Suppression or enhancement by superoxide dismutase of tumor cell killing by macrophages of normal donors and breast cancer patients

This study was designed to examine the role superoxide production by macrophages plays in tumor killing. When superoxide dismutase was added to the normal macrophage-tumor cell (MA-160 cell line) suspensions macrophage mediated tumor cytotoxicity was suppressed. In contrast, superoxide dismutase often greatly enhances the cytotoxic ability of macrophages from breast cancer patients. To determine whether the inability of breast cancer patients' macrophages to kill tumor cells might be related to decreased levels of O2- production, we measured the amount of O2- generated by both normal and breast cancer patients' macrophages. The macrophages obtained from the normal donors produced 46.0 +/- 6.7 nanomoles O2-/10(6) macrophages per 10 minutes (mean +/- S.E. of 9 donors). In contrast, macrophages from the four breast cancer patients who possessed cytotoxic macrophages produced 56.4 +/- 5.2 nanomoles O2-/10(6) macrophages per 10 minutes whereas those from the 6 breast cancer patients who possessed non-cytotoxic macrophages produced 18.6 +/- 3.2 nanomoles O2-/10(6) macrophages per 10 minutes. Thus, it appears that there is a relationship between macrophage cytotoxicity and the level of O2- produced. Furthermore, it is possible that the inability to generate sufficient amounts of O2- might explain the inability of breast cancer patients' macrophages to kill tumor cells in some cases.     

Deficiency of vitamin E in the alveolar fluid of cigarette smokers. Influence on alveolar macrophage cytotoxicity.

Cigarette smoking produces oxidant-mediated changes in the lung important to the pathogenesis of emphysema. Since vitamin E can neutralize reactive oxygen species and prevent peroxidation of unsaturated lipids, it may constitute an important component of the lung's defense against oxidant injury. To better characterize the antioxidant protective role of vitamin E, young asymptomatic smokers and nonsmokers were evaluated by bronchoalveolar lavage before and immediately after a 3-wk course of oral vitamin E (2,400 IU/d). Smoker alveolar fluid at baseline was relatively deficient in vitamin E compared with nonsmoker fluid (3.1 +/- 0.7 ng/ml vs. 20.7 +/- 2.4 ng/ml, P less than 0.005). Although smoker alveolar fluid vitamin E levels increased to 9.3 +/- 2.3 ng/ml after supplementation, the levels remained significantly lower than nonsmoker baseline levels (P less than 0.01). This deficiency was explained, in part, by the increased oxidative metabolism of vitamin E to the quinone form in the lungs of smokers compared with nonsmokers. Although the significance of a lower concentration of alveolar fluid vitamin E is unclear, it may compromise the antioxidant protection afforded by the alveolar fluid as it coats the lung's epithelial surface. The protective role of vitamin E was assessed by cytotoxicity experiments, which demonstrated that the killing of normal rat lung parenchymal cells by smoker alveolar macrophages was inversely related to the vitamin E content of the parenchymal cells. These findings suggest that vitamin E may be an important lower respiratory tract antioxidant, and that the deficiency seen in young smokers may predispose them to an enhanced oxidant attack on their lung parenchymal cells.     

Importance of hydrogen peroxide in nitrofurantoin-induced cytotoxicity: evidence from an inbred catalase-deficient strain of mice

Significant pulmonary toxicity is associated with the use of nitrofurantoin; however, the mechanism of cellular toxicity remains poorly characterized. By using a novel in vitro red blood cell (RBC) chromium 51 cytotoxicity assay, cell injury induced by nitrofurantoin was quantified with normocatalasemic BALB/c RBCs and hypocatalasemic (but otherwise genetically identical) CCN RBCs as target cell populations. Nitrofurantoin at concentrations of 2 x 10(-4) and 4 x 10(-4) mol/L resulted in significant injury to normocatalasemic RBCs with a cytotoxic index (CI) of 21.7% +/- 3.7% and 65.3% +/- 3.7% (p less than 0.05, both comparisons). This injury was substantially increased when nitrofurantoin (2 x 10(-4) and 4 x 10(-4) mol/L was incubated with hypocatalasemic RBCs, resulting in CIs of 59.0% +/- 7.4% and 91.0% +/- 2.0% respectively (p less than 0.05, both comparisons with normocatalasemic RBCs). Direct oxidant-mediated cytotoxicity induced by either H2O2 or the superoxide anion radical (as generated by xanthine-xanthine oxidase) also resulted in more significant injury to hypocatalasemic RBCs than to normocatalasemic RBCs (p less than 0.05, both comparisons). Catalase levels of CCN RBCs were approximately 7% of control BALB/c RBC values; however, the activities of superoxide dismutase and glutathione peroxidase were identical in both populations of RBCs. This model, using genetically defined target cell populations, clearly demonstrates the importance of endogenous catalase in protecting against nitrofurantoin-induced cytotoxicity, suggesting that H2O2 is a critical intermediary in the direct cell injury mediated by the drug.     

Oxidants spontaneously released by alveolar macrophages of cigarette smokers can inactivate the active site of alpha 1-antitrypsin, rendering it ineffective as an inhibitor of neutrophil elastase.

Current concepts relating to the pathogenesis of emphysema associated with cigarette smoking is that an imbalance exists within the lower respiratory tract between neutrophil elastase and the local anti-neutrophil elastase screen, enabling uninhibited neutrophil elastase to destroy the alveolar structures over time. The possible role of alveolar macrophages in contributing to this imbalance was investigated by evaluating the ability of cigarette smokers' alveolar macrophages to inactivate alpha 1-antitrypsin (alpha 1AT), the major anti-neutrophil elastase of the human lower respiratory tract. In vitro, alveolar macrophages of smokers spontaneously released 2.5-fold more superoxide anion and eightfold more H2O2 than macrophages of nonsmokers (P less than 0.01, both comparisons). Using a model system that reproduced the relative amounts of alveolar macrophages and alpha 1AT found in the epithelial lining fluid of the lower respiratory tract, we observed that smokers' macrophages caused a 60 +/- 5% reduction in the ability of alpha 1AT to inhibit neutrophil elastase. In marked contrast, under the same conditions, nonsmokers' macrophages had no effect upon the anti-neutrophil elastase function of alpha 1AT. Addition of superoxide dismutase, catalase, mannitol, and methionine prevented inactivation of alpha 1AT by smokers' macrophages, implying that the release of oxidants mediated the inactivation of alpha 1AT. In addition, by utilizing a recombinant DNA produced modified form of alpha 1AT containing an active site substitution (met358----val), the inactivation of alpha 1AT by smokers' alveolar macrophages was prevented, suggesting that the smokers' macrophages inactivate alpha 1AT by oxidizing the active site of the alpha 1AT molecule. These results suggest that in cigarette smokers, the alveolar macrophage can modulate the activity of alpha 1AT as an inhibitor of neutrophil elastase and thus play a role in the pathogenesis of emphysema associated with cigarette smoking.     

Risk factors for emphysema. Cigarette smoking is associated with a reduction in the association rate constant of lung alpha 1-antitrypsin for neutrophil elastase.

The increased risk of developing emphysema among individuals who smoke cigarettes and who have normal levels of alpha 1-antitrypsin (alpha 1AT) is hypothesized to result from a decrease in the antineutrophil elastase capacity of the lower respiratory tract alpha 1AT of smokers compared with nonsmokers. To evaluate this hypothesis we compared the time-dependent kinetics of the inhibition of neutrophil elastase by lung alpha 1AT from healthy, young cigarette smokers (n = 8) and nonsmokers (n = 12). alpha 1-antitrypsin was purified from lavage fluid using affinity and molecular sieve chromatography, and the association rate constant (k assoc) for neutrophil elastase quantified. The k assoc of smoker plasma alpha 1AT (9.5 +/- 0.5 X 10(6) M-1s-1) was similar to that of nonsmoker plasma (9.3 +/- 0.7 X 10(6) M-1s-1, P greater than 0.5). In marked contrast, the k assoc of smoker lower respiratory tract alpha 1AT was significantly lower than that of nonsmoker alpha 1AT (6.5 +/- 0.4 X 10(6) M-1s-1 vs. 8.1 +/- 0.5 X 10(6) M-1s-1, P less than 0.01). Furthermore, the smoker lower respiratory tract alpha 1AT k assoc was significantly less than that of autologous plasma (P less than 0.01). When considered in the context of the concentration of alpha 1AT in the lower respiratory tract epithelial lining fluid, the inhibition time for neutrophil elastase of smoker lung alpha 1AT was twofold greater than that of nonsmoker lung alpha 1AT (smoker: 0.34 +/- 0.05 s vs. nonsmoker: 0.17 +/- 0.05 s, P less than 0.01). Consequently, for concentrations of alpha 1AT in the lower respiratory tract it takes twice as long for an equivalent amount of neutrophil elastase to be inhibited in the smoker's lung compared with the nonsmoker's lung. These observations support the concept that cigarette smoking is associated with a decrease in the lower respiratory tract neutrophil elastase inhibitory capacity, thus increasing the vulnerability of the lung to elastolytic destruction and thereby increasing the risk for the development of emphysema.     

Bacillus Calmette-Guérin-activated murine macrophages kill syngeneic melanoma cells under strict anaerobic conditions

We have studied the spontaneous killing of B5(59) melanoma cells by Bacillus Calmette-Guerin (BCG)-elicited macrophages under strictly anaerobic conditions to investigate the role of oxygen in macrophage- mediated cytotoxicity. The number of melanoma cells capable of forming colonies after aerobic or anaerobic incubation with BCG-macrophages was used as the index of cytotoxicity. The BCG-macrophages killed melanoma cells regardless of the amount of oxygen present. The killing observed was proportional to the ratio of effector cells added; a ratio of 25:1 effector to target cells was required to achieve nearly 90% cytotoxicity both aerobically and anaerobically. This cytotoxicity was not dependent on a diffusible macrophage product nor on alteration of the medium by macrophages, since tumor cells incubated in the same culture medium, but not in contact with a mixed population of tumor cells and macrophages, were not killed. These results also indicated that macrophage-mediated cytotoxicity was dependent on macrophage-tumor cell contact. The mechanism responsible for the oxygen-independent cytotoxicity is unknown at present.     

Lung derived surface active material (SAM) inhibits natural killer cell tumor cytotoxicity

Natural killer cells have been identified in lung tissue but have been found to have significantly less tumor cytotoxicity than natural killer cells found elsewhere in the body. The natural killer cells in the lung are still functional, since their killing can be enhanced by Interleukin-2. The surface active material (SAM) of lung lining fluid has been shown to have immunomodulating activity, including the suppression of lymphocyte blast transformation and enhancement of macrophage tumor cytotoxicity. We studied the effect of SAM purified from lung lining fluid on natural killer cell tumor cytotoxicity. SAM markedly inhibited tumor killing (Percent cytotoxicity of cells alone: 41 +/- 7.3% (mean +/- SEM); cells plus SAM: 10 +/- 9.7%, p less than 0.02). Further studies demonstrated that some phospholipids, including phosphatidylcholine, the major phospholipid of SAM, also significantly inhibited natural killer cell tumor killing. This inhibition of natural killer cell tumor cytotoxicity could be reversed by the addition of Interleukin-2 to the natural killer cells. These studies demonstrated that the surfactant found in lung lining fluid significantly inhibited natural killer cell tumor cytotoxicity and this effect could be reversed by Interleukin-2.     

GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-targeted mice (GM-/-) cleared group B streptococcus (GBS) from the lungs more slowly than wild-type mice. Expression of GM-CSF in the respiratory epithelium of GM-/- mice improved bacterial clearance to levels greater than that in wild-type GM+/+ mice. Acute aerosolization of GM-CSF to GM+/+ mice significantly enhanced clearance of GBS at 24 hours. GBS infection was associated with increased neutrophilic infiltration in lungs of GM-/- mice, while macrophage infiltrates predominated in wild-type mice, suggesting an abnormality in macrophage clearance of bacteria in the absence of GM-CSF. While phagocytosis of GBS was unaltered, production of superoxide radicals and hydrogen peroxide was markedly deficient in macrophages from GM-/- mice. Lipid peroxidation, assessed by measuring the isoprostane 8-iso-PGF2alpha, was decreased in the lungs of GM-/- mice. GM-CSF plays an important role in GBS clearance in vivo, mediated in part by its role in enhancing superoxide and hydrogen peroxide production and bacterial killing by alveolar macrophages.     

The interaction of asbestos and neutrophils injures cultured human pulmonary epithelial cells: role of hydrogen peroxide

Asbestos exposure causes diffuse interstitial pulmonary fibrosis. Since alveolar epithelial cell injury is hypothesized to precede the fibrotic response in asbestosis, we investigated whether asbestos, either alone or in conjunction with neutrophils (PMNs), injured cultured human pulmonary epithelial cells (HPECs). HPEC cytotoxicity was assessed with a standard 51chromium release assay after a 16-hour incubation with asbestos and PMNs. Negligible HPEC cytotoxicity was observed after incubation with either amosite asbestos (500 micrograms/ml) or PMNs alone in serum-free media. However, incubation with both asbestos and PMNs caused significant HPEC injury, which was asbestos dose-dependent; causing 25% +/- 4% detachment and 52% +/- 8% 51chromium release with 500 micrograms/ml asbestos. The cytotoxic effects of asbestos plus PMNs were nearly completely attenuated with serum (20%) or catalase (100 micrograms/ml) but were not prevented with scavengers of superoxide anion, hydroxyl radical, or hypochlorous acid. A role for hydrogen peroxide (H2O2) in mediating HPEC injury was also suggested by the demonstration of asbestos-induced generation of H2O2 by PMNs. Furthermore, H2O2 alone (10(-4)mol/L) caused significant HPEC damage. Intimate contact between asbestos-activated PMNs and HPECs was a necessary requirement for PMN-mediated HPEC cytotoxicity. These data suggest that pulmonary epithelial cell injury is mediated in part by H2O2 release from asbestos-activated PMNs as well as intimate contact between the epithelial cell, PMNs, and asbestos.     

Reduced alveolar macrophage production of tumor necrosis factor during sepsis in mice and men

BACKGROUND AND METHODS: Tumor necrosis factor (TNF) has been implicated as a major humoral mediator of sepsis and endotoxin shock. TNF is secreted by cells of the reticuloendothelial system, including alveolar macrophages. Alveolar macrophage TNF production has been postulated to play a pathogenetic role in the development of adult respiratory distress syndrome (ARDS) in sepsis. To evaluate alveolar macrophage production of TNF during sepsis and endotoxin shock, we studied the effects of sepsis and/or in vivo lipopolysaccharide on the in vitro production of TNF by pulmonary alveolar macrophages. Human pulmonary alveolar macrophages were obtained by bronchoalveolar lavage from six septic and five nonseptic patients, cultured in the presence or absence of lipopolysaccharide (1 ng/mL), and assayed for TNF activity in a bioassay using fibroblast lysis. A murine model of sepsis was also utilized to study pulmonary alveolar macrophage TNF production under more controlled conditions. Normal mice were given ip injections of either lipopolysaccharide or saline. After 2 hrs, pulmonary alveolar macrophages were obtained and cultured in saline or various concentrations of lipopolysaccharide (0.001 to 10 micrograms/mL). RESULTS: There was no difference in baseline TNF activity, expressed as per cent lysis at 1:10 dilution, between pulmonary alveolar macrophages from control and septic patients (35.7 +/- 5.5% vs. 24.4 +/- 9.3%, respectively) (p greater than .05). However, when stimulated with lipopolysaccharide in vitro, the pulmonary alveolar macrophages from nonseptic patients produced significantly (p less than .01) more TNF (82.8 +/- 3.6%) than did pulmonary alveolar macrophages from patients with the septic syndrome (35.2 +/- 3.8%). Similar findings were obtained using the murine sepsis model. The baseline TNF activity in pulmonary alveolar macrophages from control mice was 22.9 +/- 7.0% (mean +/- SEM) and from lipopolysaccharide-injected mice was 26.8 +/- 3.3% (p greater than .05). Stimulation with 1 ng/mL lipopolysaccharide in vitro produced an increase in TNF activity in both groups, but the increase was greater in the control mice (68.1 +/- 5.7%) than in the lipopolysaccharide-injected mice (47.5 +/- 5.3%) (p less than .01). When the murine pulmonary alveolar macrophages were stimulated with higher concentrations of lipopolysaccharide (0.1 to 10 micrograms/mL), pulmonary alveolar macrophages from lipopolysaccharide-injected mice produced less than 25.5% of the TNF produced by pulmonary alveolar macrophages from control mice. CONCLUSIONS: These studies indicate that sepsis and endotoxin injection result in a rapid decrease in the ability of pulmonary alveolar macrophages from both humans and mice to produce and secrete TNF in response to lipopolysaccharide. We speculate that a downregulation of TNF production or of macrophage responsiveness to lipopolysaccharide has occurred. These results suggest that sustained TNF production by macrophages is not required for lung injury in sepsis.     

Superoxide production by phagocytic leukocytes

Mononuclear phagocytic leukocytes, as well as polymorphonuclear leukocytes, produce and release superoxide at rest, and this is stimulated by phagocytosis. Of the mouse monocytic cells studied, alveolar macrophages released the largest amounts of superoxide during phagocytosis, followed by normal peritoneal macrophages. Casein- elicited and "activated" macrophages released smaller quantities. In the guinea pig, polymorphonuclear leukocytes and casein-elicited macrophages were shown to release superoxide during phagocytosis whereas alveolar macrophages did not. Superoxide release accounted for only a small fraction of the respiratory burst of phagocytosis in all but the normal mouse peritoneal macrophage, the guinea pig polymorphonuclear leukocyte, and probably the mouse alveolar macrophage. There are obviously considerable species differences in O2- release by various leukocytes that might reflect both the production and/or destruction (e.g. by dismutase) of that substance.     

The immunotherapeutic potential of activated canine alveolar macrophages and antitumor monoclonal antibodies in metastatic canine melanoma.

A variety of immune cell activators can enhance the cytotoxic effects of monocytes/macrophages including interferon-gamma (IFN-gamma) and muramyl peptides, which are under investigation for cancer therapy in humans and dogs. Pulmonary alveolar macrophages (PAMs) in particular, are strategically located within the lung and provide a potential defense against cancer cells metastatic to the lung. For this reason, we examined the in vitro cytotoxic potential of fresh and IFN-gamma-activated PAMs from normal dogs targeted to canine malignant melanoma cells with antiganglioside monoclonal antibodies (mAbs). Antiganglioside mAbs 14.G2a (anti-GD2) and R24 (anti-GD3), both in clinical trials for human neuroectodermal tumors including melanoma, significantly enhanced the cytotoxicity of canine melanoma mediated by canine PAMs. Further, the cytotoxicity mediated by recombinant canine IFN-gamma-activated canine PAMs, in combination with anti-GD2 ganglioside mAb 14.G2a, enhanced melanoma cytotoxicity above that seen with mAb 14.G2a alone. This documentation of antibody-dependent cellular cytotoxicity mediated by activated PAMs suggests that activation and targeting of resident pulmonary immune cells be pursued as a means to control pulmonary metastases.     

Cytotoxicity of human macrophages for tumor cells. Enhancement by human lymphocyte mediators.

Human macrophages, derived from peripheral blood monocytes, acquire enhanced cytotoxicity for human target cells after incubation in mediator-rich supernates from antigen-stimulated lymphocytes. Maximum cytotoxicity was observed after 24-h incubation in mediators. In comparison to normal macrophages, mediator-activated macrophages were cytotoxic to five of the six malignant cell lines tested but had no effect on five nonmalignant cell lines. In 20 experiments with one target (SK-BR-3), mean cytotoxicity was 23 +/- 2.7% and with another target (MA-160), was 29 +/- 3.4%. Macrophages became cytotoxic after 8-h incubation with mediators and the enhanced cytotoxicity persisted for at least 40 h after the lymphocyte mediators were removed. These findings are consistent with the hypothesis that macrophages, activated by antigen-induced lymphocyte mediators, can contribute to the host resistance to tumor growth in man.     

Specificity of opsonic antibodies to enhance phagocytosis of Pseudomonas aeruginosa by human alveolar macrophages.

These studies compared the ability of specific secretory IgA (sIgA) and IgG antibodies to promote phagocytosis of viable pseudomonas aeruginosa by human alveolar macrophages. Macrophages were obtained by lung lavage of normal adult smoker and nonsmoker volunteers and were maintained as in vitro cell monolayers. Both immune sIgA and IgG agglutinating antibodies were demonstrated to coat and opsonize viable bacteria, whereas similar nonimmune immunoglobulin preparations did not. When alveolar macrophages were challenged with viable opsonized 14C-labeled Pseudomonas IgG-reacted bacteria were ingested better and killed more readily than sIgA-opsonized organisms. Phagocytic responses were not significantly different between macrophages obtained from smokers and nonsmokers. Although sIgA and IgG antibodies can be found in respiratory secretions and both are undoubtedly important in pulmonary host defense, IgG opsonic antibody was superior in enhancing the uptake of Pseudomonas by in vitro-cultured alveolar macrophages. It may be the more important respiratory antibody for certain bacterial infections.     

Different mitogen-activated protein kinase-dependent cytokine responses in cells of the monocyte lineage

Macrophages release cytokines that may contribute to the chronic inflammation observed in pulmonary conditions such as asthma and chronic obstructive pulmonary disease. Thus, inhibition of macrophage cytokine production may have a therapeutic benefit. Human lung macrophages are a rich source of the proinflammatory cytokines, tumor necrosis factor (TNF)-alpha, granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-6, and IL-8, that are elevated in the bronchoalveolar lavage and sputum of subjects with respiratory diseases. Cytokine production from both monocytes and macrophages is mediated by mitogen-activated protein kinase (MAPK) pathways. This study compared the effects of a novel p38 MAPK inhibitor, N-cyano-N'-(2-{[8-(2,6-difluorophenyl)-4-(4-fluoro-2-methylphenyl)-7-oxo-7,8-dihydropyrido[2,3-d]pyrimidin-2-yl]amino}ethyl)-guanidine (PCG), and an extracellular signal-regulated kinase (ERK) pathway inhibitor, 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD098059), on cytokine release from lipopolysaccharide (LPS)-stimulated human monocytes, monocyte-derived macrophages (MDM), and lung macrophages. Lung macrophages, MDM, and monocytes were stimulated with LPS, and cytokine release was measured by enzyme-linked immunosorbent assay. Immunoblots were performed to confirm p38 and ERK1/2 MAPK expression and activity. PCG inhibited TNF-alpha release more effectively from monocytes compared with MDM or macrophages (maximal inhibition was 99.3 +/- 1.4, 62.7 +/- 4.3, and 58.6 +/- 6.6%, respectively; n = 7-9). PD098059 was less effective at suppressing TNF-alpha release from monocytes compared with MDM and lung macrophages (maximal inhibition was 37.4 +/- 2.8, 70.1 +/- 4.5, and 68.7 +/- 5.1%, respectively; n = 7-9). The pattern of GM-CSF, IL-6, and IL-8 release was comparable with that of TNF-alpha. These data suggest a differential involvement for each of these MAPK pathways in macrophage cytokine production compared with monocytes.     

Effect of neuropeptides on macrophage mediated cytotoxicity in normal donors and cancer patients

The role of enkephalins, endorphins and other neuropeptides produced by the nervous system in the alteration of immune responsiveness is generally unknown. The present studies were undertaken to investigate the role of these neuropeptides in the modulation of cytotoxicity induced by LPS activated macrophages obtained from normal donors as well as breast cancer and Hodgkins disease patients. When the macrophages from normal donors were pretreated with these neuropeptides for 1 hr prior to co-culturing with target cells, macrophage mediated cytotoxicity was enhanced with 10(-6) M and 10(-8) M of [met]-enkephalin, 10(-6) M of [leu]-enkephalin and 10(-6) M and 10(-12) M of alpha-endorphin. However, when the macrophages were co-cultured with target cells in the presence of the neuropeptides, it was observed that 10(-6) M and 10(-12) M of alpha-endorphin enhanced cytotoxicity whereas no enhancement in cytotoxicity was observed when [met]-enkephalin or [leu]-enkephalin were added to the cultures. In fact, it appears that 10(-10) M of [met]-enkephalin and 10(-12) M of [leu]-enkephalin actually suppressed macrophage mediated cytotoxicity. When the opioid antagonist Naloxone was incubated with the neuropeptides in the presence of the macrophages the enhancement of macrophage killing produced by [met]-enkephalin, [leu]-enkephalin or alpha-endorphin was suppressed. When the breast cancer patients' macrophages were pretreated with these neuropeptides, enhancement in cytotoxicity was observed at 10(-10) M of [met]-enkephalin, and [leu]-enkephalin and at 10(-8) M and 10(-12) M of alpha-endorphin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alveolar epithelial cell chemokine expression triggered by antigen-specific cytolytic CD8(+) T cell recognition

CD8(+) T lymphocyte responses are a critical arm of the immune response to respiratory virus infection and may play a role in the pathogenesis of interstitial lung disease. We have shown that CD8(+) T cells induce significant lung injury in the absence of virus infection by adoptive transfer into mice with alveolar expression of a viral transgene. The injury is characterized by the parenchymal infiltration of host cells, primarily macrophages, which correlates with physiologic deficits in transgenic animals. CD8(+) T cell-mediated lung injury can occur in the absence of perforin and Fas expression as long as TNF-alpha is available. Here, we show that the effect of TNF-alpha expressed by CD8(+) T cells is mediated not exclusively by cytotoxicity, but also through the activation of alveolar target cells and their expression of inflammatory mediators. CD8(+) T cell recognition of alveolar cells in vitro triggered monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) expression in the targets, which was mediated by TNF-alpha. Antigen-dependent alveolar MCP-1 expression was observed in vivo as early as 3 hours after CD8(+) T cell transfer and depended upon TNF-R1 expression in transgenic recipients. MCP-1 neutralization significantly reduced parenchymal infiltration after T cell transfer. We conclude that alveolar epithelial cells actively participate in the inflammation and lung injury associated with CD8(+) T cell recognition of alveolar antigens.     

Effect of short-term enteral feeding with eicosapentaenoic and gamma-linolenic acids on alveolar macrophage eicosanoid synthesis and bactericidal function in rats.

OBJECTIVES: Because vasoactive eicosanoids derived from arachidonic acid present in immune cell phospholipids promote lung inflammation in critically ill patients, novel experimental diets containing eicosapentaenoic acid from fish oil and gamma-linolenic acid from borage oil have been designed to limit arachidonic acid metabolism. However, excess dietary eicosapentaenoic acid impairs superoxide formation and bacterial killing by immune cells. The present study determined whether short-term enteral feeding with diets enriched with either eicosapentaenoic acid alone or in combination with gamma-linolenic acid would modulate alveolar macrophage eicosanoid synthesis without compromising bactericidal function. DESIGN: Prospective, randomized, controlled, blinded study. SETTING: University medical center. SUBJECTS: Adult male Sprague-Dawley rats. INTERVENTIONS: Rats underwent surgical placement of a gastroduodenal feeding catheter and were randomly assigned to receive one of three high-fat (55.2% of total calories), low-carbohydrate diets containing isocaloric amounts of lipids for 4 days. The control diet was enriched with linoleic acid, whereas the two test diets were low in linoleic acid and enriched with either 5 mole % eicosapentaenoic acid alone or in combination with 5 mole % gamma-linolenic acid. Alveolar macrophages were then procured to assess phospholipid fatty acid composition, eicosanoid synthesis after stimulation with endotoxin, superoxide formation and phagocytosis by flow cytometry, and killing of Staphylococcus aureus MEASUREMENTS AND MAIN RESULTS: Alveolar macrophage levels of arachidonic acid were significantly (p < .01) lower and levels of eicosapentaenoic and dihomo-gamma-linolenic acids were higher after feeding the eicosapentaenoic and gamma-linolenic acid diet vs. the linoleic acid diet. Ratios of thromboxane B2,/B3, leukotriene B4/B5, and prostaglandin E2/E1 were reduced in the macrophages from rats given either the eicosapentaenoic acid or eicosapentaenoic acid with gamma-linolenic acid diet compared with ratios from rats given the linoleic acid diet. Macrophages from rats given the eicosapentaenoic with gamma-linolenic acid diet released 35% or 24% more prostaglandin E1 than macrophages from rats given either the linoleic acid or the eicosapentaenoic acid diet, respectively. Macrophage superoxide generation, phagocytosis of opsonized zymosan, and killing of S. aureus were similar irrespective of dietary treatment. CONCLUSION: Short-term enteral feeding with an eicosapentaenoic acid-enriched or eicosapentaenoic with gamma-linolenic acid-enriched diet rapidly modulated the fatty acid composition of alveolar macrophage phospholipids, promoted a shift toward formation of less inflammatory eicosanoids by stimulated macrophages, but did not impair alveolar macrophage bactericidal function relative to responses observed after feeding a linoleic acid diet.     

Cytotoxic effects of polymorphonuclear leukocytes and macrophages in patients undergoing lymph depletion via thoracic duct drainage

It has been previously reported that both human peripheral blood monocyte derived macrophages and polymorphonuclear leukocytes acquire enhanced cytotoxicity for tumor cells. Lymphocyte depletion by thoracic duct cannulation prior to renal transplantation has been shown to suppress allograft rejection. However, the effects of thoracic duct drainage on macrophage and polymorphonuclear leukocyte function is not known. When the macrophages obtained from thoracic duct drainage patients were studied prior to cannulation, four of the five patients possessed cytotoxic macrophages. At 1 to 2 weeks post thoracic duct drainage, cytotoxicity was significantly depressed whereas by 3 weeks post thoracic duct cannulation most of the patients' macrophages exhibited maximal cytotoxicity. Approximately 3 1/2 weeks after cannulation these five patients received cadaveric renal transplants. The cytotoxic effects of the macrophages were tested again after transplantation and it was found that the macrophages became incapable of killing the tumor targets. In contrast to our findings with macrophage mediated cytotoxicity, the polymorphonuclear leukocytes generally retained their cytotoxic capabilities at all time points tested. However, it was noted that cytotoxic activity reached maximal levels within the first 3 weeks after cannulation but fell to low cytotoxic levels at approximately 4 to 5 weeks after cannulation. When tested several months post cannulation and transplantation, polymorphonuclear leukocyte mediated cytotoxicity increased dramatically in four of the five patients studied.     

Gamma interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis.

Gamma interferon (IFN gamma) is a potent immune mediator that plays a central role in enhancing cellular immune processes. This study demonstrates that while lung mononuclear cells from normal individuals spontaneously release little or no interferon (less than 10 U/10(6) cells per 24 h), those from patients with pulmonary sarcoidosis spontaneously release considerable amounts (65 +/- 20 U/10(6) cells per 24 h, P less than 0.02 compared to normals). Furthermore, cells from patients with active disease release far more interferon than those from patients with inactive disease (101 +/- 36 compared to 24 +/- 8 U/10(6) cells per 24 h, P less than 0.02). Characterization of this interferon using acid sensitivity, specific antibody inhibition, and target cell specificity criteria demonstrated that it was almost entirely IFN gamma. This spontaneous release of IFN gamma appeared to be compartmentalized to the lung of these patients in that their blood mononuclear cells spontaneously released little or no IFN gamma (P less than 0.02, compared to sarcoidosis lung mononuclear cells) and no IFN gamma was detected in their serum. Both lung T lymphocytes and alveolar macrophages contributed to the spontaneous release of IFN gamma by lung mononuclear cells from sarcoid patients; purified preparations of T lymphocytes and alveolar macrophages from these patients spontaneously released similar amounts of IFN gamma (56 +/- 21 and 32 +/- 11 U/10(6) cells per 24 h, respectively, P greater than 0.3). At least one role for IFN gamma in the pathogenesis of sarcoidosis appeared to be related to activation of alveolar macrophages, as alveolar macrophages recovered from patients with active disease spontaneously killed [3H]uridine-labeled tumor cell targets (17.7 +/- 4.5% cytotoxicity compared with 2.8 +/- 0.9% in normals, P less than 0.02) and purified IFN gamma enhanced the ability of alveolar macrophages from sarcoidosis patients with inactive disease to kill similar targets (P less than 0.001, compared to alveolar macrophages cultured in medium alone). Treatment of sarcoid patients with corticosteroids, a therapy known to suppress the activity of the disease, caused a marked reduction in the level of spontaneous IFN gamma release by lung mononuclear cells compared with untreated patients (P less than 0.02), which suggests that the effectiveness of corticosteroid therapy in controlling active pulmonary sarcoidosis may, at least in part, be due to suppression of IFN gamma release.