Effects of exogenous surfactant and recombinant human copper-zinc superoxide dismutase on oxygen-dependent antimicrobial defenses.

The use of human recombinant CuZn superoxide dismutase (rhSOD) in addition to exogenous surfactant has been studied as a therapeutic strategy to prevent acute and chronic lung injury in premature infants with blood monocytes (MO). However, scavenging of superoxide by rhSOD may compromise bacterial killing by phagocytes. In the present study, we investigated the interaction of exogenous surfactant and rhSOD with the antibacterial activity of human blood MO. MO were preincubated in the presence or absence of: (1) modified natural surfactant (Curosurf); 1 mg/ml); (2) rhSOD (2,500 U/ml) and (3) bovine catalase (25,000 U/ml). Bacteria (Legionella pneumophila or Escherichia coli) were then added and incubated for 6 h. Viable bacteria were determined by counting colony-forming units. The ability of the MO to generate superoxide anions (O2-) in response to bacterial infection was also investigated. The antibacterial capacity of MO was not impaired by the presence of rhSOD either alone or combined with Curosurf. In some instances, bactericidal activity was even potentiated by the addition of rhSOD. Exposure of MO to catalase interfered with the increased bacterial killing of MO and rhSOD, suggesting that hydrogen peroxide (H2O2) production was critically important in the process of bacterial killing. Both bacterial species were also found to induce the generation of intra- and extracellular O2- by MO. Data indicate that rhSOD potentiates the killing of bacteria by human MO. The mechanism of action appears to be related to the ability of bacteria to induce the generation of O2-, which in turn is converted to H2O2 in the presence of rhSOD. This has important implications in the development of therapeutic intervention strategies using antioxidant therapy in premature infants with respiratory distress syndrome.

Expression and localization of heat shock proteins in rat basophilic leukemia cells: differential modulation by degranulation, thermal or oxidative stress.

BACKGROUND: Rat basophilic leukemia (RBL-2H3) cells are well characterized in terms of morphological and biochemical changes upon activation, and have been extensively used as a model system for studying the mechanisms of the immediate hypersensitivity reaction. To investigate whether overexpression of heat shock/stress proteins (HSP) is involved in the mast cell-dependent reactivity, we examined the adaptive responses of RBL-2H3 cells to classical stress conditions such as heat shock or oxidative injury produced by an aqueous extract of tobacco smoke. METHODS: HSP were determined by flow cytometry and immunocytochemistry. Degranulation was confirmed as the release of beta-hexosaminidase, determined spectrophotometrically, and by electron microscopy experiments. RESULTS: We found that RBL-2H3 cells respond to heat shock or oxidative injury by the synthesis of both the inducible 72 kDa HSP (Hsp70), and the oxidation-specific 32 kDa heme oxygenase (HO)-1. Heat shock induced mainly Hsp70 in a cell growth-dependent manner, whereas oxidative stress induced mainly HO-1 in a cell growth-independent manner. However, heat shock or oxidative stress had no significant effects on degranulation. CONCLUSION: Stress-mediated synthesis of HSP was not associated with RBL-2H3 degranulation and likewise, degranulation did not induce HSP.

Geranylgeranylacetone protects human monocytes from mitochondrial membrane depolarization independently of Hsp70 expression.

The anti-ulcer drug geranylgeranylacetone (GGA) has been shown to induce the expression of heat shock proteins (HSPs), in particular of Hsp70, in gastric and small intestine cells. In this study, we investigated whether GGA was able to induce Hsp70 in another cell type, human monocytes, which represent a well-established model of Hsp70 expression under oxidative stress. In these cells, GGA had no significant effect either on basal or tobacco smoke-induced Hsp70 expression. We further investigated the effects of GGA on mitochondria, a key organelle of oxidant-mediated cell injury and a putative target for GGA-mediated protection. GGA significantly increased basal mitochondrial membrane polarization and inhibited the decrease in mitochondrial membrane potential of human monocytes exposed to distinct sources of clinically relevant oxidants such as tobacco smoke and y-irradiation. Our results indicate that mitochondria are targets for GGA-mediated protection against oxidative stress in human monocytes, independently of Hsp70.

Mitochondrial membrane potential and apoptosis peripheral blood monocytes in severe human sepsis.

UNLABELLED: Reduced mitochondrial membrane potential (Delta(Psi)m), which is considered as an initial and irreversible step towards apoptosis, as well as cell death regulating proteins, such as Fas, Hsp70, or Bcl-2, may play an important role in sepsis. We studied the relationship between sepsis severity and peripheral blood monocyte Delta(Psi)m, cell death (necrosis and apoptosis), soluble Fas ligand, Hsp70, and Bcl-2 expression over time in 18 patients with sepsis, and compared these data with those of a group of 17 healthy control subjects. All measurements were performed within 3 d of the onset of severe sepsis (T1), then 7 to 10 d later (T2), and finally at hospital discharge (T3). Delta(Psi)m was expressed as the percent monocytes with altered Delta(Psi)m (%Delta(Psi)m). Patients with sepsis had greater %Delta(Psi)m at T1 and T2 but not at T3 (14.6 +/- 2.6% and 15.9 +/- 2%, respectively, versus control 6.6 +/- 0.2%, p < 0.01). Septic patients exhibited greater cell death in their monocytes and had greater Hsp70 expression only at T1. Bcl-2 levels were similar in septic and control subjects. Comparing survivors with non-survivors of sepsis, nonsurvivors had a greater %Delta(Psi)m at T1 (26.4 +/- 5.3% versus 10.1 +/- 2.7%, p < 0.01) and a significant decrease in Bcl-2 expression, whereas no difference was found in Hsp70 levels. These results indicate that mitochondrial dysfunction and subsequent cell death occur in severe sepsis and suggest that %Delta(Psi)m is a marker of severity in human sepsis. KEYWORDS: mitochondria; apoptosis; sepsis; heat-shock protein 70; proto-oncogene protein c-Bcl-2

Increased eosinophil cationic protein levels in bronchoalveolar lavage from wheezy infants.

Although studies examining the serum suggest a role for eosinophils in wheezing episodes in infants and toddlers, the presence of a chronic eosinophilic inflammation within their airways remains to be demonstrated. In this study we investigated whether eosinophil cationic protein (ECP) levels are increased in BAL fluid (BALF) from infants and toddlers with recurrent wheezing episodes, during an asymptomatic period. The levels of ECP in BALF were quantitated by radioimmunoassay in 61 children (36 with severe recurrent episodes of wheezing and 25 who were non-wheezy), aged 6-36 months, in whom flexible bronchoscopy was clinically indicated. BALF eosinophil counts were < or = 1% in all patients and did not differ in wheezers, compared to non-wheezers. In contrast, ECP levels in BALF were > or = 2.2 micrograms/l in 18 of 36 (50%) wheezy infants but in only three of 25 (12%) control infants (p < 0.01). Neutrophil counts were significantly higher in the wheezer group than in the non-wheezer group (8.1 x 10(3) cells/ml vs. 3.0 x 10(3) cells/ml). ECP levels in the BALF were not correlated with the absolute number of eosinophils (r = 0.03; p = 0.8) but were correlated with the absolute number of neutrophils (r = 0.54; p = 0.001). There was no association between high ECP levels in BALF and the atopic status of the wheezers. In conclusion, ECP levels are increased in BALF from young children with recurrent wheezing episodes, even during relatively quiescent periods, suggesting a chronic increased cell activation in the lower airways.

Treatment with 815-nm diode laser induces long-lasting expression of 72-kDa heat shock protein in normal rat skin.

BACKGROUND: We previously reported that skin closure is improved by photoirradiation of the wound margins with an 815-nm diode laser system. OBJECTIVES: To determine whether the beneficial effects of laser treatment involve the overexpression of the inducible 72-kDa heat shock protein, Hsp70. METHODS: Expression of Hsp70 was investigated by immunocytochemistry in normal hairless rat dorsal skin and compared with its expression after laser photoirradiation. RESULTS: Constitutive expression of Hsp70 was mainly confined to the upper epidermal layer. Laser irradiation further increased epidermal expression of Hsp70 while inducing de novo synthesis of the protein in dermal structures, particularly around blood vessels, hair follicles and sebaceous glands. Laser-induced expression of Hsp70 was still present 7 days after photoirradiation. CONCLUSIONS: Laser-induced expression of Hsp70 might contribute to improved tissue regeneration and wound healing.

Laser assisted skin closure (LASC) by using a 815-nm diode-laser system accelerates and improves wound healing.

BACKGROUND AND OBJECTIVE: This study aimed to evaluate a 815-nm diode-laser system to assist wound closure to accelerate and improve healing process. STUDY DESIGN/MATERIALS AND METHODS: A total of 25 male hairless rats (mutant OFA Sprague-Dawley rats, IFFA-CREDO, L'Arbresle, France) with four dorsal skin incisions were used for the study. For each wound, the good apposition of the edges was obtained with buried absorbable suture. In the laser group, the laser beam was applied spot by spot through a transparent adhesive dressing along two incisions with the following parameters: 1.5 W; 3 seconds; spot diameter, 2 mm; fluence, 145 J/cm(2). Both control wounds were closed with conventional suture techniques. The duration of the closure procedure was noted for each group. Clinical examination, histologic study, and measurement of tensile strength were performed at 3, 7, 15, and 21 days after surgery. Determination of activation of heat shock protein 70 (Hsp70) through immunocytochemistry was performed at days 1 and 7. RESULTS: LASC was 4 times faster to process than conventional suture: 1 minute 49 +/- 20.6 seconds vs. 7 minutes 26 +/- 62.2 seconds. In the laser group, healing was accelerated resulting in a more indiscernible scar than in the control groups. Histologic aspect was better with earlier continuous epidermis and dermis and a thinner resulting scar. Tensile strength was 30 to 58% greater than in control groups at 7 and 15 days (P < 0.001). Expression of Hsp70 was markedly induced in skin structures examined after laser exposure. CONCLUSIONS: This study shows the ability of the 815-nm diode-laser system to assist wound closure leading to an acceleration and an improvement of wound healing with indiscernible resulting scar. The mechanisms of this phenomenon are still unclear but further investigations are in progress to attempt to explain them.

Differential basal synthesis of Hsp70/Hsc70 contributes to interindividual variation in Hsp70/Hsc70 inducibility.

The source of intraspecies variation in the expression of heat shock proteins (HSPs) remains unresolved but could shed light on differential stress tolerance and disease susceptibility. This study investigated the influence of variable basal HSP synthesis on differential inducibility of HSP synthesis. Basal and heat-induced synthesis of the major HSP families in peripheral blood monocytes from healthy donors (n = 42) were analysed using biometabolic labelling and densitometry. Basal Hsp70/Hsc70 synthesis and percentage induction of Hsp70/Hsc70 synthesis were significantly correlated (r = -0.57, p < 0.0001), and described most accurately by an exponential decay equation (R = 0.68, R2 = 0.46). This regression equation suggests that increasing levels of basal Hsp70/Hsc70 synthesis are accompanied by an exponential decrease in the percentage induction of Hsp70/Hsc70 synthesis. The model fits data from European and non-European population groups independently, although both coefficients in the regression equation were larger for non-Europeans. This implies population group as an additional factor influencing differential HSP expression. The differential inducibility of Hsp70/Hsc70 due to variable basal synthesis of Hsp70/Hsc70 and based upon population group may contribute to differential stress tolerance or disease susceptibility.

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes.

The free radicals nitric oxide (.NO) and superoxide (O(2)(-).) react to form peroxynitrite (ONOO(-)), a highly toxic oxidant species. In this study we investigated the respective effects of NO and ONOO(-) in monocytes from healthy human donors. Purified monocytes were incubated for 6 or 16 h with a pure NO donor (S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an.NO/ONOO(-) donor (3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and without superoxide dismutase (200 IU/ml), or pure ONOO(-). We provide evidence that 3-morpholinosydnonimine chlorhydrate alone represents a strong stress to human monocytes leading to a dose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis and necrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO(-), but not.NO, was responsible for the observed effects. This observation was further strengthened by the absence of a stress response in cells exposed to S-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO(-) alone also induced mitochondrial membrane depolarization and cell death by apoptosis and necrosis. Thus ONOO(-) formation may well explain the toxic effect generally attributed to.NO.

Adaptive responses of human monocytes infected by bordetella pertussis: the role of adenylate cyclase hemolysin.

The activation/adaptive responses of human monocytes exposed to Bordetella pertussis parental or mutant strains were evaluated and correlated to the expression of two bacterial toxins: adenylate cyclase-hemolysin and pertussis toxin. The marked rise in intracellular cyclic adenosine monophosphate (cAMP) observed in monocytes infected by B. pertussis parental strain, inversely correlated with (1) the production of tumor necrosis factor alpha; (2) the release of superoxide anion; and (3) the expression of the 72-kDa heat shock/stress protein, Hsp70. Experiments performed with mutants deficient in adenylate cyclase-hemolysin or with purified bacterial toxins confirmed the key role of adenylate cyclase-hemolysin in the control of monocytes' response to infection by B. pertussis. This bacterial strategy primarily involves evasion from antimicrobial defenses and, eventually, the sacrifice of the host cell.

Inducibility of the 70 kD heat shock protein in peripheral blood monocytes is decreased in human acute respiratory distress syndrome and recovers over time.

The heat shock/stress proteins (HSP), and, in particular, the inducible, cytosolic Hsp70, represent an extremely conserved response to many different cellular injuries, including reactive oxygen species (ROS). Hsp70 has been shown to confer to cells and tissues protection against the deleterious effects of ROS or cytokines, both in vitro and in animal models of acute respiratory distress syndrome (ARDS). We hypothesized that Hsp70 expression levels in peripheral blood monocytes (PBM) of patients with ARDS, would correlate with disease severity. We prospectively included 13 patients with previous ARDS (50 +/- 17 yr; range, 20 to 76 yr), nine ventilated patients with non-ARDS/ALI disease (45 +/- 20 yr; range, 19 to 76 yr), and 14 healthy volunteers (45 +/- 20 yr; range, 22 to 77 yr). PBM activation state was evaluated according to their membrane expression of CD16, and oxidative status according to plasma lipid peroxidation products. Both baseline expression and Hsp70 inducibility (after in vitro heat shock) were examined in PBM, using flow cytometric analysis. We found that basal expression of Hsp70 in PBM was similar for patients and control subjects, whereas Hsp70 inducibility- a reflection of the ability to mount a stress response-was significantly reduced in the patients with ARDS (p = 0. 02). Among all correlation analyses we considered between Hsp70 inducibility on the one hand, clinical and laboratory biomarkers for disease severity and outcome in the patients with ARDS on the other, only the duration of ventilatory support was significant (p < 0.003). As an approach to distinguish between disease and ventilation, we also analyzed a group of, ventilated patients without ARDS. Our results indicate that in patients with ARDS, Hsp70 inducibility in PBM is decreased, but it recovers over time with duration of ventilatory support.

Curosurf modulates cAMP accumulation in human monocytes through a membrane-controlled mechanism.

The cellular mechanisms by which pulmonary surfactant exerts its effects, including anti-inflammatory or proinflammatory effects, have remained elusive. To address the issue of whether plasma membrane modifications represent a target for these mechanisms, we designed an experimental protocol involving the determination of changes in cAMP levels under membrane-dependent or -independent stimulatory pathways. The effects of a modified natural porcine surfactant, Curosurf, and the major surfactant protein A were evaluated on resting and stimulated cAMP levels of human monocytes. We found that agents that elevate intracellular cAMP exhibit different susceptibilities toward a preexposure to Curosurf. The rise in cAMP induced by membrane-active agents such as cholera toxin or the diterpene forskolin was significantly inhibited by monocyte preexposure to Curosurf. In contrast, the rise in cAMP induced by the membrane-permeant phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine or by the Bordetella pertussis toxin adenylate cyclase-hemolysin was unaffected by Curosurf. Surfactant protein A did not affect either cAMP levels or the inhibitory capacity of Curosurf. We suggest that a plasma membrane-associated event affecting the mechanism underlying the effects of cholera toxin or forskolin is involved in the inhibition of cAMP accumulation caused by Curosurf.

Modified natural porcine surfactant modulates tobacco smoke-induced stress response in human monocytes.

Tobacco smoke (TS) is a potent source of oxidants and oxidative stress is an important mechanism by which TS exerts its toxicity in the lung. We have shown that TS induces heat shock (HS)/stress protein (HSP) synthesis in human monocytes. Pulmonary surfactant (PS) whose major physiological function is to confer mechanical stability to alveoli, also modulates oxidative metabolism and other pro-inflammatory functions of monocytes-macrophages. In order to determine whether PS alters the stress response induced by TS, we incubated human peripheral blood monocytes overnight with modified natural porcine surfactant (Curosurf) (1 mg/ml) before exposure to TS. Curosurf decreased TS-induced, but not HS-induced, expression of the major cytosolic, inducible 72 kD HSP (Hsp70). Furthermore, TS-generated superoxide anions production was significantly decreased by Curosurf in an acellular system, suggesting a direct scavenging effect of PS. We also examined the effects of TS and PS on monocytes ultrastructure. Monocytes incubated with Curosurf presented smoother cell membranes than control monocytes, while TS-induced monocyte vacuolization was, at least in part, prevented by Curosurf. Taken together, our data suggest that PS plays a protective role against oxygen radical-mediated, TS-induced cellular stress responses.

Tobacco smoke induces coordinate activation of HSF and inhibition of NFkappaB in human monocytes: effects on TNFalpha release.

Tobacco smoke (TS) exposure is a major risk factor for human disease, and macrophages of healthy smokers have a depressed capacity to release cytokines, including tumor necrosis factor (TNF)alpha. TS induces the synthesis of heat shock (HS)/stress proteins (HSP), and, in particular, of Hsp70. We determined whether Hsp70 induction by TS was mediated by the activation of the HS transcription factor, HSF. HSF activation has been shown to inhibit NFkappaB. Thus, we also determined the effects of TS on NFkappaB. U937 cells and human peripheral blood monocytes were exposed to TS, binding activities of the respective transcription factors were analyzed, and Hsp70 expression and TNFalpha release were determined in parallel. TS activated HSF, which was associated with Hsp70 overexpression and inhibition of NFkappaB binding activity and TNFalpha release. The altered cytokine profile observed in smokers may relate to an HSF/Hsp70-mediated inhibition of NFkappaB activity.

Flow cytometry is a rapid and reliable method for evaluating heat shock protein 70 expression in human monocytes.

The increasing interest in stress/heat shock proteins (Hsps) as markers of exposure to environmental stress or disease requires an easily applicable method for Hsp determination in peripheral blood cells. Of these cells, monocytes preferentially express Hsps upon stress. An appropriate fixation/permeabilization procedure was developed, combined with immunofluorescence staining and flow cytometry for the detection of the inducible, cytosolic, 72 kDa Hsp (Hsp70) in human monocytes. Higher relative fluorescence intensity was observed in cells exposed to heat shock (HS), reflecting a higher expression of Hsp70 in these cells as compared with cells kept at 37 degrees C. The heat-inducible increased Hsp70 expression was temperature- and time-dependent. Expression of Hsp70 was not uniform within the monocyte population, indicating the presence of subpopulations expressing variable levels of Hsp70 in response to HS. Simultaneous measurements of intracellular Hsp70 and membrane CD14 expression revealed that the higher Hsp70 inducibility coincided with the higher CD14 expression. Comparisons performed with biometabolic labelling, Western blotting, immunofluorescence and immunoperoxidase microscopic analysis, showed a high concordance between these different methods; however, cytometry was more sensitive for Hsp70 detection than Western blotting. Flow cytometric detection of intracellular Hsp70 is a rapid, easy and quantitative method, particularly suited for the determination of protein levels in individual cells from an heterogeneous population such as peripheral mononuclear blood cells, and applicable to cohort studies.