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 natural killer cytotoxicity in vitro by clinical grade serine protease inhibitors

The effects of clinical grade serine protease inhibitors on natural killer (NK) activity were compared. Cytotoxicity was measured with the Calcein-AM release method, using K562, Raji as a target. There is a significant correlation between measurements of NK activity by the Calcein-AM method and the 51Cr release assay. Cytotoxicity was inhibited with a calcium chelating agent or a perform inhibitor. Although up to 65% of cytotoxicity was inhibited by nafamostat mesilate with an E/T ratio of 10:1, and by 55% by ulinastatin, neither gabexate mesilate nor antithrombin III inhibited any cytotoxicity. None of these agents inhibited lymphokine-activated killer cell activity. In clinical applications, it should be noted that some protease inhibitors have been proven to have immunosuppressive effects.     

Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin.

Cytolytic granules purified from natural killer lymphocytes (NK) contain a pore-forming protein (perforin) and a number of serine proteases. When these proteases are inhibited by serine protease-specific isocoumarin reagents the serine proteases are inactivated and the cytolytic activity of the granules is decreased. Paradoxically, it has been found that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) frequently cannot block killing even though it inhibits many of the serine proteases. At the same time it has been reported that "purified" perforin alone can lyze cells. To address these inconsistencies we first compared the ability of PMSF and four new sulfonyl fluoride serine protease inhibitors to inhibit proteases and cell lysis. We determined the effects on lysis and the second order inhibition rate constants for five granule protease activities: ly-tryptase, ly-chymase, Met-ase (methionine cleaving), Ser-ase (serine cleaving) and Asp-ase (aspartic acid cleaving). One compound, 2-(Z-NH(CH2)2CONH)C6SO2F, was a potent inhibitor of Met-ase activity (k(obsd)/[I] = 162 M-1 s-1), ly-chymase activity (k(obsd)/[I] = 147 M-1 s-1), and granule-mediated as well as perforin-mediated lysis. PMSF was a poor inhibitor of granule proteases (k(obsd)/[I]'s less than 7 M-1 s-1 for four activities and no inhibition of Ser-ase); the lack of reactivity is consistent with the failure of PMSF to block granule lytic activity. We also prepared enriched perforin by anion exchange chromatography and showed that a ly-chymase and a Met-ase associated with perforin. By inhibiting these proteases we also inhibited lytic activity.     

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.     

Apoptosis of endothelial cells induced by the neutrophil serine proteases proteinase 3 and elastase.

The pathogenesis of vasculitis associated with anti-neutrophil cytoplasmic antibodies is not established. The anti-neutrophil cytoplasmic antibody autoanigens proteinase 3 (PR3) and elastase induce detachment and cytolysis of endothelial cells in vitro. We investigated whether PR3 and elastase trigger endothelial cell apoptosis. Primary bovine pulmonary artery endothelial cells were treated with either PR3, elastase, or myeloperoxidase (MPO) and apoptosis assessed by four different methods. By the cell death detection enzyme-linked immunosorbent assay, DNA fragmentation increased to 208 +/- 84% or 153 +/- 27% of control with 1 micrograms/ml PR3 or elastase at 24 hours. By ultraviolet light microscopy, the percentage of apoptotic cells significantly increased (P < 0.05) with 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase at 6, 12, or 24 hours. Values at the 24-hour time point are 15.3 +/- 6.4% or 25.8 +/- 6.6% for 5 or 10 micrograms/ml PR3 and 13.9 +/- 3.6% or 20.7 +/- 1.8% for 25 or 50 micrograms/ml elastase compared with 2.2 +/- 1.2% for control. Similarly, with flow cytometry, 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase for 6, 12, or 24 hours demonstrated increasing apoptosis in a dose- and time-dependent manner with the highest values achieved at 24 hours (23.4 +/- 4.0% and 35.6% for 5 and 10 micrograms/ml PR3 and 31.8 +/- 4.0% and 47.8% for 25 and 50 micrograms/ml elastase compared with 7.9 +/- 2.2% in control). Typical DNA laddering was apparent from 6 to 24 hours at 5 or 10 micrograms/ml PR3 and 25 or 50 micrograms/ml elastase. Myeloperoxidase did not induce cell apoptosis. Release of PR3 and elastase by activated neutrophils during acute inflammation, including anti-neutrophil cytoplasmic antibody-associated vasculitis, may result in vascular damage by endothelial cell apoptosis.     

Inhibition of neutrophil shape change by an inhibitor of chemotaxis

Human mononuclear cells exposed to staphylococcal peptidoglycan in serum-free culture rapidly produce an inhibitor of neutrophil chemotaxis which we have previously described. We found that this inhibitor of chemotaxis has its most potent effect on the inhibition of neutrophil shape change from a spherical to a polarized configuration. In order to quantify this shape change inhibition, we developed an assay using flow cytometric techniques. Neutrophils exposed to a chemoattractant simultaneously change their shape and decrease their forward angle light scattering intensity (delta FLS) with a correlation coefficient of 0.886 (p less than 0.001). In 51 experiments, neutrophils pretreated with the inhibitor of chemotaxis decreased their FLS by only 6.8 +/- 1.3 channels, while neutrophils pretreated with medium or control culture supernatants decreased theirs by 26.4 +/- 1.9 and 20.5 +/- 3.0 channels respectively (p less than 0.001). The factor which causes inhibition of shape change was indistinguishable from the inhibitor of chemotaxis by physical properties and chromatography. We conclude that this inhibitor of chemotaxis may act by inhibiting a physiologic step at or before shape change.     

Inhibition by histamine of platelet-activating-factor-induced neutrophil chemotaxis in bronchial asthma

Circulating human polymorphonuclear neutrophils are involved in asthma after their migration into the lung by local chemotactic factors. Investigation of the locomotion of neutrophils in Boyden chambers, showed that the chemotactic intensity of the platelet-activating factor (PAF) was similar in cells from healthy subjects and allergic asthmatics, although the optimal effect of the mediator was observed at 10(-6) M and 10(-8) M, respectively. Histamine had no direct chemoattractant effect on neutrophils but inhibited PAF-induced chemotaxis of neutrophils from healthy subjects and allergic asthmatics. This study provides additional evidence that neutrophils are involved in asthma, and points out the interaction between PAF and histamine in the migration of neutrophils to the lung.     

SPINK家族与人类疾病

丝氨酸蛋白酶抑制物Kazal型(SPINK)家族与慢性胰腺炎、Netherton综合征、食管癌等疾病关系密切。目前对SPINK1、SPINK5、SPINK7等亚族的研究比较清楚,其他一些亚族还有待深入研究。本文综述了SPINK家族的基因定位、蛋白质结构、生理功能以及与人类疾病的关系。     

Inhibitory effect of serine protease inhibitors on neutrophil-mediated endothelial cell injury

To investigate the inhibitory effect of serine protease inhibitors (SPI) on neutrophil-mediated endothelial cell (EC) injury, we analyzed the in vitro cytotoxicity of radiolabeled human umbilical vein EC (HUVEC) mediated by neutrophils in the presence of SPI. The EC injury was inhibited dose-dependently by urinary trypsin inhibitor (ulinastatin, UTI) and ONO-5046, which have the ability to inactivate neutrophil elastase, but not by gabexate mesilate, nafamostat mesilate, aprotinin, and argatroban, which have no ability to inactivate neutrophil elastase. In addition, when UTI and ONO-5046 were added to the tumor necrosis factor alpha-primed neutrophils alone, they showed a dose-dependent inhibition of the intracellular elastase activity, but the other SPI did not, for either flow cytometry or confocal microscopy. Therefore, UTI and ONO-5046 may protect EC against the neutrophil-mediated injury not only by inactivating the extracellular elastase secreted by neutrophils, but also by acting directly on neutrophils and suppressing the production and secretion of activated elastase from them.     

A genomic analysis of rat proteases and protease inhibitors

Proteases perform important roles in multiple biological and pathological processes. The availability of the rat genome sequence has facilitated the analysis of the complete protease repertoire or degradome of this model organism. The rat degradome consists of at least 626 proteases and homologs, which are distributed into 24 aspartic, 160 cysteine, 192 metallo, 221 serine, and 29 threonine proteases. This distribution is similar to that of the mouse degradome but is more complex than that of the human degradome composed of 561 proteases and homologs. This increased complexity of rat proteases mainly derives from the expansion of several families, including placental cathepsins, testases, kallikreins, and hematopoietic serine proteases, involved in reproductive or immunological functions. These protease families have also evolved differently in rat and mouse and may contribute to explain some functional differences between these closely related species. Likewise, genomic analysis of rat protease inhibitors has shown some differences with mouse protease inhibitors and the expansion of families of cysteine and serine protease inhibitors in rodents with respect to human. These comparative analyses may provide new views on the functional diversity of proteases and inhibitors and contribute to the development of innovative strategies for treating proteolysis diseases.     

Inhibition of chemotaxis of neutrophil leukocytes to interleukin-8 by endotoxins of various bacteria.

The effects of endotoxins from various bacteria (Escherichia coli, Klebsiella pneumoniae, Vibrio cholerae, Shigella flexneri, Salmonella typhosa, and Pseudomonas aeruginosa) on chemotaxis of neutrophil leukocytes to formyl peptide and interleukin-8 were tested in an improved chemotaxis assay involving a "sparse-pore" polycarbonate (Nuclepore) membrane in a Boyden-type chamber. The possible chemotactic activity of the endotoxins themselves were tested by the same technique. In addition, the effects of these substances on random motility of neutrophils were tested with a corresponding assay involving similar chambers fitted with membranes of standard pore density. Possible activation of the complement system of serum by each endotoxin was tested with sheep erythrocyte assays and the maximum endotoxin concentration (100 micrograms/ml) used in the chemotaxis and motility assays. All endotoxins inhibited chemotaxis of neutrophils to interleukin-8. No endotoxin affected chemotaxis to formyl peptide or was itself chemotactic for neutrophils. Endotoxin of S. flexneri inhibited random motility of neutrophils, while the others had no such effect. Endotoxins of K. pneumoniae and of P. aeruginosa produced moderate and marked inhibition, respectively, of total complement, as measured by hemolysis of sheep erythrocytes, without affecting the levels of C3c and C4 in these assays. Endotoxins of the other bacteria had no demonstrable effect in any of these assays of complement activation. These results suggest that chemotaxis to interleukin-8 may be mediated by cellular mechanisms different from those involved in chemotaxis to formyl peptide. Furthermore, the presence of these endotoxins could be significant for the suppression of neutrophil accumulation in inflammatory lesions mediated by interleukin-8.     

Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.     

Inhibition of neutrophil chemotaxis and activation following decentralization of the superior cervical ganglia.

Recent studies have shown that bilateral decentralization (sympathectomy) of the superior cervical ganglia (SCG) of rats sensitized to the parasite Nippostrongylus brasiliensis attenuated the development of pulmonary inflammation following allergen challenge. Sympathectomy inhibited total leukocyte infiltration into lung lavage fluids, particularly neutrophil infiltration. To define the effects of decentralization of the SCG on neutrophil responses, peripheral blood neutrophils of rats were isolated and tested in in vitro chemotaxis and phagocytosis assays. Neutrophils from rats that were sympathectomized 7 days previously displayed a marked reduction in chemotaxis to N-formyl-methionyl-leucyl-phenylalanine and leukotriene B4 compared to neutrophils from sham-operated or unoperated groups. Although the degree of chemotaxis was greater in blood neutrophils from parasite-infected rats than from uninfected rats, sympathectomy markedly reduced the chemotactic responses of both groups. In addition, neutrophils of sympathectomized rats were unresponsive to lipopolysaccharide-induced metabolic activation as assessed by in vitro phagocytosis and oxidative reduction of nitroblue tetrazolium. Thus, decentralization of the SCG of rats affects the chemotactic responses and functions of neutrophils. Understanding the role of the sympathetic nervous system in modulating the behavior of neutrophils will shed light on the interactions between the nervous and immune systems.     

Assessment of neutrophil chemotaxis by laser and video densitometry.

A laser densitometer and a video densitometer were used to evaluate neutrophil chemotaxis slides which were also assessed by the standard microscopic technique. A linear relationship was observed between cell number per high power field (HPF) and peak height by laser densitometry (r2 = 0.66) or peak area by video densitometry (r2 = 0.81). For wells containing more than 20 cells/HPF the least variability was observed with video densitometry. Quantitative results from neutrophil chemotaxis assays performed in 48-well microchambers can be obtained rapidly and conveniently by the use of commercially available video densitometers.     

Suppressive effect of IgA soluble immune complexes on neutrophil chemotaxis.

The effect of IgA and IgG soluble immune complexes (SIC) on neutrophil chemotaxis was investigated. Six types of SIC were prepared from kappa-type IgA and IgG myeloma proteins: IgA-anti-kappa chain antibody SIC in IgA excess, IgA-anti-kappa chain antibody in SIC in anti-kappa chain antibody excess, IgA-anti-alpha chain antibody SIC in IgA excess, IgG-anti-kappa chain antibody SIC in IgG excess, IgG-anti-kappa chain antibody SIC in anti-kappa chain antibody excess, and IgG-anti-gamma chain antibody SIC in IgG excess. Three types of IgA SIC had a marked suppressive effect on neutrophil chemotaxis, while IgG SIC, free IgA, free anti-kappa chain antibody, and free anti-gamma chain antibody showed no inhibitory activity. This suppressive effect on neutrophil chemotaxis caused specifically by polymerized IgA was a cell-directed one and was expressed in a concentration dependent manner.     

Polymorphonuclear neutrophil chemotaxis induced and inhibited by Bacteroides spp.

A possible virulence factor for Bacteroides subcutaneous abscesses has been found. The effect of Bacteroides culture filtrate and outer membrane on chemotaxis of rabbit peritoneal polymorphonuclear (PMN) neutrophils was assayed in Boyden chambers and via exocytosis of N-acetyl-beta-glucosaminidase. Both Bacteroides culture filtrate and outer membrane elicited some chemotaxis, as measured in the Boyden chamber; however, they had little effect upon exocytosis in cytochalasin B-treated PMN neutrophils. In the presence of serum complement, they completely abolished PMN neutrophil movement in the Boyden chamber assay, yet they gave a definite positive response for the exocytosis assay in the presence of serum complement or activated complement fragment.     

Activities of the MBL-associated serine proteases (MASPs) and their regulation by natural inhibitors

There has been rapid progress in determining the mechanism by which complement is activated by the complex formed between Mannose-Binding Lectin and its associated proteases (MASPs). MBL and the MASPs are of low abundance, but are similar to the more abundant C1q-C1r2s2 complex (C1), which has been extensively investigated. In this review we summarise recent findings on MBL-MASPs' structure. enzymic activity and regulation, and compare MBL-MASPs with C1.