Inhibition by dexamethasone of human neutrophil apoptosis in vitro

The efficacy of collagen-sponge to reduce postoperative scar formation was investigated in 65 Japanese white rabbits that received laminectomy in the 7th and 8th thoracic vertebra. The defect after laminectomy was filled by collagen-sponge in 25 rabbits, by free fat in 20 rabbits, and by nothing in 20 rabbits as controls. The animals were sacrificed 2, 4, 8 and 12 weeks and additional 5 rabbits of which defects were filled with collagen-sponge were sacrificed after 24 weeks. All the defects were examined histologically. At 4 weeks after laminectomy, the defects filled by collagen-sponge showed that fibrous tissue had invaded into the sponge, but there was no remarkable adhesion to the dura mater. At 8 weeks, the defect with collagen sponge showed foaming cells, and no thickening of the dura mater was observed. At 12 weeks, the grouping of foaming cells was partially replaced by fat cells. At 24 weeks, most of the foaming cells were replaced by fat cells, and the defect was then similar to that filled by free fat at 12 weeks. In contrast, the defect with no interposed membrane was already filled with fibrous tissue at 4 weeks, and adhesion to the dura mater was observed. Although the free-fat graft at 12 weeks postoperatively showed no remarkable adhesion around the dura mater, infiltration of fat tissue into the spinal canal was observed in 2 of 5 rabbits. These results indicated that collagen-sponge can be utilized as a new biomaterial to effectively prevent scar formation after laminectomy.     

Modulation of neutrophil apoptosis by psychological stress and glucocorticoid

Neutrophils play a pivotal role in host defense against bacterial infection, however, these cells may have a harmful effect on normal tissues under certain pathological conditions. We demonstrate here that apoptosis of these cells is modulated by psychophysical stress and its related hormones, suggesting that psychoneural systems may exert an effect on host defense through modulating neutrophil survival.     

Thiol-mediated redox regulation of neutrophil apoptosis

BACKGROUND: Intracellular glutathione, an endogenous antioxidant, protects cellular function against oxidative stress. Because oxidative stress has been implicated in neutrophil apoptosis, we hypothesized that reduced thiol levels may induce apoptosis through an alteration in cellular redox state. METHODS: Human polymorphonuclear leukocytes (PMNs), were incubated with medium or with increasing concentrations of the reduced glutathione (GSH)-depleting agents diethylmaleate and diamide and buthionine sulfoximine, an inhibitor of GSH synthesis. Apoptosis was assessed by means of flow cytometry with propidium iodide DNA staining and confirmed morphologically. GSH was measured colorimetrically, and tyrosine phosphorylation was assessed by means of immunoblotting. RESULTS: Diethylmaleate and diamide induced a dose-dependent reduction in GSH and a corresponding increase in PMN apoptosis. This effect could be reversed with N-acetylcysteine, suggesting that diethylmaleate induces apoptosis through the depletion of GSH. The antioxidant pyrolidine dithiocarbamate had no effect. Because oxidants can mediate intracellular signaling via tyrosine phosphorylation, we therefore evaluated the effects of the tyrosine kinase inhibition on diethylmaleate-induced PMN apoptosis. Both genistein and herbimycin A reduced diethylmaleate-induced apoptosis and tyrosine phosphorylation. CONCLUSIONS: Sulfhydryl oxidation by diethylmaleate alone induces apoptosis, providing evidence of a redox-sensitive, thiol-mediated pathway of apoptosis. Furthermore, tyrosine phosphorylation appears to play an important role in this process. Because apoptosis is a critical mechanism regulating PMN survival in vivo, manipulation of PMN intracellular thiols may represents a novel therapeutic target for the regulation of cellular function.     

Calpain and calpastatin regulate neutrophil apoptosis

The average polymorphonuclear neutrophil (PMN) lives only a day and then dies by apoptosis. We previously found that the calcium-dependent protease calpain is required for apoptosis in several mouse models of cell death. Here we identify calpain, and its endogenous inhibitor calpastatin, as regulators of human neutrophil apoptosis. Cell death triggered by the translation inhibitor cycloheximide is calpain-dependent, as evidenced using either a calpain active site inhibitor (N-acetyl-leucyl-leucyl-norleucinal) or agents that target calpain's calcium binding sites (PD150606, PD151746). No significant effect on cycloheximide-triggered apoptosis was found by using inhibitors of the proteasome or of other papain-like cysteine proteases, providing further evidence that the active site calpain inhibitor prevents apoptosis via its action on calpain. In addition, we find that potentiation of calpain activity by depleting its endogenous inhibitor, calpastatin, is sufficient to cause apoptosis of neutrophils. Nevertheless, apoptosis signalled via the Fas antigen proceeds regardless of the presence of calpain inhibitor. These experiments support a growing body of work, indicating an upstream regulatory role for calpain in many, but not all, forms of apoptotic cell death. They also identify calpastatin as a participant in apoptotic cell death and suggest that for at least one cell type, a decrease in calpastatin is a sufficient stimulus to initiate calpain-dependent apoptosis.     

Critical role of Lyn kinase in inhibition of neutrophil apoptosis by granulocyte-macrophage colony-stimulating factor

The signal pathway for control of apoptosis in human neutrophils is currently unknown. In this study, we provide the first evidence that a Src family tyrosine kinase, Lyn, plays a key role in inhibition polymorphonuclear (PMN) cell death. Several nuclear proteins associated with apoptosis, i.e., p53, cdc2, and Rb, were absent from PMN. Bcl-2, known to inhibit apoptosis, was also not expressed. Programmed cell death that rapidly occurred in PMN could be arrested by granulocyte-macrophage CSF (GM-CSF), but this activation did not induce p53, cdc2, retinoblastoma, or Bcl-2 expression. Instead, GM-CSF produced a rapid activation of Lyn and Hck, but not Fgr, tyrosine phosphorylation within 1 min. Co-immunoprecipitation studies indicated that only Lyn, but not Hck, was physically coupled to GM-CSF receptor. By histologic assessment and evaluation of DNA fragmentation, only antisense Lyn, but not antisense Hck or antisense Fgr, could reverse the cell survival advantage provided by GM-CSF. Therefore, the physical coupling of Lyn to GM-CSF receptor and its early activation are required for inhibition or delay of apoptosis in PMN.     

Inflammatory response during cardiopulmonary bypass: neutrophil apoptosis

Cardiopulmonary bypass (CPB) is essential to open heart surgery. However, CPB induces many types of inflammatory response, and may contribute to the tissue injury and the development of postoperative complications. On the other hand, the neutrophil responses to injury and infection immediately and secretes elastases and cytokines followed by prolongation of inflammatory changes, and programmed cell death (apoptosis) of neutrophils is delayed by inflammatory response. In this study, we evaluated the alternation of the neutrophil life span during CPB. Peripheral blood was obtained from eight adult patients before CPB, 1 hr and 2 hr after CPB start. After separation of neutrophils, and incuvation in the presence of TNF-alpha for 3 hr, we measured fluorescence-microscopically apoptosis rate (%A). %A significantly decreased with time (before 9.7 +/- 2.3%, 1 hr 3.0 +/- 1.0%, 2 hr 1.5 +/- 0.6%, p < 0.05). We conclude that neutrophil apoptosis was suppressed significantly during CPB. Systemic inflammatory change induced by CPB may be prolonged with extended life span of neutrophil.     

Interleukin-6 and a delay of neutrophil apoptosis after major surgery

Surgical treatments affect host immune systems. Apoptosis of the circulatory lymphocytes and an elevation of the number of neutrophils are induced by surgery. These alterations are not dependent only on the stress hormone. The course of neutrophilia and the serum concentration of interleukin-6 are given after various surgical treatments. Neutrophil-mediated lung injury is discussed.     

Delayed neutrophil apoptosis after total body irradiation in mice. The role of granulocyte-macrophage colony-stimulating factor in neutrophil function

We performed an in vitro study of the long-term effects of a sublethal dose (5 Gy) of x-irradiation on the survival and function of neutrophils in adult mice. For this purpose, we incubated control neutrophils harvested from long-term bone marrow cultures (LTBMCs) with supernatant withdrawn from cultures obtained in adult mice 6 or 9 months postirradiation. We noted a significant increase in superoxide anion production, NADPH, and protein levels in these cells after 3, 6, and 15 hours of incubation compared with the same cells incubated with supernatant from control LTBMCs. We also observed a delay in apoptosis that was correlated with maintenance of adenosine triphosphate levels and survival. Similar differences were found when control LTBMC neutrophils were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) (1.3 nM). Indeed, enzyme-linked immunosorbent assay revealed a significant overproduction of this cytokine, together with higher interleukin (IL)-6 and IL-3 levels, in the supernatant from cultures of irradiated mice. Our results suggest that GM-CSF is one of the cytokines responsible for promoting the survival and activation of neutrophil function after total body irradiation.     

Endothelial activation in monosodium urate monohydrate crystal-induced inflammation: in vitro and in vivo studies on the roles of tumor necrosis factor alpha and interleukin-1

OBJECTIVE: There is relatively little direct evidence for the roles of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) in activating endothelium in vivo. The aim of this study was to use in vitro and in vivo models to investigate the contribution of these cytokines to both E-selectin expression and the recruitment of polymorphonuclear cells (PMN) in monosodium urate monohydrate (MSU) crystal-induced inflammation. METHODS: MSU crystals were incubated with freshly isolated mononuclear cells, after which the harvested supernatants were tested for their ability to induce E-selectin expression during coculture with human umbilical vein endothelial cells. Subsequent experiments were performed with the addition of neutralizing anticytokine antibodies/antisera. The role of TNF alpha was then studied in an MSU crystal-induced monarthritis model, in the presence or absence of anti-TNF alpha (5 mg/kg intravenously). 99mtechnetium (99mTc)-labeled PMN cells and (111)indium (111In)-labeled anti-E-selectin monoclonal antibody (MAb) 1.2B6 were intravenously administered 4 hours after intraarticular injection to quantify PMN recruitment and E-selectin expression in inflamed joints. RESULTS: MSU crystals were a potent stimulus for IL-1 and TNF alpha production by monocytes in vitro, and these cytokines fully accounted for MSU crystal-stimulated, monocyte-mediated endothelial activation. In the MSU crystal-induced monarthritis model, TNF alpha blockade was very effective in suppressing both E-selectin expression and PMN emigration into the inflamed joints, as judged by gamma-camera image analysis and postmortem tissue counting following the intravenous injection of 99mTc-PMN and 111In-anti-E-selectin MAb. CONCLUSION: IL-1 and TNF alpha appear to be the only factors released by monocytes following incubation with MSU crystals, which induce E-selectin expression in vitro. Anti-TNF alpha is effective in suppressing endothelial activation and PMN recruitment in vivo E-selectin imaging can be used to assess the endothelial response to therapy and may prove useful for clinical studies.     

Caspases mediate tumor necrosis factor-alpha-induced neutrophil apoptosis and downregulation of reactive oxygen production

BACKGROUND: Cyclosporine (CSA) has improved patients and organ-graft survival rates, but its chronic nephrotoxicity is still an issue. Although prolonged vasoconstriction could contribute to chronic CsA tubulointerstitial changes by producing chronic ischemia, this relationship has been difficult to demonstrate thus far, and cellular origin and mediators of these structural alterations remain ill-defined. METHODS: As a part of a clinical trial in kidney transplant recipients on triple immunosuppressive therapy (CsA, azathioprine and steroid), which includes renal biopsy as "per protocol," 22 patients enrolled between 12 and 24 months posttransplantation underwent renal hemodynamic evaluation by measuring glomerular filtration rate and renal plasma flow by the plasma clearance of unlabeled iohexol and the renal clearance of para-aminohippuric acid, respectively. In parallel, the CsA pharmacokinetic profile was also determined. A week later, a protocol biopsy of kidney graft was performed. Light microscopy examination and localization of endothelin-1, RANTES, monocyte chemoattractant protein-1 gene expression by in situ hybridization in the graft specimens were evaluated and related to the pattern of histologic lesions. RESULTS: Ten out of 22 kidney transplant recipients who underwent the protocol biopsy had CsA nephrotoxicity, eight had chronic rejection, and four had no lesions at histological examination. The total daily exposure to CsA was higher in patients with CsA nephrotoxicity than in those with chronic rejection or no lesions at biopsy. Renal function was preserved in the CsA toxicity group as compared with the chronic rejection group, despite some degree of renal hypoperfusion. Tubular atrophy and striped interstitial fibrosis were found in all patients with light microscopical evidence of CsA nephrotoxicity, whereas glomerular and arteriolar lesions were less frequent. Intense staining for endothelin-1, RANTES, and monocyte chemoattractant protein-1 mRNAs selectively localized at tubular epithelial cells was found in biopsies taken from patients with CsA nephrotoxicity, but not in the chronic graft rejection group, whose tubuli had only minimal staining for RANTES mRNA on a few occasions. CONCLUSION: Long-term CsA administration to kidney allograft recipients leads to tubulointerstitial injury independently of its vascular effect. The possible contribution to the development of interstitial fibrosis of inflammatory and growth factors released by tubular cells in which CsA accumulates is proposed.     

Inhibition of neutrophil apoptosis and modulation of the inflammatory response by granulocyte colony-stimulating factor in healthy and ethanol-treated human volunteers

Nitrogen dioxide levels were measured in 80 homes in the Latrobe Valley, Victoria, Australia, using passive samplers. Some 148 children between 7 and 14 yr of age were recruited as study participants, 53 of whom had asthma. Health outcomes for the children were studied using a respiratory questionnaire, skin prick tests, and peak flow measurements. Nitrogen dioxide concentrations were low, with an indoor median of 11.6 microgram/m3 (6.0 ppb), and a maximum of 246 microgram/m3 (128 ppb). Respiratory symptoms were more common in children exposed to a gas stove (odds ratio 2.3 [95% CI 1. 0-5.2], adjusted for parental allergy, parental asthma, and sex). Nitrogen dioxide exposure was a marginal risk factor for respiratory symptoms, with a dose-response association present (p = 0.09). Gas stove exposure was a significant risk factor for respiratory symptoms even after adjusting for nitrogen dioxide levels (odds ratio 2.2 [1.0-4.8]), suggesting an additional risk apart from the average nitrogen dioxide exposure associated with gas stove use. Atopic children tended to have a greater risk of respiratory symptoms compared with nonatopic children with exposure to gas stoves or nitrogen dioxide, but the difference was not significant.     

Hypertonic saline resuscitation reduces neutrophil margination by suppressing neutrophil L selectin expression

Hypertonic saline (HS) reduces hemorrhage-induced lung injury by suppressing the neutrophil oxidative burst and reducing lung neutrophil influx. This study investigated whether this is caused by the effects of HS on endothelial adhesion molecule expression, the production of chemoattractants in the lung, or a direct effect of HS on neutrophil selectin expression. METHODS: BALB/c mice were made to hemorrhage to 40 mm Hg for 1 hour and resuscitated with shed blood and either 4 mL/kg 7.5% HS or two times the shed blood volume of lactated Ringer's solution (LRS). Neutrophil L selectin expression was determined by flow cytometry, total neutrophil counts were obtained by differential staining, and pulmonary endothelial P and E selectin expression was evaluated by immunohistochemistry. Chemoattractants in lung lavages were determined with a modified Boyden chamber migration assay. RESULTS: Chemotactic activity of lavage fluid of HS-treated animals was not significantly different from that of LRS-treated animals, and endothelial P and E selectin expression was not altered by HS resuscitation. Neutrophils of HS-treated animals, however, expressed significantly less L selectin than those of LRS-treated mice. Concomitantly, circulating neutrophil counts of LRS-treated animals were significantly decreased compared with those of HS-treated mice. CONCLUSION: HS had little effect on endothelial selectin expression and chemoattractant production in the lung. HS significantly decreased neutrophil L selectin expression, however. This suggests that HS resuscitation may reduce lung injury by preventing neutrophil L selectin expression and endothelial adhesion.     

Production and regulation of monocyte chemoattractant protein-1 in lipopolysaccharide- or monosodium urate crystal-induced arthritis in rabbits: roles of tumor necrosis factor alpha, interleukin-1, and interleukin-8

The production of monocyte chemoattractant protein-1 (MCP-1) and its regulation by TNFalpha, IL-1, and IL-8 were investigated in two rabbit models of arthritis induced by intra-articular injection of lipopolysaccharide (LPS) or monosodium urate (MSU) crystals. We first prepared recombinant rabbit MCP-1 and antibodies and then developed an immunoassay. The immunoassay detected 3 pg/ml rabbit MCP-1 and did not cross-react with other rabbit chemokines such as IL-8 or GRO. MCP-1 was first detected in synovial fluid (SF) at 1 hour, and peaked at 4 or 2 hours after the injection of LPS or MSU crystals, respectively. Immunohistochemically, MCP-1 was detected in synovial lining cells and infiltrating neutrophils. The amounts of MCP-1 detected in SF from neutrophil-depleted rabbits were similar to those in normal rabbits, suggesting that synovial lining cells were the main source of MCP-1 detected in SF. The peak level of MCP-1 in SF after LPS-injection was inhibited by 57% with anti-TNFalpha mAb and by 41% with IL-1 receptor antagonist (IL-1Ra). Coadministration of anti-TNFalpha mAb and IL-1Ra inhibited 90% of MCP-1 production. In contrast, the peak level of MCP-1 in SF after MSU crystal-injection was not affected by any cytokine inhibitor, but was reduced by 52% with coadministration of anti-TNFalpha mAb and IL-1Ra. Anti-IL-8 IgG had no effect on the production of MCP-1 in either model. Thus, the production of MCP-1 in LPS-induced arthritis was mostly regulated by TNFalpha and IL-1, whereas half the extent of MCP-1 production in MSU crystal-induced arthritis was independent of TNFalpha or IL-1. IL-8 does not seem to regulate the production of MCP-1 in SF either directly or indirectly. Finally, administration of neutralizing anti-MCP-1 antibody inhibited LPS- and MSU crystal-induced monocyte infiltration by 58.4% and 44.9%, respectively, suggesting that synovial production of MCP-1 plays an important role in the recruitment of monocytes in these arthritis models.     

Renal transport of urate during diuretic-induced hypouricemia

The effect of two weeks administration of a uricosuric diuretic (SKF-62698) on renal urate handling has been examined in 11 normal men. Plasma urate concentrations had declined by more than 60 per cent after two weeks. Urate excretion per unit of glomerular filtration rate and urate clearance (Curate) per unit of glomerular filtration rate were increased after the administration of SKF-62698. The importance of intact tubular secretion of urate in producing these changes was assessed by administering pyrazinamide, an agent that curtails urate secretion, to each participant. The decrements in urate excretion and clearance produced by pyrazinamide both increased significantly, whereas the residual urate excretion rates and clearances not suppressible by pyrazinamide were only minimally altered by SKF-62698 treatment. These results suggest that the excretion of secreted urate was enhanced by prolonged administration of SKF-62698, probably secondary to the inhibition of postsecretory urate reabsorption. In addition, because the nonsuppressible urate excretion did not decline despite a 63 per cent reduction in the plasma urate, it is likely that the reabsorption of filtered urate also was impaired by SKF-62698.     

Prevention from hypoxia-induced apoptosis by pre-conditioning: a mechanistic approach in cultured neurons from fetal rat forebrain

Molecular effects of pre-conditioning by 1-h hypoxia were investigated in cultured neurons from fetal rat forebrain, submitted the following day to a 6-h hypoxia that induces apoptosis. While preventing from apoptosis, pre-conditioning led to increased number of living neurons, DNA synthesis, with persistent overexpression of Bcl-2 and proliferating cell nuclear antigen (PCNA). Adaptative mechanisms would involve anti-apoptotic proteins and regulators of the cell cycle, to finally promote neuronal proliferation.     

Proteolytic cleavage of ICAM-1 by human neutrophil elastase

Human leukocyte elastase (HLE) participates in tissue destruction in a number of inflammatory disorders, including rheumatoid arthritis and cystic fibrosis. Since HLE has been shown to bind to Mac-1, and ICAM-1 plays a key role during the recruitment and the activation of leukocytes at inflamed sites, we investigated the capacity of HLE to cleave ICAM-1. Flow-cytometric analyses showed a dose-dependent cleavage of ICAM-1 by HLE on different human cell lines. The cleavage was completely inhibited by alpha1-antitrypsin, a natural HLE protease inhibitor. The ability of HLE to degrade ICAM-1 was further confirmed by electrophoretic analysis using a soluble form of ICAM-1 (D1-D5). Enzymatic removal of N-linked glycosylation did not significantly modulate ICAM-1 cleavage by HLE, while removal of sialic acid residues partially reduced the sensitivity of ICAM-1 to HLE. We further showed that sputum of cystic fibrosis patients contains high levels of HLE activity capable of cleavage of cell surface ICAM-1. The cleavage induced by incubation of cells with the sputum sample was totally inhibited by alpha1-antitrypsin and the specific peptidic HLE inhibitor N-methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone. Moreover, the cleavage of ICAM-1 was concomitant to that of CD4 at the surface of the same cell, at the same amplitude, and at all HLE concentrations. The capacity of HLE to modulate the expression of ICAM-1 on the surface of leukocytes by proteolytic cleavage brings support to the hypothesis that overproduction of HLE can cause severe immunologic lung disorders by affecting intercellular adhesion.