Quartz-dust-induced production of reactive oxygen metabolites by human granulocytes

We studied the effect of quartz on the production of reactive oxygen species by human polymorphonuclear leukocytes (PMN) in vitro by a chemiluminescence (CL) assay. Quartz caused a rapid dose-dependent CL response in the cells. Diamond dust used as an inert control did not stimulate the production of reactive oxygen metabolites by PMN. The quartz-induced activation of oxygen metabolism was also demonstrated by measuring oxygen consumption, nitroblue tetrazolium reduction, and superoxide and hydrogen peroxide production by PMN. Poly-vinyl-pyridine N-oxide (a quartz surface modifying agent) completely abolished the quartz-induced response, but had no effect on opsonized zymosan-induced CL response of PMN. The effect of N-acetylcysteine (a known antioxidant) was inhibitory to the CL formation induced by both quartz and opsonized zymosan. Our results are in agreement with the hypothesis that quartz-induced production of reactive oxygen metabolites is a possible mechanism by which quartz dust produces chronic inflammation and tissue injury of the lung. Agents interfering with the generation of reactive oxygen metabolites may provide a rationale for treatment of mineral-dust-induced pulmonary disease.     

Production of oxygen free radicals by phagocytes in patients on hemodialysis

In order to elucidate the vulnerability to infection in patients on chronic hemodialysis, as one of the host defense mechanisms, the production of oxygen free radicals by phagocytes was studied in patients by luminol- or lucigenine-enhanced chemiluminescence (CL). Whole blood CL of the patients, in both luminol- or lucigenine-enhanced was significantly higher than that in healthy adults after stimulation by zymosan, PMA, and Staphylococcus aureus (S. aureus) 209 P. However, the CL response of the patients' polymorphonuclear neutrophils (PMNs) with the same stimuli was slightly lower than that in healthy adults. There were no differences in the levels of opsonins, such as complements and immunoglobulins, between the patients and healthy adults. It appears that any factor in the patients' serum enhances CL response, because of the PMN CL response after addition of patients' serum was higher than that after addition of healthy controls' serum, and the PMN CL response after the addition of patients' serum obtained after hemodialysis was higher than that before hemodialysis. The addition of erythrocytes to PMNs from healthy adults caused a reduction in the PMN CL response, but the addition of urea and creatinine had no effect. The CL response induced by microsphere-bound luminol (lumisphere), which makes possible the direct measurement of highly reactive oxygen within phagosomes, was studied in the patients and controls. The CL response in the patients was slightly lower than those in controls, but not significant. These results suggest that not only CL response of phagocytes but also other defense mechanisms should be studied further to make clear the vulnerability to infection in these patients. In addition, the effect of three antibiotics, cefbuperazone, cefminox and latamoxef on luminol-enhanced CL of whole blood was studied in healthy adults and the patients. After 3-hour exposure to those drugs at subinhibitory concentration (1/4 MIC), Klebsiella pneumoniae (K. pneumoniae) 163 treated by drugs induced higher CL response of whole blood than that by untreated bacteria in both healthy adults and the patients, and the peak time of the CL response induced by the drug-treated bacteria was shorter than that by untreated bacteria. This study suggests that the three drugs at sub-MIC work in partnership with host defense against infection due to K. pneumoniae even in patients on chronic hemodialysis.     

Increased apoptotic neutrophils and macrophages and impaired macrophage phagocytic clearance of apoptotic neutrophils in systemic lupus erythematosus

OBJECTIVE: To evaluate whether patients with systemic lupus erythematosus (SLE) have a higher rate of apoptosis in and secondary necrosis of polymorphonuclear neutrophils (PMNs) and macrophages compared with controls; to compare the rate of macrophage phagocytic clearance of apoptotic PMNs in patients with SLE and healthy controls; to evaluate whether in vitro PMN and macrophage apoptosis and secondary necrosis, and the ability of macrophages to phagocytose apoptotic bodies, are correlated with lupus disease activity; and to determine whether macrophage clearance of apoptotic bodies in patients with SLE and normal controls is related to certain serum factors. METHODS: Thirty-six patients with SLE and 18 healthy, nonsmoking volunteers were studied. PMNs and monocytes were isolated from fresh blood and cultured in the presence of different sources of serum. Apoptotic PMNs and macrophages were examined by annexin V binding and morphology on May-Giemsa-stained cytopreparations, at different time points. The presence of secondary necrotic PMNs and macrophages was verified by staining with trypan blue. Macrophage phagocytosis of apoptotic PMNs was measured using a coded, observer-blinded, microscopically quantified phagocytosis assay. Cells were cultured in the presence of serum obtained from healthy subjects or from patients with SLE. RESULTS: At 5 and 24 hours, the percentage of apoptotic PMNs from patients with SLE was significantly higher than that of PMNs from healthy subjects. At 24 and 48 hours, the percentage of secondary necrotic PMNs from patients with SLE was also significantly higher than the percentage of necrotic PMNs from controls. Serum from patients with SLE accelerated the rate of apoptosis in and secondary necrosis of PMNs from healthy subjects. Macrophages from SLE patients were less capable of phagocytosing apoptotic PMNs compared with macrophages obtained from controls. Macrophages from patients with active SLE were less capable of phagocytosing apoptotic PMNs than were macrophages from patients with inactive SLE, but the difference was not statistically significant. The percentage of phagocytosis of apoptotic PMNs by macrophages from SLE patients correlated negatively with the SLE Disease Activity Index, serum levels of anti-double-stranded DNA, and the erythrocyte sedimentation rate, and correlated positively with serum levels of C3, C4, and albumin, the hemoglobin level, and the leukocyte count. Serum from SLE patients not only significantly increased macrophage apoptosis in cells from healthy subjects but also remarkably down-regulated the clearance of apoptotic PMNs by macrophages from healthy subjects. In contrast, serum from healthy subjects significantly increased phagocytosis of apoptotic PMNs by macrophages from SLE patients. CONCLUSION: The observed increase of apoptotic PMNs and macrophages and the poor ability of macrophages from patients with SLE to phagocytose apoptotic bodies may indicate an impaired clearance mechanism, which may be mediated by factors in a patient's serum.     

Defective natural killer and phagocytic activities in chronic obstructive pulmonary disease are restored by glycophosphopeptical (inmunoferón)

We have investigated both modifications in natural (innate) immunity caused by chronic obstructive pulmonary disease (COPD) and the effects of a glycophosphopeptical immunomodulator (Inmunoferón) treatment on COPD-associated immunoalterations. In a double-blinded clinical trial, 60 patients with COPD received glycophosphopeptical or placebo during 90 consecutive days at oral doses of 3 g/d. Fifty-six sex- and age-matched healthy control subjects were included as a reference group for immunologic parameters. Peripheral blood natural killer (PBNK) cell cytotoxic activity and phagocytic activity of peripheral monocytes/macrophages (Mo/Ma) and polymorphonuclear (PMN) cells were assessed at baseline and then again at the end of treatments. We found both PBNK activity and phagocytic activity to be significantly decreased in patients with COPD compared with levels in healthy volunteers. The treatment with glycophosphopeptical provoked significant stimulatory effects on PBNK cytotoxic activity. This stimulation was not mediated by an increase in CD3(-)CD56(+) NK cells. Further, glycophosphopeptical significantly increased the percentage of monocytes and PMNs that phagocytize Escherichia coli in vitro, as well as increased phagocytic indices. We conclude that peripheral blood cells of patients with COPD show clear defects in natural immunity that are partially rescued by glycophosphopeptical.     

Blood polymorphonuclear behavior in patients with ankylosing spondylitis

The oxidative metabolism and chemotaxis of polymorphonuclear leukocytes (PMNs) collected from patients with ankylosing spondylitis and healthy subjects were studied in parallel. The responses to opsonized zymosan were significantly lowered considering oxygen consumption and release of superoxide anions, whereas no modification of these parameters to phorbol myristate acetate and calcium ionophore (A 23187) stimulations were observed. A seric factor was not involved but the characterization of a specific intrinsic abnormality of the PMNs needs further investigations. PMN chemotaxis, assessed by two methods performed in parallel, remained unchanged.     

Phenotype of blood monocytes and alveolar macrophages in interstitial lung disease

The immunologic phenotype of the monocyte-macrophage cell populations in bronchoalveolar lavage (BAL) fluid and monocytes in peripheral blood (PB) were studied in 20 patients with sarcoidosis, 18 with idiopathic pulmonary fibrosis (IPF), seven with extrinsic allergic alveolitis (EAA), and 12 healthy volunteers. There were no significant differences in expression of the immunologic markers CD13(My7), CD14(My4), and Monocyte-2 on blood monocytes between the patient groups and healthy volunteers, but there were marked differences between groups in the expression of the three markers on BAL macrophages. The percentage of Monocyte-2+ macrophages was increased in BAL in subjects with sarcoidosis, EAA, and IPF compared with healthy volunteers, greatest in EAA. This increase is probably due to increased recruitment of blood monocytes into alveoli, since the cells had a monocytic morphology on phase contrast microscopy (in normal subjects the majority of blood monocytes, but few alveolar macrophages, express the Monocyte-2 antigen). Patients with IPF had a significantly lower percentage of CD13(My7)+ macrophages in BAL than the other three groups. Compared with IPF patients and healthy volunteers, patients with EAA had a significantly higher percentage of CD14(My4)+ macrophages, whereas in sarcoidosis patients the numbers were reduced. These observations suggest an increased influx of blood monocytes into the alveoli in interstitial lung disorders. Phenotypic differences were found between the BAL macrophage populations of the various interstitial diseases. These differences in alveolar macrophage phenotype may be due to local factors, depending on the type of inflammation.     

Digestive vacuoles of Plasmodium falciparum are selectively phagocytosed by and impair killing function of polymorphonuclear leukocytes

Sequestration of parasitized erythrocytes and dysregulation of the coagulation and complement system are hallmarks of severe Plasmodium falciparum malaria. A link between these events emerged through the discovery that the parasite digestive vacuole (DV), which is released together with infective merozoites into the bloodstream, dually activates the intrinsic clotting and alternative complement pathway. Complement attack occurs exclusively on the membrane of the DVs, and the question followed whether DVs might be marked for uptake by polymorphonuclear granulocytes (PMNs). We report that DVs are indeed rapidly phagocytosed by PMNs after schizont rupture in active human serum. Uptake of malaria pigment requires an intact DV membrane and does not occur when the pigment is extracted from the organelle. Merozoites are not opsonized and escape phagocytosis in nonimmune serum. Antimalarial Abs mediate some uptake of the parasites, but to an extent that is not sufficient to markedly reduce reinvasion rates. Phagocytosis of DVs induces a vigorous respiratory burst that drives the cells into a state of functional exhaustion, blunting the production of reactive oxygen species (ROS) and microbicidal activity upon challenge with bacterial pathogens. Systemic overloading of PMNs with DVs may contribute to the enhanced susceptibility of patients with severe malaria toward invasive bacterial infections.     

Increased Spontaneous Production and Generation of Superoxide Anion by Blood Neutrophils in Patients with Asthma

Spontaneous production and PMA- and opsonized zymosan- stimulated generation of superoxide anion by blood cells in asthmatic patients were compared with those in normal volunteers and chronic obstructive pulmonary disease (COPD) patients using a lucigenin-dependent chemiluminescence method. Superoxide anion generation by 100 μl of blood in patients with asthma and/or COPD was significantly greater than that in normal subjects [asthma: 5684 ± 253 chemiluminescence (CI); COPD: 4994 ± 240 CL; normal: 2543 ±213 CL]. This is consistent with the increased superoxide generation per leukocyte (PMN) in these patients (Asthma: 1.56 ± 0.08 CL/PMN; COPD: 1.31 ± 0.08 CL/PMN; normal: 0.83 ± 0.07 CL/PMN. However, spontaneous production of superoxide by individual PMNs was increased only in asthmatic patients, compared with that in normal subjects (Asthma: 0.14 ± 0.02 CL/PMN; COPD: 0.07 ± 0.01 CL/PMN; normal: 0.07 ± 0.01 CL/PMN. These results indicate that the respiratory burst is enhanced in both asthmatic patients and COPD patients, and that superoxide production by resting neutrophils is also increased in asthmatic patients, but not in COPD patients, compared with normal subjects.     

Iprindole-induced phospholipidosis in rat alveolar macrophages: alterations in oxygen consumption and release of oxidants

Rats chronically treated with the cationic amphipilic drug iprindole developed a phospholipid storage disorder in their pulmonary alveolar macrophages (AMs). AMs from these iprindole-treated rats (IP-AMs) were compared to AMs from control rats (C-AMs) regarding oxygen consumption and the release of two reactive oxygen species, superoxide anion and hydrogen peroxide. Responses of C-AMs and IP-AMs were compared at rest and when stimulated by unopsonized or opsonized zymosan. Opsonization was not necessary in order to induce respiratory burst-associated phenomena in either cell type; in fact in all cases, for given cell type responses to unopsonized zymosan were virtually identical to those of opsonized zymosan. When at rest, IP-AMs consumed oxygen at a rate nearly identical to that of C-AMs. When stimulated with zymosan particles, IP-AMs consumed more oxygen than controls. However, when superoxide anion and hydrogen peroxide, two products of the respiratory burst were measured, IP-AMs released less of these species than C-AMs when at rest and when particle stimulated. Despite the lower total release of these species by the IP-AMs, the zymosan-induced release (stimulated minus resting levels) was greater for these cells than C-AMs. Therefore, the IP-AMs were found to be more responsive to the zymosan particle than C-AMs. The results indicate marked changes in the release of reactive oxygen species from the AMs following induction of phospholipidosis.     

Polytrauma induces increased expression of pyruvate kinase in neutrophils

Polytrauma (PT) leads to systemic activation of polymorphonuclear neutrophils (PMNs). Organ damage commonly found in these patients is ascribed to respiratory bursts of activated PMNs. With the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, PMN extracts from PT patients were found to contain a clear protein band not seen in control PMNs from healthy volunteers. This band was identified by amino acid sequencing and Western blotting as pyruvate kinase (PK). Enzymatic assays revealed a 600-fold increase in PK activity in PMNs of PT patients, with the highest levels occurring between the fifth and seventh posttraumatic day. In lymphocytes, no such increase was detectable. As PK is a major regulatory enzyme in glycolysis, glucose-dependent lactate production in PMNs from PT patients was assayed. These cells showed a higher glycolytic lactate production than controls. It was additionally demonstrated that acute activation of respiratory burst activity depends mainly on breakdown of glucose to lactate via the pentose-phosphate pathway and glycolysis. In PMNs from PT patients, this glucose-dependent respiratory burst activity was more than twofold higher than in controls. The increase in expression and activity of PK in PMNs from PT patients may contribute to the high glucose-dependent respiratory burst activity seen in these cells.     

Expression of Fcgamma and complement receptors on peripheral blood monocytes in systemic lupus erythematosus and rheumatoid arthritis

OBJECTIVES: Fcagamma and complement receptors play an important role in the interaction between immune complexes (IC) and monocytes/macrophages. Recent work has demonstrated that their relative expression on these cells may be modified by cytokines, including TNF-alpha and IL-4. Furthermore, cytokines may alter the expression of adhesion molecules such as ICAM-1. However, little data exist on the in vivo expression of specific Fcgamma and complement receptors in systemic lupus erythematosus (SLE) or rheumatoid arthritis (RA), two diseases in which IC are important in pathogenesis. METHODS: Venous blood was obtained from 30 patients with SLE, 25 with RA and 25 healthy controls. Monocyte phenotype was determined by flow cytometric analysis of whole blood samples, with selective gating using forward and side scatter signals. Surface expression of Fcgamma receptors RI (CD64), RII (CD32) and RIII (CD16), complement receptors CR1 (CD35) and CR3 (CD11b/CD18), and adhesion molecules ICAM-1 (CD54) and CD11a (LFA-1) was determined. The effects of disease activity and corticosteroid therapy on the expression of these molecules were also examined. RESULTS: The expression of FcgammaRII was reduced on monocytes from patients with SLE compared with healthy controls and patients with RA (P = 0.002). This did not correlate with disease activity using conventional indices [SLEDAI (SLE disease activity index), C3/C4 levels and anti-double-stranded DNA antibody titres], and was independent of prednisolone therapy. There was no significant difference in FcgammaRI or RIII expression on SLE monocytes compared with healthy controls. In contrast, the expression of FcgammaRIII was increased on RA monocytes (P = 0.01), this being highest in patients with active disease. The proportion of FcgammaRIII-positive monocytes was also increased in RA, and prednisolone therapy was associated with a lower proportion of FcgammaRIII-positive cells. An increase in CR3 expression was seen on RA monocytes (P = 0.002), whilst CR1 was increased on monocytes from patients with active SLE or active RA. ICAM-1 expression was reduced on monocytes from patients with SLE (P = 0.002), although high-dose prednisolone therapy was associated with the lowest level of surface ICAM-1 on monocytes. CONCLUSIONS: Peripheral blood monocytes from patients with SLE or RA display significantly altered phenotypes compared with those from healthy controls. The observed reduction in SLE of FcgammaRII may represent a mechanism by which monocytes are protected from IC-mediated activation. Prednisolone therapy and disease activity had little effect on phagocytic receptor expression. The observed changes may reflect the different cytokine profiles seen in SLE and RA.     

Monocyte dependent excited oxygen radical generation in rheumatoid arthritis: inhibition by gold sodium thiomalate

A chemiluminescent assay was used to measure generation of excited oxygen species by peripheral blood monocytes obtained from normal controls (NC) and patients with rheumatoid arthritis (RA). Whole peripheral blood mononuclear cells (PBMC) and adherent cells (AdhC) from RA patients showed a significantly higher chemiluminescent response to opsonized zymosan at certain observation times than cells from NC. There was no significant difference however in response to stimulation by calcium ionophore (CI). Gold sodium thiomalate (GSTM) significantly inhibited the peak response of fresh PBMC and AdhC (from both RA and NC) to zymosan and CI. GSTM inhibited only the peak response of fresh cells to zymosan; by contrast cells cultured with GSTM for 72 h showed inhibition at all time periods. The effect of GSTM was dependent on dosage and duration of incubation with cells. The addition of GSTM after zymosan stimulation did not inhibit chemiluminescence. GSTM appears to act upon cellular events following membrane activation which culminates in the generation of excited oxidative species. These results suggest a mechanism of action for GSTM in RA by inhibition of monocyte and macrophage dependent generation of oxy radicals and related excited oxygen species.     

Preactivated monocytes from hypertensive patients as a factor for atherosclerosis?

Recently, we reported our findings regarding the elevated secretion patterns of proinflammatory cytokines obtained from peripheral blood monocytes of hypertensive patients. To investigate the direct impact of these preactivated monocytes, the adhesion of monocytes from normal controls and hypertensive patients to vascular endothelial cell monolayers was determined spontaneously and after in vitro stimulation with either lipopolysaccharide (LPS) or angiotensin II (Ang II), with or without preincubation with the AT1 receptor antagonist eprosartan. Peripheral blood monocytes from 20 patients and 20 healthy individuals were isolated by density gradient centrifugation and plastic adherence; endothelial cells were obtained from human umbilical cords by collagenase digestion. The adhesion was determined by an assay with 51Cr-radiolabeled monocytes. Oxygen species release induced by phorbol myristate acetate (PMA) as a further activation marker was analyzed for monocytes and HUVEC by chemiluminescence (CL). Spontaneous adhesion of monocytes from patients and the adhesion after stimulation with Ang II were significantly increased compared with normal controls (P<0.05). Preincubation with eprosartan diminished the adhesion in both groups to comparable levels. In monocytes, peak levels of PMA and Ang II induced CL analysis were significantly higher in patients (P<0.005). These data indicate that preactivated monocytes from hypertensives may be of pathogenic importance in atherosclerosis.     

Effects of calcium-regulating hormones and drugs on human monocyte chemiluminescence

Chemiluminescence (CL) associated with phagocytosing monocytes has been used as an index of the oxygen dependent metabolic activity of these cells. Because of the relationships between monocytes and cells involved in bone resorption, we studied the effects on human monocyte CL of hormones and drugs active in mineral metabolism. Two-hour preincubations of monocytes with human PTH-(1-34), bovine PTH, or prostaglandin E2 caused significant decreases in peak CL during phagocytosis stimulated by the addition of latex particles. Similar studies with dichloromethylene diphosphonate, ethane hydroxydiphosphonate, or salmon calcitonin (sCT) caused significant increases in CL, whereas preincubation with human CT at the same molar concentration did not. CL was also decreased after preincubations with methylprednisolone or bacterial endotoxin. The effect of bovine PTH was dose dependent to concentrations as low as 10 ng/ml, was not fully present after a shorter 1-min preincubation with the hormone, and differed in an otherwise identical system using polymorphonuclear leukocytes instead of monocytes. The production of hydrogen peroxide by phagocytosing monocytes was also significantly affected by each of the drugs and hormones. The direction and magnitude of these changes were similar to those in CL experiments, except for the effects of sCT. These studies relate the oxygen-dependent function of phagocytes to mediators of bone resorption and provide a new system for studying the effects of hormones and drugs on the cellular elements of bone and blood.     

乙型肝炎e抗原对外周血单核细胞Toll样受体2表达的影响

目的 探讨HBeAg对外周血单核细胞表面Toll样受体2(TLR2)表达的影响.方法 对68例慢性乙型肝炎(CHB)患者(其中HBeAg与HBV DNA刚性37例,HBeAg阴性、HBVDNA阳性14例,HBeAg与HBV DNA阴性17例)和16名健康人的外周血肝素抗凝,加入Pam3CSK4在37℃、5%CO2条件下刺激3 h,用特异性TLR2单克隆抗体标记,经流式细胞仪检测CD14+细胞表面TLR2表达的百分率;比较刺激前后各组之间的差异.定量检测HBV DNA和血清HBV标志物,分析TLR2表达水平与HBeAg、HBV DNA的关系.用HBeAg与健康人及HBeAg阴性CHB患者的抗凝血在37℃、5%CO2条件下共培养6 h,用流式细胞仪定量分析HBeAg刺激后CD14+细胞表面TLR2的表达水平. 结果 HBeAg阳性CHB患者外周血CD14+细胞TLR2的表达率为47.57%±21.40%,明显低于健康对照组(76.51%±7.46%)和HBeAg阴性组(HBV DNA阳性组为73.2%±14.2%、HBV DNA阴性组为75.2%±11.3%,P<0.05);TLR2的表达水平在HBeAg阴性的HBV DNA阳性或阴性CHB患者组与健康对照组的差异无统计学意义.HBeAg阳性CHB患者外周血单核细胞经Pam3CSK4刺激后,TLR2的表达率明显升高,大部分患者TLR2的表达水平能够达到健康人或HBeAg阴性患者未经配体刺激的水平.健康人或HBeAg阴性CHB患者的外周血与HBeAg共孵育6 h后,CD14+细胞表面TLR2表达水平较HBeAg刺激前明显下降(P<0.05).结论 HBeAg阳性的CHB患者外周血CD14+细胞TLR2的表达水平明显低于HBeAg阴性患者和健康人,HBeAg能够抑制CD14+细胞TLR2的表达,提示HBeAg能够引起CHB患者外周血单核细胞表面TLR2表达的下调,这可能是HBV持续感染的重要因素之一;CHB患者HBVDNA水平可能不影响单核细胞表面TLR2的表达;Pam3CSK4可以明显提高HBeAg阳性CHB患者单核细胞表面TLR2的表达水平,TLR2配体有可能成为CHB免疫治疗的一个潜在靶位.     

Increased adhesion molecules expression and production of reactive oxygen species in leukocytes of sleep apnea patients

Obstructive sleep apnea (OSA) is associated with increased cardiovascular morbidity and mortality. Free radicals and adhesion molecules were implicated in the pathogenesis of atherosclerosis leading to cardiovascular disorders. Therefore, we investigated the link between CD15, CD11c, CD11b, and CD64 expression on leukocytes and their ability to generate reactive oxygen species (ROS) in patients with OSA and control volunteers. We also studied the effects of hypoxia in vitro on monocytes from control subjects and the ability of monocytes from both groups to adhere to human endothelial cells in culture. The effect of nasal continuous positive airway pressure (nCPAP) treatment was studied as well. We found that OSA was associated with increased expression of adhesion molecules CD15 and CD11c on monocytes, increased adherence of monocytes in culture to human endothelial cells, increased intracellular ROS production in some monocyte and granulocyte subpopulations, and upregulation of CD15 expression due to hypoxia in vitro in monocytes of control subjects. Furthermore, nCPAP treatment was associated with downregulation of CD15 and CD11c monocyte expression and decreased basal ROS production in CD11c+ monocytes. Monocyte adherence to endothelial cells decreased as well. Our findings provide one of the possible mechanisms for explaining the high rate of cardiovascular morbidity in patients with sleep apnea.     

狼疮肾炎患者循环单核细胞表达Fas和血浆可溶性Fas Ligand水平的研究

目的 探讨狼疮肾炎（LN）病人单核细胞凋亡加速的机制。方法 对17例LN病人进行观察,以15名正常人为对照。采用流式细胞仪检测单核细胞Fas和FasLigand(FasL)的表达;酶联免疫吸附法（ELISA）测定血浆中可溶性FasLigand(sFasL）的水平;单核细胞与重组人FasL共孵育,磺化丙啶染色、流式细胞仪检测单核细胞凋亡,四甲基偶氮唑蓝（MTT）法观察单核细胞体外存活率。结果 LN病人单核细胞Fas的表达较正常人明显上调（P＜0．05）;处于狼疮活动期的LN病人与狼疮静止期的LN病人之间单核细胞Fas的表达差异无显著性（P＞0．05）;处于狼疮活动期的LN病人与狼疮静止期的LN病人之间单核细胞Fas的表达差异无显著性（P＞0．05）。正常人和LN病人的单核细胞表面均未检测出FasL的表达。正常人血浆中未能检测出sFasL,但LN病人血浆存在有sFasL;sFasL的水平在狼疮活动期和静止期的LN病人之间差异无显著性（P＞0．05）;单核细胞与重组人FasL在体外共同培养2h后,LN病人单核细胞的凋亡较正常人明显增高（P＜0．05）,单核细胞与重组人FasL在体外共同培养6h后,LN病人单核细胞的存活率较正常人明显低下（P＜0．05）。结论 LN病人单核细胞表面功能在Fas表达上调,血浆中存在sFasL,这可能是LN病人单核细胞凋亡加速的原因之一。     

The production of reactive oxygen species in mice infected or immunized with Plasmodium berghei

The capacity of peritoneal exudate cells (PEC) obtained from mice infected or immunized with Plasmodium berghei to produce reactive oxygen species (ROS) and the biological basis of this response was investigated, using luminol-dependent zymosan-triggered chemiluminescence (CL). CL response of PEC from infected mice increased at the early stage but was significantly depressed at the later course of the infection. A similar biphasic activity of peroxidase was also observed in PEC from infected mice. On the other hand, PEC from immunized mice exhibited concomitant increases of the ability to produce CL, the activity of peroxidase and the expression of Fc and C3 receptors on cell surface. Compared with the controls, PEC from immunized mice showed an elevated background CL, responded more rapidly to the stimulation and generated considerably higher CL when triggered with opsonized zymosan. The data suggest that phagocytes in immunized mice are active in the production of ROS while those in infected mice are less active, and the inhibition of the ability of phagocytes to produce ROS may be one of the mechanisms for the parasites to escape from host immune system.     

Human Neutrophil Functions Are Inhibited In Vitro by Clinically Relevant Ethanol Concentrations

Neutrophils [polymorphonuclear neutrophils (PMNs)] play a pivotal role in host defense in man. These defenses may be compromised, however, in alcohol users and abusers. We therefore evaluated the effect of ethanol levels (12.5 to 500 mg/dl), on key functions of human PMNs—chemotaxis and production of reactive oxygen species—and on changes in cytosolic-free calcium ([Ca²⁺]_i), a pivotal intracellular mechanism of PMN activation. Ethanol significantly inhibited chemotaxis as evaluated by formyl-methionyl-leucyl-phenylalanine (fMLP)-induced upregulation of surface adhesion molecules (CD11b), fMLP-induced PMN elongation was only inhibited by a very high ethanol concentration of 500 mg/dl. Production of reactive oxygen species by normal PMNs was assessed by either chemiluminescence (CL) for hypochlorous acid or ferricytochrome c reduction (FCR) for superoxide anions. For PMN stimulated by fMLP, ethanol inhibited CL but not FCR. For PMNs activated by phorbol myristate acetate, ethanol inhibited both CL and FCR. Ethanol did not alter baseline [Ca²⁺]_i, as assessed by videomicroscopy using the Ca²⁺-sensing fluorescent dye Fura-2-AM, but did significantly potentiate the increase in peak [Ca²⁺]_i, levels that occurs in response to stimulation by fMLP. Calcium channel blockers attenuated ethanol's inhibition of CL. Thus, acute in vitro ethanol, at clinically relevant concentrations, can inhibit several critical aspects of PMN functions. But, in PMNs, unlike neural cells, these inhibitory effects do not seem to be mediated by decreases in Ca²⁺ influx or in [Ca²⁺]_i.