A comparison of the changes in the non‐neuronal cell populations of the superior cervical ganglia following decentralization and axotomy

Transecting the axons of neurons in the adult superior cervical ganglion (SCG; axotomy) results in the survival of most postganglionic neurons, the influx of circulating monocytes, proliferation of satellite cells, and changes in neuronal gene expression. In contrast, transecting the afferent input to the SCG (decentralization) results in nerve terminal degeneration and elicits a different pattern of gene expression. We examined the effects of decentralization on macrophages in the SCG and compared the results to those previously obtained after axotomy. Monoclonal antibodies were used to identify infiltrating (ED1+) and resident (ED2+) macrophages, as well as macrophages expressing MHC class II molecules (OX6+). Normal ganglia contained ED2+ cells and OX6+ cells, but few infiltrating macrophages. After decentralization, the number of infiltrating ED1+ cells increased in the SCG to a density about twofold greater than that previously seen after axotomy. Both the densities of ED2+ and OX6+ cells were essentially unchanged after decentralization, though a large increase in OX6+ cells occurred after axotomy. Proliferation among the ganglion's total non‐neuronal cell population was examined and found to increase about twofold after decentralization and about fourfold after axotomy. Double‐labeling experiments indicated that some of these proliferating cells were macrophages. After both surgical procedures, the percentage of proliferating ED2+ macrophages increased, while neither procedure altered the proliferation of ED1+ macrophages. Axotomy, though not decentralization, increased the proliferation of OX6+ cells. Future studies must address what role(s) infiltrating and/or resident macrophages play in regions of decentralized and axotomized neurons and, if both are involved, whether they play distinct roles. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 68–79, 2002     

Antibody to myelin constituents: A possible factor in induction of cell-mediated demyelination

Results from this laboratory have demonstrated that¹⁴C-labeled myelin opsonized with antibodies raised to purified CNS myelin in rabbit is phagocytized by cultured macrophages in larger amounts than untreated myelin or myelin opsonized with preimmune serum. The cultured macrophages produced high amounts of radioactive cholesterol ester and triglyceride from the antibody-treated myelin while much less was formed from preimmune serum-treated or untreated myelin. Antiserum to galactocerebroside also greatly enhanced the formation of radioactive cholesterol ester, while that to myelin basic protein as well as to other myelin constituents had little or no effect. Serum from Lewis rats with acute EAE 13–14 days after immunization with whole CNS myelin also stimulated radioactive cholesterol ester formation compared to serum from Freund's adjuvant-injected controls (FAC). Serum from EAE rats as a result of myelin basic protein injection was as active as that from rats with whole myelin injection. No galactocerebroside antibody could be demonstrated in the EAE sera, although a strong immunostaining to myelin basic protein and proteolipid protein was demonstrated. IgG prepared from EAE serum also showed stimulatory effects compared to IgG from FAC serum, but much of the activity was lost, and the possibility that other factors may be involved is discussed. These experiments provide evidence that myelin phagocytosis and digestion by macrophages is enhanced by the presence of antibody to myelin. In EAE this antibody may leak into CNS with the breakdown of the blood-brain barrier. A humoral involvement in demyelination in EAE is implicated, and these findings may be extended eventually to the demyelinative mechanism in multiple sclerosis where IgG is found in large amounts in the CNS.Special Issue Dedicated to Dr. Abel Lajtha.     

Experimental pulmonary candidiasis

The initial interaction of Candida albicans with pulmonary tissue of B6D2/F₁ mice was investigated. The LD₅₀ for mice challenged intravenously (IV) was approximately 3 × 10⁵ yeasts, whereas the LD₅₀ by the intratracheal (IT) route was in excess of 10⁸ yeasts. Mice challenged IV died of progressive yeast growth in the kidneys. In contrast, mice challenged IT rapidly eliminated the entire inoculum by the first day after challenge. Resident pulmonary alveolar macrophages (PAM) killed upwards of 70% of C. albicans in an in vitro killing assay. At effector: target ratios favoring the effector cell population resident PAM were able to restrict the formation of yeast germ tubes to only 30% of the yeasts, whereas at equivalent ratios virtually all of the intracellular yeasts produced germ tubes. Evaluation of the ability of PAM, harvested from genetically different strains of inbred mice, to kill C. albicans in vitro showed that killing ability was a property of resident PAM from mice with the black 6 background. It was discovered that during the initial stages of infection in vivo, the expression of the F4/80 surface molecule was down regulated, and the expression of the Mac 1 surface molecule upregulated. There were no quantitative changes in expression of either Mac 2, Mac 3, Ly 5 or the 5C6 surface epitopes. Taken together, the data show that pulmonary tissue is quantitatively very resistant to C. albicans infection, because of the ability of resident PAM to rapidly phagocytize and kill yeasts. Killing of C. albicans by resident PAM may be a property of a subset of this mononuclear phagocyte population and was accompanied by alterations in the expression of surface molecules.Presented as part of the Everett S. Beneke Symposium in Mycology, May 27, 1988.     

The cytotoxicity of chrysotile asbestos fibers to pulmonary alveolar macrophages I. Effects of inhibitors of ADP-ribosyl transferase

Pulmonary alveoler macrophages exposedto very short chrysotile asbestos fibers present a typical cytotoxic response: extracellular releases of lactate dehydrogenase and -galactosidase, and a decrease in cellular ATP content. The objective of this study was to determine if nicotinamide and 3-aminobenzamide, two inhibitors of the ADP-ribosyl transferase, could modify the in vitro toxicity of chrysotilee fibers. After 30 min of pre-exposure with each of the two inihibitors, pulmonary alveolar macrophage monolayers were concominantly exposed for 18 hours to 50g of fibers. It was observed that, in a dose-effect relationship (5 to 30 mM), nicotinamide was very effective in reducing the extracellular liberation of the marker enzymes. At 30 mM, the enzyme releases in the medium had returned to control values; the restoration of cell viability was confirmed by ATP levels. Up to 5 mM 3-aminobenzamide did not provide any protection against chrysotile cytotoxicity. Nicotinic acid, a structural analogue of nicotinamide, but not an inhibitor of the ADP-ribosyl transferase, also showed no protective effect. Nicotinamide and 3-aminobenzamide increased the intracellular NAD⁺ pools, respectively by 350% and 250%. However, with or without additives, the chrysotile fibers caused a constant and significant decrease in NAD⁺ levels (40–55 pmoles). These results suggest that the inhibition of the nuclear ADP-ribosyl transferase is not the major mechanism by which nicotinamide protects pulmonary alveolar macrophages against the chrysotile asbestos fibers.Abbreviations 3-AB 3-aminobenzamide - ADPRT ADP-ribosyl transferase - -GAL -galactosidase - DTT dithiothreitol - FBS fetal bovine serum - FMN flavin mononucleotide - HEPES N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid - LDH lactate dehydrogenase - NAD⁺ nicotinamide adenine dinucleotide (oxidized form) - NADH nicotimide adenine dinucleotide (reduced forms) - NADPH nicotimide adenine dinucleotide phosphate (reduced form) - NAM nicotinamide - NIC nicotinic acid - ORS oxygen radical species - PAM pulmonary alveolar macrophages - S.E. standard error of the mean - TAPS tris (hydroxymethyl) methylamino-propane sulfonic acid - TRIS tris (hydroxymethyl) aminomethane - VSF very short chrysotile fibers     

Prevention by N-acetylcysteine of benzo[a]pyrene clastogenicity and DNA adducts in rats

The daily i.t. administration of benzo[a]pyrene (BP) to Sprague-Dawley rats, for 3 consecutive days, did not cause any toxicity or clastogenicity in bone marrow cells, as evaluated by monitoring the ratio of polychromatic to normochromatic erythrocytes and the frequency of micronucleated polychromatic erythrocytes. However, BP produced a considerable enhancement of binucleated and micronucleated pulmonary alveolar macrophages, as well as a significant increase in polymorphonucleates recovered by bronchoalveolar lavage. These effects were prevented by administering the thiol N-acetylcysteine (NAC) by gavage 5 h before each BP instillation. In addition, the i.t. treatment with BP resulted in the formation of BP diolepoxide (BPDE)-DNA adducts in lungs and liver, as assessed by synchronous fluorescence spectrophotometry, with fluorescence peaks of similar magnitude in the 2 tissues. Pretreatment with NAC by gavage completely prevented BPDE adducts to liver DNA and significantly decreased those to lung DNA.     

Role of Ca2+ ion on Leishmania-macrophage attachment

Summary Leishmania donovani, the etiological agent for the disease visceral leishmaniasis, attach themselves to the macrophages for initiation of the disease. The attachment process has been found to be regulated by Ca²⁺ ions. Verapamil, a Ca²⁺-channel blocker inhibits Leishmania-macrophage attachment. The inhibitory effect is increased with time. Nifedipine, another Ca²⁺-channel blocker exhibits the same effect. The attachment process is stimulated by Ca²⁺-ionophore alone. The inhibitory effects of the calcium channel blockers are reversed by the ionophore.     

Bidirectional effect of met-enkephalin on macrophage effector functions

Summary Met-enkephalin (ME) exerts a bimodal effect on functional activities of rat peritoneal macrophages (PM); in a range of low concentration (10^-9-10^-7 M) antibody dependent cellular cytotoxicity (ADCC)was markedly stimulated with a simultaneous decrease of Fc receptor (FcR) mediated phagocytosis while the opposite was observed at 10^-6-10^-5 M concentrations.Studying the possible underlying mechanism(s) the followings were recorded: (1) ME in all applied concentrations induced an early Na⁺ influx which was followed by a Ca²⁺ efflux in the range of low concentrations. In the range of high concentrations Na⁺ influx was accompanied by a Ca²⁺ influx. (2) ME at 10^-8 M concentration induced a rise in cGMP level with a plateau in the 60–120th min of incubation. This effect was prevented by 10^-5 M of naloxone. At 10^-6 M concentration a transient rise of cAMP level was recorded which was not affected by naloxone. (3) Verapamil in 10^-6 M abolished both the Ca²⁺ influx and the rise in cAMP level induced by 10^-6-10^-5 M ME but not the rise in cGMP level induced by lower ME concentrations. (4) cAMP elevation by high ME concentrations was abolished by enkephalinase inhibitory puromycin. (5) PM-enkephalinase as assessed by the cleavage of fluorogenic substrate L-alanine beta naphthylamide (ABNA), was inhibited by 10^-6-10^-5 M of ME. This inhibition was abolished by verapamil, but not affected by naloxone. In the range of low concentrations ME appears to act on specific delta opioid receptors and its action is positively coupled to guanylate cyclase. In relatively higher concentrations ME-action is not mediated by specific delta opioid receptors and it appears to involve Ca²⁺ influx, adenylate cyclase activation as well as the processing of hormone by PM-enkephalinase.     

ESR检测大鼠肺巨噬细胞释放的活性氧自由基

用ESR捕捉技术检测大鼠AM释放的活性氧自由基的性质表明:1.PMA和BCG均能刺激AM产生较强的OH·;能刺激人末稍血白细胞释放活性氧自由基的ConA和顺铂在本实验条件下未能使AM产生活性氧自由基信号。2.经膜活性剂PMA刺激的AM所释放活性氧自由基的高峰在刺激后2min,而经颗粒性物质BCG刺激,AM释放自由基的高峰时间明显后移。3.测试体系中的AM数过多或过少都不适合捕捉ESR信号。在本实验条件下,捕捉到最高自由基信号的AM终浓度为5×10⁷AM/ml。4.测试体系中存在DETAPAC或EDTA,可使捕捉到的自由基信号明显增强。