neutrophil migration by aggregated gammaglobulin.

Heat-aggregated human gammaglobulin has been shown to inhibit the random migration of human neutrophils in serum-containing medium. This inhibition was not due to metabolic exhaustion or deactivation of the cells, since migration in the presence of aggregated gammaglobulin and casein as a chemotactic stimulus was not inhibited. The inhibition of migration was not mediated by a negative chemotactic gradient produced as a result of complement activation, and could be demonstrated in complement-depleted serum. Sera obtained from patients with rheumatoid arthritis with evidence of circulating immune complexes were able to significantly inhibit neutrophil migration, indicating that this phenomenon may be a useful means for the detection of circulating immune complexes. It is suggested that aggregated gammaglobulin or immune complexes can inhibit the chemokinetic effect of serum on neutrophils by a reversible interaction with the neutrophil surface, and that this inhibition could contribute to the accumulation of neutrophils at sites of immune complex deposition in vivo.     

Inhibition of human neutrophil secondary granule discharge by antiinflammatory agents

Human neutrophil cobalamin binding protein (NCBP) is located exclusively in the neutrophil secondary granules. The soluble stimuli formly-methionyl-leucyl-phenylalanine and the low-molecular-weight complement fragment C5a both promote the does-dependent release of NCBP from cytochalasin B-treated neutrophils in vitro. The extracellular discharge of NCBP induced by eigher secretagogue is inhibited by prior exposure of neutrophils to the corticosteroids hydrocortisone and methylprednisolone and the nonsteroidal anti-inflammatory agents indomethacin and ibuprofen. The four antiinflammatory agents function as competitive antagonists of neutrophil secondary granule discharge with a site of action at or near the cell surface. These findings support the hypothesis that antiinflammatory agents prevent neutrophil activation in vitro by inhibition of stimulus-receptor coupling. The significance of these observations with regard to the in vivo actions of these agents remains uncertain, however.     

Inhibition of human neutrophil degranulation by transforming growth factor-beta1

Neutrophils enter tissues including the uterus and are found in the endometrium in increased numbers prior to menses. In this environment, they are exposed to transforming growth factor (TGF)-beta1 produced by endometrial stromal and epithelial cells. We observed that incubation of neutrophils in vitro with TGF-beta1 at 1 pg/ml significantly reduced their secretion of lactoferrin in response to lipopolysaccharide (LPS). This effect was achieved with as little as 15 min of pretreatment with TGF-beta1. Inhibition of lactoferrin release by TGF-beta1 was observed irrespective of whether neutrophils were stimulated by ligands for Toll-like receptor (TLR)-2, TLR-4 or FPR, the G protein-coupled receptor for formylated peptides. Inhibition by TGF-beta1 was negated by SB-431542, a small molecule inhibitor that specifically blocks the kinase activity of the type I TGF-beta receptor (ALK5) In contrast to lactoferrin release, another important neutrophil function, interleukin (IL)-8 driven chemotaxis, was not affected by TGF-beta1 at 1 pg/ml or 100 pg/ml. We conclude that in tissues of the female reproductive tract, TGF-beta1 inhibition of neutrophil degranulation may prevent these cells from initiating an inflammatory response or releasing degradative enzymes that could potentially damage the oocyte or fetus.     

Inhibition of neutrophil superoxide anion generation by platelet products: role of adenine nucleotides

Previously, we demonstrated the stimulus-specific ability of platelet lysate or release products to inhibit neutrophil functions in response to f-met-leu-phe (fMLP) or phorbol myristate acetate (PMA). The present study further characterizes these activities and identifies one of the platelet inhibitors of neutrophil superoxide anion (O2-) generation. Sephadex G-200 chromatography revealed two fMLP-specific inhibitory activities in platelet lysate. One component eluted with materials less than 13 kilodaltons (kD) molecular weight, whereas the second fractionated at the void volume (Mr greater than 200 kD). PMA-specific inhibitor(s) cofractionated at the void volume with the lysate's fMLP-inhibitory activity. Release products of activated platelets contained low-molecular-weight (less than 13 kD), fMLP-specific inhibitor activity but failed to demonstrate PMA-specific inhibition of neutrophil O2- generation. Biochemical characterization identified two distinct high-molecular-weight inhibitors in platelet lysate. Pretreatment with adenosine deaminase (ADA) completely abrogated the maximum inhibitory effect (43.8 +/- 7.5%) of the high-molecular-weight, fMLP-specific lysate component but failed to reduce maximum PMA-specific inhibition (93.0 +/- 2.0%) of neutrophil O2- generation. Likewise, while the lysate's fMLP-specific inhibitor was heat stable, heating reduced the PMA-specific inhibition 56.5 +/- 10.4%. Equal sensitivity to ADA, resistance to trypsin, and heat stability were demonstrated by the low-molecular-weight inhibitor(s) in platelet lysate and release products. Neither high- nor low-molecular-weight platelet inhibitors scavenged O2-. High-performance liquid chromatography (HPLC) of the low-molecular-weight inhibitory activity from both platelet lysate and release products revealed physiological levels of adenine nucleotides. Addition of pure adenine nucleotides at physiological levels (1-10 microM) mimicked the effectiveness of the platelet inhibitor. We conclude that stimulus-specific limitation of neutrophil O2- generation by platelet adenine nucleotides may represent a mechanism for reducing inadvertent tissue damage during inflammation.     

CD14 receptor-mediated uptake of nonopsonized Mycobacterium tuberculosis by human microglia.

This study was carried out to determine the role of CD14 receptors in the uptake of nonopsonized Mycobacterium tuberculosis by human microglia. Treatment of microglial cell cultures with antibodies to CD14 or with soluble CD14 significantly blocked infection by M. tuberculosis H37Rv, suggesting that CD14 receptors could facilitate entry of nonopsonized tubercle bacilli into macrophages within the brain.     

Potentiation of neutrophil aggregation by human leukocyte inhibitory factor

The current studies were designed to extend our investigations on the ability of the lymphokine leukocyte inhibitory factor (LIF) to function as a neutrophil activator. Specifically, we investigated whether LIF could modulate neutrophil (PMN) aggregation. Aggregation was measured as the increase in light transmission using a Payton aggregometer. We found that up to 16 units of LIF was not able to directly induce PMN clumping. However, when preincubated with 0.5–16 units LIF for 10 min, PMN aggregation was significantly enhanced in a dose-dependent manner after stimulation with 10^–7 M FMLP (94.5±3.1%), 20 nM leukotriene B₄ (183.1±8.2%), and 100 g guinea pig serum-opsonized zymosan (29.8 ± 8.6%). While the LIF preparation used in these studies was highly purified, specificity for the LIF effect was demonstrated by the ability of several treatments to prevent augmentation of aggregation including: (1) the competitive binding of LIF to one of its substrates: benzoyl-arginine-ethyl-ester; (2) the blocking of PMN LIF receptors withN-acetyl-d-glucosamine; and (3) phenylmethylsulfonylfluoride treatment of the LIF preparation. As aggregation is thought to represent the in vitro correlate to adherence, these studies provide further evidence for a proinflammatory role for LIF in vivo.     

Inhibition of formylmethionyl-leucyl-phenylalanine-stimulated neutrophil chemiluminescence by human immunoglobulin A paraproteins.

Immunoglobulin A (IgA) paraproteins from patients with myeloma have been shown to inhibit human neutrophil chemotaxis to C5a, casein, and chemotactic factors produced by Escherichia coli. This study demonstrates that these paraproteins also inhibit neutrophil chemotaxis in response to the synthetic peptide formylmethionyl-leucyl-phenylalanine (f-MLP). Furthermore, the neutrophil chemiluminescence response stimulated by f-MLP was markedly suppressed by the presence of IgA paraprotein. Maximal inhibition of chemiluminescence was observed when the paraprotein was present during the chemiluminescence response. The inhibitory activity was substantially reduced by removal of the Fc region of IgA or by conversion of polymeric IgA to monomeric IgA by limited reduction and alkylation. Additional experiments showed that these IgA paraproteins inhibited C5a but not phorbol myristate acetate-stimulated chemiluminescence. These observations are constant with the hypothesis that polymeric forms of IgA bind to human neutrophils and interfere with the binding of chemotactic factor to its receptor or the consequent receptor-mediated oxidative burst or both.     

Inhibition of human neutrophil locomotion by the polyamine oxidase-polyamine system.

The polyamines, spermine and spermidine, in the presence of either bovine serum [containing polyamine oxidase (PAO) activity] or partially purified PAO, inhibited human neutrophil locomotion. This effect could not be produced by either bovine serum, PAO, or the polyamines alone. The results suggested that at least two of the products generated during the oxidation of polyamines by PAO, namely H2O2 and ammonia, are not responsible for the effects on neutrophils. Acrolein, a breakdown product of other products (aminoaldehydes), was found to inhibit the neutrophil functions. Since polyamines, and possibly PAOs, accumulate at inflammatory sites, products of the PAO-polyamine reaction could function as regulators of the inflammatory response.     

Inhibition of human neutrophil chemotaxis and chemiluminescence by amphotericin B.

The effect of the antifungal drugs amphotericin B, fluocytosine, miconazole, griseofulvin, and nystatin on the chemotactic responsiveness of human neutrophils was studied. Amphotericin B in a concentration of 2 mug/ml inhibited chemotatic responsiveness, and in a concentration of 5 mug/ml it also inhibited chemiluminescence. The inhibition of chemotaxis could be reversed by washing the cells. The other antifungal drugs did not inhibit chemotaxis even in concentrations much higher than those obtained in human serum during treatment.     

Inhibition of neutrophil response by curcumin

Blood neutrophils, when exposed to appropriate stimuli, aggregate, degranulate and generate superoxide anion. Curcumin, a non-steroidal antiinflammatory agent, modulated these functions, depending upon the kind of stimulus used. It inhibited monkey neutrophil aggregation induced by chemotactic peptide fmlp and zymosan activated plasma (ZAP) but did not affect that induced by serum treated zymosan (STZ) and arachidonic acid (AA). Generation of O ₂ ^– radical was inhibited by curcumin, when cells were stimulated by AA, STZ and fmlp. Curcumin inhibited the release of myeloperoxidase, an azurophilic granule marker enzyme. Release of lysozyme was less susceptible to inhibition by curcumin. The results suggest that curcumin interferes with neutrophil responses to various physiological stimuli and a part of its antiinflammatory action is mediated via inhibition of neutrophil function. Inhibition of neutrophil function by curcumin appears to be mediated via calcium dependent mechanisms.     

Inhibition of neutrophil activation by fibrinogen

Physiological levels of human fibrinogen markedly inhibited the chemotactic activity of human neutrophils triggered by zymosan-activated serum (ZAS), C5a, or IL-8 in a Boyden chamber assay. Fibrinogen also slightly inhibited theN-formyl-methionyl leucyl-phenylalanine (FMLP) -induced migration of human neutrophils. Albumin was devoid of the inhibitory activities displayed by fibrinogen in this system. The inhibition of chemotaxis by fibrinogen was dose-dependent and saturable. Fibrinogen placed in the upper compartment of the Boyden chamber produced a larger inhibition than that obtained with fibrinogen placed in the lower compartment. Lysine as well as the lysine analog 6-aminohexanoic acid (AHA) decreased the inhibitory capacity of fibrinogen. In contrast, both arginine and glutamine failed to suppress the fibrinogen-mediated inhibition of neutrophil chemotaxis. AHA counteracts the inhibition of ZAS-induced chemotaxis by anti-CD18 monoclonal antibody, suggesting that lysine binding sites are required for integrin function in chemotaxis. Fibrinogen also inhibited, in a dose-dependent manner, the oxygen consumption of neutrophils activated by opsonized zymosan. Taken together, the present results indicate that fibrinogen modulates neutrophil functions and suggest that in addition to its role in blood coagulation, circulating fibrinogen may be involved in regulation of the inflammatory response.     

半乳糖受体介导的c-myc反义核酸对人肝癌细胞

目的 :探讨半乳糖 (Gal) -聚乙烯亚胺 (PEI) -c -myc反义寡核苷酸 (ASODN)交联复合物对人肝癌细胞增殖的影响。方法 :Gal-PEI-ASODN复合物作用于人肝癌Bel- 74 0 2细胞 ,台盼蓝染色法检测不同时间、不同浓度作用下细胞增殖的变化 ,倒置显微镜观察细胞形态 ,流式细胞仪分析细胞亚二倍体的百分率 ,透射电镜观察细胞超微结构的改变。结果 :在 0 - 96h的不同时间点 ,与ASODN组 (含ASODN 2 0 μmol/L)相比 ,Gal-PEI-ASODN复合物 (含ASODN 0 75 μmol/L)明显抑制Bel- 74 0 2细胞的增殖 ,Gal-PEI -ASODN复合物的起效浓度更低 ,起效时间更短 ;不同浓度的单纯ASODN与Bel- 74 0 2细胞作用 72h ,测得ASODN的IC50 为 2 0 9μmol/L ,而在半乳糖受体的介导下 ,ASODN与Bel- 74 0 2细胞作用 4 8h ,测得ASODN的IC50 为 0 2 94 μmol/L ,ASODN的抑制效果提高 70 9倍 ;Gal-PEI -ASODN复合物与Bel- 74 0 2细胞作用 4 8h ,与单纯ASODN组相比 ,细胞亚二倍体百分率更高 ,并且电镜下可见明显的细胞凋亡形态。结论 :半乳糖受体介导的投递系统可明显提高ASODN对Bel- 74 0 2细胞的增殖抑制效应     

Inhibition of tumour necrosis factor alpha (TNF-alpha)-induced neutrophil respiratory burst by a TNF inhibitor.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in microbial defence and tissue damage by activating neutrophils. Therefore the ability of natural molecules to regulate the activity of TNF-alpha is likely to be of major importance in our understanding of the mechanisms of inflammation. We have examined the effects of a highly purified urine-derived TNF inhibitor (TNF inh) on the TNF-alpha-induced respiratory burst in human neutrophils. TNF-alpha inh-treated TNF-alpha was unable to stimulate a neutrophil lucigenin-dependent chemiluminescence response and superoxide formation. Treatment of TNF with the inhibitor also significantly reduced the priming ability of TNF-alpha for a response to the peptide f-met-leu-phe. These results show that the ability of TNF-alpha to induce a key neutrophil response is amenable to regulation by the TNF-alpha inh.     

Inhibition of neutrophil superoxide production by fanetizole

The effects on neutrophil function of the new immunomodulatory agent fanetizole mesylate were studied. Fanetizole did not affect random or stimulated migration, phagocytosis, or degranulation by normal human neutrophils. Production of superoxide in response to the chemotactic factor formyl-methionyl-leucyl-phenylalanine (f-Met-Leu-Phe) was markedly inhibited (41.3±3.9%) by 250M fanetizole. This inhibition was not due to scavenging of superoxide by fanetizole, as there was no impairment of superoxide detection in a cell-free xanthine-xanthine oxidase system. Inhibition was dose dependent (no effect seen with 1 or 10M fanetizole) and stimulus specific (no impairment of superoxide production in response to phorbol myristate acetate). Washing the cells after fanetizole treatment partially restored their superoxide response to f-Met-Leu-Phe. Suppression of neutrophil production of toxic oxygen metabolites may partially explain the antiarthritic effect of fanetizole, and study of such selective inhibitors may be useful in probing the contribution of neutrophils to inflammatory tissue damage.Part of this work has been published in abstract form (Clinical Research 32:469A, 1984). Supported in part by grant AI16732 from the National Institutes of Health and a Charlton Fund grant from Tufts University School of Medicine.     

Inhibition of human peripheral blood neutrophil respiratory burst by alcohol-based venipuncture site disinfection

Ethanol inhibits the respiratory burst of neutrophils. Therefore, the effects of alcohol-based skin disinfection on oxygen metabolism in neutrophils were tested using 70% ethanol or an ethanol-isopropanol-n-propanol mixture. Neutrophil respiratory burst activity as assessed fluorometrically by oxidation of 2', 7'-dichlorofluorescein diacetate increased at 10 min after disinfection with 70% ethanol compared to the activity at 30 s. The increase was significant for triggering oxidative burst with formyl-peptide but not with phorbol myristate acetate.     

Inhibition of neutrophil oxidative metabolism by nimesulide

Oxygen derived free radical release from activated neutrophils may be in part responsible of tissue damage in the acute phase of inflammation. We have shown that the methane sulfonanilide antiinflammatory agent nimesulide inhibits the respiratory burst of phagocytosing neutrophils without affecting their phagocytic or chemotactic responsiveness. In fact, chemiluminescence and superoxide anion generation by polymorphonuclear leukocytes (PMN) stimulated with zymosan particles or with the synthetic peptide FMLP are inhibited by nimesulide and its 4-OH metabolite in a dose dependent fashion without affecting cell viability. The control of the extracellular flux of radical species by phamacological compounds may affect the course of inflammation reducing tissue damage. Our data suggest that the inhibition of superoxide anion production by neutrophils is an additional mechanism of action of the antiinflammatory agent nimesulide.     

Inhibition of human neutrophil chemotaxis in vitro by phenothiazines and related compounds.

Chlorpromazine (CPZ) and three other phenothiazines and the structurally related antidepressant drugs imipramine and amitriptyline were found to depress human neutrophil chemotactic responsiveness. A 7 X 10(-6)M solution of CPZ inhibited chemotaxis, whereas concentrations of the other tested drugs 10 to 1,000 times greater than this were needed to inhibit chemotaxis. This effect of CPZ could not, however, be demonstrated when testing neutrophils from patients treated with the drug. The inhibition of chemotaxis was reversible when CPZ-incubated neutrophils were washed before testing for chemotactic responsiveness. CPZ affects neutrophil funcjtion as well as other aspects of immune response.     

Prolonged hypoxia modulates platelet activating factor receptor-mediated responses by fetal ovine pulmonary vascular smooth muscle cells

Hypoxia augments PAF receptor (PAFr) binding and PAFr protein expression in venous SMC (SMC-PV). We compared effect of acute and prolonged hypoxia (pO₂ < 40 torr) on PAFr-mediated responses in arterial SMC (SMC-PA) and SMC-PV. Cells were studied for 30 min (acute) or for 48 h (prolonged) hypoxia and compared to normoxic (pO₂ ~ 100 torr) conditions. PAF binding was quantified in fmol/10⁶ cells (mean ± SEM). PAF binding in normoxia were SMC-PA, 5.2 ± 0.2 and in SMC-PV, 19.3 ± 1.1; values in acute hypoxia were SMC-PA, 7.7 ± 0.4 and in SMC-PV, 27.8 ± 1.7. Prolonged hypoxia produced 6-fold increase in binding in SMC-PA, but only 2-fold increase in SMC-PV, but binding in SMC-PV was still higher. Acute hypoxia augmented inositol phosphate release by 50% and 40% in SMC-PA and SMC-PV, respectively. During normoxia, PAFr mRNA expression by both cell types was similar, but expression in hypoxia by SMC-PA was greater. In SMC-PA, hypoxia and PAF augmented intracellular calcium flux. Re-exposure of cells to 30 min normoxia after 48 h hypoxia decreased binding by 45–60%, suggesting immediate down-regulation of hypoxia-induced PAFr-mediated effects. We speculate that re-oxygenation immediately reverses hypoxia effect probably due to oxygen tension-dependent reversibility of PAFr activation and suggest that exposure of the neonate to prolonged state of hypoxia will vilify oxygen exchange capacity of the neonatal lungs.