Comparison of alveolar macrophage and blood monocyte oxidative burst response in pulmonary sarcoidosis

The alveolitis of sarcoidosis is dominated by lymphocytes and mononuclear phagocytes and chronic macrophage activation may play a role in the pulmonary interstitial fibrosis of sarcoidosis. We measured superoxide anion release of alveolar macrophages from sarcoidosis patients after in vitro stimulation, as toxic oxygen radicals have been proposed as mediators of chronic tissue damage. In untreated patients alveolar macrophage activity was normal, but significantly lower than in blood monocytes. However, a negative correlation between lymphocytosis in bronchoalveolar lavage fluid and macrophage oxidative metabolism was observed, showing that only patients with high intensity alveolitis have a decreased oxidative burst response after in vitro stimulation. This may reflect in vivo activation of the cells with subsequent reduced ability to respond after additional stimulation in vitro. Patients with radiological stage I had lower alveolar macrophage response than patients in stage II or III. There was no correlation with SACE levels, lung function tests or smoking habits. Nine patients were reinvestigated after treatment with prednisolone. Although the lymphocytosis of lavage fluid was only insignificantly changed, all but one patient showed improved macrophage release of superoxide anion. Blood monocyte oxidative burst response was normal in all patients before and after treatment. In conclusion, only mononuclear phagocytes of the target organ (lung) showed an altered function and the most pronounced decrease was observed in sarcoid patients with active alveolitis. Chronic low grade toxic oxygen radical release of alveolar macrophages may be involved in the pathology of pulmonary sarcoidosis.     

Interleukin 6 primes human neutrophil and monocyte oxidative burst response

Interleukin 6 (IL-6), a 26-kDa inducible protein, is a cytokine with multiple biological activities. This paper reports on the regulatory role of rIL-6 on the function of human polymorphonuclear and mononuclear leukocytes, a property not described previously. rIL-6 by itself did not exhibit any chemotactic activity and it could not activate these cells for an oxidative burst response. Preincubation of both cell types with rIL-6 at concentrations of 5 and 50 ng/ml primed the cells for enhanced generation of oxygen radicals following stimulation with the chemotactic peptide f-Met-Leu-Phe or the phorbol ester PMA. The enhancement of the oxidative burst response occurred both at the level of superoxide anion generation, an early step in the activation pathway, and at the level of the hydrogen peroxide-myeloperoxidase mediated response, a later step in the oxidative burst pathway. The priming ability was abolished by heat treatment of rIL-6 at 100 degrees C but not at 70 degrees C. Stimulation of B cell growth and immunoglobulin production combined with enhancement of oxidative burst response of phagocytic cells by IL-6 provide an effective mechanism of fighting against invading micro-organisms.     

Modulation of human neutrophil and monocyte oxidative burst byLegionella pneumophila sonic extract

The effect of Legionella pneumophila sonic extract on human neutrophil and monocyte oxidative burst was studied by Superoxide anion release and luminol-enhanced chemiluminescence assays.Legionella pneumophila sonic extract by itself did not stimulate neutrophils and monocytes. The sonic extract at 8–2000g/ml primed neutrophils for enhanced Superoxide release and, at 8–62.5 g/ml, for enhanced chemiluminescence. Monocytes were only primed for enhanced chemiluminescence at very low extract concentrations (below 16g/ml). Monocyte Superoxide release was suppressed by extract concentrations higher than 2000g/ml and the chemiluminescence response of neutrophils and monocytes by concentrations higher than 250 and 125 g/ml, respectively. The priming activity was heat stable and present in fractions below 5 kDa. On the basis of these findings it is suggested that enhanced production of oxygen metabolites by neutrophils in contact with legionella components at low concentrations could contribute to the lung tissue damage seen in Legionnaires' disease, whereas the suppression of phagocyte oxidative burst by higher extract concentrations may be one of the mechanisms by which Legionella pneumophila survives intracellularly.     

Blood monocyte oxidative burst activity in acute P. falciparum malaria

The release of superoxide anion from blood monocytes was studied in eight patients with acute primary attack P. falciparum malaria. Before treatment a significant enhancement of the oxidative burst prevailed, which contrasts with previous findings of a depressed monocyte chemotactic responsiveness. During treatment and after clinical recovery the activity of superoxide anion release normalized in all patients.     

Pneumocystis carinii induces an oxidative burst in alveolar macrophages.

There is evidence that alveolar macrophages (AM) play a role in the clearing of Pneumocystis carinii from the lungs. To investigate the mechanisms involved in this process, we studied in vitro the induction of an oxidative burst by P. carinii in a cell line of macrophages (NR8383) and AM from normal rats. P. carinii was added to macrophage monolayers (10(6) cells), and the H2O2 produced after 4 h of incubation was measured. Both NR8383 macrophages and normal rat AM produced H2O2 in response to P. carinii cysts and trophozoites isolated from dexamethasone-treated rats, although the amount of H2O2 induced in AM from normal rats was larger. NR8383 macrophages bound and phagocytized both P. carinii cysts and trophozoites and produced increasing amounts of H2O2 as a dose-related response to cysts and trophozoites. Opsonization of P. carinii with normal rat serum increased H2O2 production by both types of macrophages; this enhancement was decreased, but not abolished, when the serum was first depleted of complement by heat treatment. These findings demonstrate that NR8383 macrophages and normal rat AM produce an oxidative burst in response to P. carinii and that this response is enhanced by complement.     

Pseudomonas aeruginosa exotoxin A primes human monocyte oxidative burst response in vitro

Modulation of the oxidative burst responsiveness of human blood monocytes and neutrophils after incubation with purified exotoxin A from Pseudomonas aeruginosa was studied in a lucigenin- and luminol-enhanced chemiluminescence system. Exotoxin A alone caused a dose-dependent stimulation of monocyte chemiluminescence responses, whereas neutrophil responses were inconsistent. Preincubation of monocytes with exotoxin A primed the cells for a significantly higher oxidative burst response when N-f-methionyl-leucyl-phenylalanine (fMLP) was used as a secondary stimulus, especially in the lucigenin-enhanced system. Heat-treatment at 100 degrees C for 15 min completely abolished the priming activity of the exotoxin A preparation. These findings demonstrate that exotoxin A modulates monocyte responsiveness in the chemiluminescence assay and suggest that increased release of toxic oxygen radicals from mononuclear phagocytes may contribute to the tissue damage in lungs with chronic P. aeruginosa infections.     

Targeting monocyte recruitment in CNS autoimmune disease

Monocytes and macrophages play a pathogenic role in a number of autoimmune inflammatory diseases. Recent studies in experimental autoimmune encephalomyelitis (EAE), the animal model of multiple sclerosis, have identified a critical chemokine-mediated mechanism of monocyte homing to the central nervous system (CNS). Here, we summarize the current findings in EAE, develop a rationale for targeting the chemokine axis in order to treat CNS inflammatory disease, and review currently available molecule-specific therapeutics that inhibit monocyte trafficking to the CNS.     

Stress preconditioning attenuates oxidative injury to the alveolar epithelium of the lung following haemorrhage in rats

Inhibition of cAMP-dependent stimulation of vectorial fluid transport across the alveolar epithelium following haemorrhagic shock is mediated by reactive nitrogen species released within the airspaces of the lung. We tested here the hypothesis that the prior activation of the cellular heat shock or stress response, via exposure to either heat or geldanamycin, would attenuate the release of airspace nitric oxide (NO) responsible for the shock-mediated failure of the alveolar epithelium to respond to catecholamines in rats. Rats were haemorrhaged to a mean arterial pressure of 30–35 mmHg for 60 min, and then resuscitated with a 4 % albumin solution. Alveolar fluid clearance was measured by change in concentration of a protein solution instilled into the airspaces 5 h after the onset of haemorrhage. Stress preconditioning restored the cAMP-mediated upregulation of alveolar liquid clearance after haemorrhage. The protective effect of stress preconditioning was mediated in part by a decrease in the expression of iNOS in the lung. Specifically, stress preconditioning decreased the production of nitrite by endotoxin-stimulated alveolar macrophages removed from haemorrhaged rats or by A549 and rat alveolar epithelial type II cell monolayers stimulated with cytomix (a mixture of TNF-α, IL-1β and IFN-γ) for 24 h. In summary, these results provide the first in vivo evidence that stress preconditioning restores a normal fluid transport capacity of the alveolar epithelium in the early phase following haemorrhagic shock by attenuating NO-mediated oxidative stress to the lung epithelium.     

Human alveolar macrophage and blood monocyte interleukin-6 production

Interleukin-6 (IL-6) modulates a number of processes relevant to host immunity and inflammation. We investigated the capacity of the human alveolar macrophage to elaborate IL-6 in response to lipopolysaccharide (LPS), recombinant interleukin-1 (rIL-1), and recombinant tumor necrosis factor (rTNF), and compared macrophage IL-6 production to that of blood monocytes and lung fibroblasts. Unstimulated and TNF-stimulated alveolar macrophages and monocytes produced little or no detectable IL-6. In contrast, macrophages and monocytes produced large amounts of IL-6 in response to LPS and monocytes produced lesser but readily detectable amounts in response to rIL-1. Monocytes and alveolar macrophages differed significantly in their capacity to produce IL-6, with macrophages making more IL-6 in response to LPS and less IL-6 in response to rIL-1 than autologous blood monocytes. Monocytes aged in vitro produced little detectable IL-6 in response to LPS or rIL-1, suggesting that differences in cell maturity may account for the diminished capacity of the alveolar macrophage to produce IL-6 in response to IL-1 but not its enhanced capacity to produce IL-6 in response to LPS. Mononuclear phagocytes and lung fibroblasts also differed in their ability to produce IL-6. Lung fibroblasts produced more IL-6 in response to rIL-1 and less IL-6 in response to LPS than monocytes and macrophages. In addition, monocytes and macrophages elaborated electrophoretically identical IL-6 moieties that differed from those produced by lung fibroblasts. These differences could be at least partially attributed to differences in sialylation and/or glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphofunctional characteristics of the alveolar epithelium and alveolar macrophages in chronic bronchitis

Quantitative histoenzymatic determination based on early autopsy material was introduced for evaluation of oxidoreductase and hydrolytic enzymes activity in alveolar macrophages (AM) and alveolar epithelial cells (AEC) of the lungs in chronic catarrhal (CC) and chronic purulent (CP) bronchitis with bronchopneumonia development in sites other than acute inflammatory lesions. AM hydrolase hypoactivity was recorded in both CC and CP bronchitis, whereas inhibition of dehydrogenase activity stood out in CP bronchitis along with disturbed activity of the key enzymes of energy and carbohydrate metabolism in AEC. Characteristic features were elucidated for relationships between the cells of the alveolar lining in various forms of chronic bronchitis. The findings suggest a conclusion on inhibited activity of AM in chronic bronchitis.     

Mechanisms of monocyte recruitment in vascular repair after injury

The inflammatory response to acute vessel wall injury has been increasingly recognized to play a decisive role in neointima formation. In particular, the exuberant infiltration with monocytes aggravates neointimal growth and can thereby promote restenosis. The adhesion of circulating monocytes to the site of mechanical injury represents the key event in monocyte recruitment, and this review highlights recent insights into the molecular mechanisms of monocyte adhesion throughout the course of neointimal growth. An acute and a chronic phase of monocyte recruitment after vascular injury can be discerned. The adhesion of platelets to the denuded subendothelial matrix is the hallmark of the acute phase providing an adhesive substrate for monocytes, whereas chronic monocyte recruitment is regulated by the interaction with neointimal smooth muscle cells and recovering endothelial cells. Clearly, the mechanisms of monocyte rolling and adhesion differ considerably between these diverse substrates. This review is particularly focused on the contribution of chemokines and adhesion molecules to monocyte recruitment to injured vessels according to the different stages of neointimal growth, and on closely related functions of the chemokine-like molecule macrophage migration inhibitory factor. Understanding the complex molecular interactions of the injured vessel wall with circulating monocytes may enable therapeutic targeting to prevent the development of restenosis.     

Recombinant interleukin 1 alpha and beta prime human monocyte superoxide production but have no effect on chemotaxis and oxidative burst response of neutrophils

We report that recombinant human IL 1 alpha and beta and the two synthetic short fragments 163-171 and 226-254 do not exhibit chemotactic activity for human peripheral blood neutrophils and monocytes. These products up to concentrations of 4 X 10(4) units/ml failed to show chemotactic activity. Furthermore, IL 1 alpha and beta failed to generate chemotactic factors from human serum. Recombinant IL 1 alpha, IL 1 beta, or IL 1 beta fragments 163-171 and 226-254 did not induce any superoxide response by monocytes or neutrophils. However, exposure of monocytes to recombinant IL 1 alpha or IL 1 beta resulted in enhanced generation of superoxide response following stimulation with PMA. No priming was observed in neutrophils. These results suggest that IL 1 alpha and beta are involved in regulation of monocyte oxidative burst response, but play no direct regulatory role on neutrophil function.     

Neuronal MCP-1 mediates microglia recruitment and neurodegeneration induced by the mild impairment of oxidative metabolism

Chemokines are implicated in the neuroinflammation of several chronic neurodegenerative disorders. However, the precise role of chemokines in neurodegeneration is unknown. Thiamine deficiency (TD) causes abnormal oxidative metabolism in the brain as well as a well-defined microglia activation and neurodegeneration in the submedial thalamus nucleus (SmTN), which are common features of neurodegenerative diseases. We evaluated the role of chemokines in neurodegeneration and the underlying mechanism in a TD model. Among the chemokines examined, TD selectively induced neuronal expression of monocyte chemoattractant protein-1 (MCP-1) in the SmTN prior to microglia activation and neurodegeneration. The conditioned medium collected from TD-induced neurons caused microglia activation. With a neuron/microglia co-culture system, we showed that MCP-1-induced neurotoxicity required the presence of microglia, and exogenous MCP-1 was able to activate microglia and stimulated microglia to produce cytokines. A MCP-1 neutralizing antibody inhibited MCP-1-induced microglia activation and neuronal death in culture and in the thalamus. MCP-1 knockout mice were resistant to TD-induced neuronal death in SmTN. TD selectively induced the accumulation of reactive oxygen species in neurons, and antioxidants blocked TD-induced MCP-1 expression. Together, our results indicated an induction of neuronal MCP-1 during mild impairment of oxidative metabolism caused by microglia recruitment/activation, which exacerbated neurodegeneration.     

Leptin stimulates the oxidative burst in control monocytes but attenuates the oxidative burst in monocytes from HIV-infected patients

Leptin, the 16 kDa product of the ob gene, is a an adipocyte-secreted hormone that centrally regulates weight. However, the physiological role of leptin is not limited to the regulation of food intake and energy expenditure, and leptin has a variety of effects in peripheral tissues, such as a regulatory role modulating the immune system. Thus, leptin receptor is expressed in human peripheral blood mononuclear cells, mediating the leptin stimulation of proliferation and activation, the production of proinflammatory cytokines from cultured monocytes, and the prevention of apoptotic death in serum-deprived monocytes. Because leptin can stimulate monocytes and the production of reactive oxygen species (ROS) are the result of monocyte activation, we investigated the effect of leptin on ROS production by human monocytes in vitro. Oxidative burst was measured by oxidation of the redox-sensitive dye 2',7'-dichlorofluorescein diacetate, and analysed by flow cytometry. We have found that stimulation with leptin produces oxygen radical formation by monocytes. This effect is dependent on the dose and maximal response is achieved at 10 nM leptin. Because HIV infection induces the production of ROS, we next investigated the effect of leptin on ROS production in monocytes from HIV-positive (HIV+) subjects. We have also found that monocytes from HIV+ subjects spontaneously produced increased amounts of free radicals. In contrast, leptin stimulation of monocytes from these patients partially inhibited the production of ROS. This effect of leptin was also dependent on the dose and maximal effect was achieved at 10 nM. The effect of leptin stimulating the production of ROS is consistent with the proinflammatory role in the immune system. On the other hand, the inhibitory effect on monocytes from HIV+ subjects may be explained by the attenuation of the oxidative burst by a delayed activation of monocytes in a hyperinflammatory state.     

Mannose inhibits the human neutrophil oxidative burst

Stimulated human neutrophils (PMNs) increase their oxygen consumption and secrete reactive oxygen species that are involved in bactericidal activity and inflammation. While studying lectin-mediated bacterial adherence, we observed that D-mannose appeared to inhibit PMN metabolism. Further studies showed that 100 mM mannose inhibited oxygen consumption by 82%, superoxide secretion by 84%, luminol-enhanced chemiluminescence (CL) by 98%, and hexose-monophosphate shunt activity by 100% when PMN were stimulated with 1 microM phorbol myristate acetate (PMA). Inhibition was also seen with 0.1 microM formyl-methionyl-leucyl-phenylalanine (fMLP), and 0.1 microM A23187, reagents thought to stimulate the respiratory burst by different transductional mechanisms. Inhibition was dose-responsive and specific since 100 mM D-galactose, alpha-D-glucose, or alpha-L-fucose only minimally affected PMN oxidative metabolism. Inhibition of PMA-induced superoxide production was seen almost immediately upon the addition of 50 mM mannose and was reversed by washing. Neutrophils remained viable as measured by trypan blue exclusion. These data suggest that mannose inhibits the neutrophil oxidative burst at the level of the hexose monophosphate shunt. Further investigation should elucidate the specific mechanism(s) of this burst inhibition as well as define uses for it as a tool to study oxidative as well as nonoxidative killing by PMN.     

cAMP and β-adrenergic stimulation of rat alveolar epithelium

The absorption of fluid (bicarbonate-buffered Ringer with 10 mmol/l glucose) instilled into rat lung is a Na⁺-coupled process that takes place through two apical transport systems: an amiloride-sensitive Na⁺ transport and a Na⁺-glucose co-transport [5]. Fluid absorption in isolated, perfused rat lungs and the permeability to³H-mannitol of alveolar epithelium were studied in control conditions and during stimulation of the alveolar epithelium by cAMP or isoproterenol. cAMP led to a threefold increase in the rate of fluid absorption and to an increase in the paracellular permeability. A similar response was found following \-adrenergic stimulation obtained with isoproterenol in the perfusate. The increase in fluid transport was due to enhancement of the amiloride-sensitive component of Na⁺ transport. The Na⁺-glucose co-transport which accounts for about 60% of fluid absorption in control conditions was depressed, possibly as a consequence of a depolarization of the apical alveolar cell membrane. Fluid absorption was reduced by 40% by apical amiloride (10^–4 mol/l) in control lungs and to an even larger extent in isoproterenol-stimulated lungs; it was completely abolished by amiloride in cAMP stimulated lungs. Since the Na⁺-glucose co-transport was still operative, this suggests that a secretory process was triggered. This was confirmed in experiments in which both kinds of transport were inhibited with a combination of amiloride and glucose-free Ringer. In these conditions fluid balance was zero in unstimulated lungs whilst fluid entry into alveoli was observed in isoproterenol and cAMP stimulated lungs.     

Differential recruitment of accessory molecules by FcgammaRI during monocyte differentiation

Aggregation of the human high-affinity receptor for immunoglobulin G, FcgammaRI, results in initiation of intracellular signaling cascades. However, as the receptor contains no known signaling motif, it is required to recruit an accessory molecule. The gamma chain has been proposed to fulfil this role. Here, we show that in U937 cells differentiated to a more macrophage-like phenotype with dibutyryl cAMP, FcgammaRI no longer signals through the gamma chain but rather uses FcgammaRIIa to initiate tyrosine phosphorylation. Expression of the gamma chain is, however, increased in the dbcAMP-induced cells, but here the gamma chain specifically associates with the IgA receptor, FcalphaRI. Recruitment of the gamma chain either by FcgammaRI in cytokine-primed cells or by FcalphaRI in dbcAMP-induced cells couples ligand binding to the activation of phosphatidyl choline-specific phospholipase D.     

N-acetylcysteine inhibits human neutrophil and monocyte chemotaxis and oxidative metabolism

The effect of N-acetylcysteine (NAC) on human neutrophil and monocyte cell viability, chemotaxis, oxygen consumption and chemiluminescence was studied. It was found that NAC at concentrations higher than 3 X 10(-2) M resulted in neutrophil and monocyte cytotoxicity. The studies on the effect of NAC on neutrophil and monocyte chemotaxis showed that NAC inhibited chemotaxis of both cell types in a concentration dependent manner. NAC at 3 X 10(-2) M inhibited chemotaxis of both cell types by about 50% and at 10(-1) M inhibited PMN chemotaxis by 95% and MNL chemotaxis by 85%. The studies on the effect of NAC on neutrophil chemiluminescence demonstrated that NAC at concentrations of 1.5 X 10(-2) M, or higher, inhibited the response of the activated cells totally. When pH adjusted NAC or Mucomyst was used the inhibition was observed with higher concentrations of the drug (1.5 X 10(-1) M). NAC exhibited a similar pattern of inhibition on monocyte chemiluminescence response. These findings demonstrate that NAC, at concentrations obtainable in vivo by inhalation, impairs the chemotaxis and generation of oxygen radicals by human phagocytic cells. This property of NAC could have important implications concerning the prevention of tissue damage caused by these cells in inflammatory areas.     

A pore transport model for pulmonary alveolar epithelium

Hydrodynamic heteropore flow models for transport of solutes across alveolar epithelial tissue have been developed. A two-size cylindrical pore model and a similar parallel-plate model were formulated, tested and used to predict effective pore sizes from literature data on transport in bullfrog, canine and rat lungs. The best fit equivalent pore-size estimates were obtained using a modified, nonlinear least squares procedure, with alveolar surface area to volume ratio (S/V) and small-pore area fraction of total pore area as parameters. Small-pore and large-pore width estimates of 4 nm (84% of total flow area) and 10 nm, respectively, with an average deviation of 20% from experimentally derived permeabilities were obtained from the bullfrog alveolar epithelium parallel-plate pore model (13 solutes, diameters 0.3 to 2.8 nm). The equivalent cylindrical pore model diameter estimates were 5 nm and 10 nm, with small-pore area fraction and percentage deviations similar to the parallel-plate model estimates. Eighty-eight percent of the bulk water driven by a sucrose osmotic gradient was predicted to be transported through the small pores. The rat alveolus parallel-plate pore model (6 solutes) yielded small-pore and large-pore widths of 0.4 nm and 50 nm, respectively. Clearance rate-constant data for dextran macromolecules (3,000 to 250,000 Daltons), using a single parallel-plate pore model, resulted in a pore width estimate of 98 nm for canine alveoli with an average deviation of the predicted rate constants of 18% from literature experimental values. In all cases tested, the parallel-plate pore model predicted lower small-pore size estimates than did the cylindrical pore model, and both models had appreciably smaller percentage deviations from experimental data than previous models.     

Increased chemoattractant induced neutrophil oxidative burst, accelerated apoptosis, and dysregulated tyrosine phosphorylation associated with lifelong bacterial infections

A boy with lifelong recurrent bacterial infection at cutaneous and mucosal sites was investigated. PMN oxidative burst to phorbol myristate acetate (PMA) and zymosan was normal but was increased 20- to 50-fold upon C5a or formyl-met-leu-phe (fMLP) chemoattractant stimulation, accompanied by accelerated PMN apoptosis. His PMNs showed increased constitutive tyrosine phosphorylation of 21-, 25-, and 44-kDa proteins, and of src-family kinases (p59(hck), p58(fgr), and p53/56(lyn)). Phosphorylation was abnormally enhanced following fMLP stimulation. Expression and activity of the major PMN tyrosine phosphatases, i.e., CD45, CD148, and SHP-1 and -2, was normal. However, dephosphorylation of phospho-p58(fgr) and phospho-p53/56(lyn) by lysates of patient's PMNs was enhanced. Thus, another phosphatase may be overactive, perhaps dephosphorylating a regulatory (inhibitory) site on a protein tyrosine kinase, accounting for the abnormal PMN tyrosine phosphorylation and function. With age (now 13 years), T-cell lymphopenia and loss of T-cell responses developed. This appears to be a unique primary immunodeficiency with abnormal PMN oxidative and apoptotic responses to chemoattractants, dysregulated protein tyrosine phosphorylation, serious bacterial infection, and T-lymphocyte attrition.