Apoptosis inhibitor of macrophage (AIM) expression in alveolar macrophages in COPD

Background

Marked accumulation of alveolar macrophages (AM) conferred by apoptosis resistance has been implicated in pathogenesis of chronic obstructive pulmonary disease (COPD). Apoptosis inhibitor of macrophage (AIM), has been shown to be produced by mature tissue macrophages and AIM demonstrates anti-apoptotic property against multiple apoptosis-inducing stimuli. Accordingly, we attempt to determine if AIM is expressed in AM and whether AIM is involved in the regulation of apoptosis in the setting of cigarette smoke extract (CSE) exposure.

Methods

Immunohistochemical evaluations of AIM were performed. Immunostaining was assessed by counting total and positively staining AM numbers in each case (n = 5 in control, n = 5 in non-COPD smoker, n = 5 in COPD). AM were isolated from bronchoalveolar lavage fluid (BALF). The changes of AIM expression levels in response to CSE exposure in AM were evaluated. Knock-down of anti-apoptotic Bcl-xL was mediated by siRNA transfection. U937 monocyte-macrophage cell line was used to explore the anti-apoptotic properties of AIM.

Results

The numbers of AM and AIM-positive AM were significantly increased in COPD lungs. AIM expression was demonstrated at both mRNA and protein levels in isolated AM, which was enhanced in response to CSE exposure. AIM significantly increased Bcl-xL expression levels in AM and Bcl-xL was involved in a part of anti-apoptotic mechanisms of AIM in U937 cells in the setting of CSE exposure.

Conclusions

These results suggest that AIM expression in association with cigarette smoking may be involved in accumulation of AM in COPD.     

Activation of a suicide process of thymocytes through DNA fragmentation by calcium ionophores and phorbol esters

Calcium ionophore, A23187, is known to be a comitogen, but it activates a suicide process characterized by DNA fragmentation at linker regions in mouse immature thymocytes. It did not induce DNA fragmentation in T lymphocytes prepared from lymph node and spleen cells. Induction of DNA fragmentation by A23187 depends on protein phosphorylation and synthesis of mRNA and protein, because an inhibitor of protein kinase, 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride (H-7), actinomycin D, and cycloheximide, respectively, inhibits the DNA fragmentation and cell death. Studies adding the inhibitors at various times show that protein phosphorylation and mRNA synthesis occur within a few hours after incubation with A23187 followed by the protein synthesis responsible for inducing DNA fragmentation. Phorbol esters, 12-O-tetradecanoyl 13-acetate (TPA) and phorbol 12,13-dibutyrate (PBD), which are capable of activating protein kinase C, also induced similar DNA fragmentation in immature thymocytes, followed by cell death. PBD committed the suicide process after 6 h of incubation, because the DNA fragmentation above the control level was not induced when PDB was removed from the medium before 6 h of incubation. A23187 or a phorbol ester alone induced DNA fragmentation followed by cell death, whereas the addition of TPA at low concentration inhibited the DNA fragmentation induced by A23187 accompanied with an increase in DNA synthesis. The result suggests that TPA switched a suicide process induced by A23187 to an opposite process: stimulation of DNA synthesis. Physiologic factors and mechanisms which regulate cell proliferation and death in the thymus are not known at present, but the signals by protein kinases and calcium ions may regulate both cell proliferation and death, independently, synergistically or antagonistically.     

Murine macrophages produce secretory leukocyte protease inhibitor during clearance of apoptotic cells: implications for resolution of the inflammatory response

Macrophage-derived secretory leukocyte protease inhibitor (SLPI) can be induced locally as well as systemically in response to microbial products such as LPS and lipotechoic acid. It is not known whether phagocytosis of apoptotic cells, an essential function of macrophages, can regulate expression and secretion of SLPI. In this study, we report that exposure of peritoneal macrophages of BALB/c mice or murine macrophage cell lines RAW264.7 and J774.1 to apoptotic target cells induced an elevation in SLPI secretion. Secreted SLPI retained its antichymotrypsin activity. SLPI expression in thymuses from BALB/c mice that had been injected with anti-CD3 Ab to induce apoptosis of thymocytes was also elevated both at the mRNA and protein levels. Colchicine, a microtubular inhibitor, blocked the internalization of apoptotic cells by macrophages but not SLPI secretion, suggesting that surface recognition of apoptotic cells is sufficient for the induction of SLPI. Exposure of RAW264.7 cells to apoptotic CTLL-2 cells induced both SLPI and TNF-alpha, and addition of IFN-gamma inhibited SLPI but augmented TNF-alpha production. Transfection of either the secreted or a nonsecreted form of SLPI into RAW264.7 cells led to suppression of TNF-alpha production in response to apoptotic cells. Thus, macrophages secrete an increased amount of SLPI when encountering apoptotic cells, which may help to attenuate potential inflammation during clearance of these cells.     

Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation-induced cell death

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time-dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation-induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.     

Single site hydrolysis of 2',3'-O-(2,4,6-trinitrophenyl)-ATP by the F1-ATPase from thermophilic bacterium PS3 is accelerated by the chase-addition of excess ATP.

The interaction of 2',3'-O-(2,4,6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP) and TNP-ADP to F1-ATPase from a thermophilic bacterium PS3 (TF1) was investigated. When TNP-ADP or TNP-ATP was added to the isolated alpha or beta subunit of TF1, characteristic difference spectra were generated for each subunit. Difference spectra generated on addition of these analogs to TF1 resembled those observed for the beta subunit, indicating TNP analogs bind to the beta subunits in the molecule of TF1. Results of equilibrium dialysis showed that TNP-ADP binds to a single high affinity site on TF1 in the presence of Mg2+ with a dissociation constant of 2.2 nM. When TNP-ATP was added to TF1 in a substoichiometric molar ratio, it rapidly bound to TF1 and was slowly hydrolyzed. The hydrolysis proceeded nearly to completion without showing stable equilibrium between bound species of TNP-ATP and TNP-ADP. Similar to beef heart mitochondrial F1, this hydrolysis was greatly accelerated by the chase-addition of 100 microM ATP. However, the hydrolyzed product, TNP-ADP, remained bound on the beta subunit even after the chase.     

Inheritance of Susceptibility to Friend Mouse Leukemia Virus: V. Introduction of a Gene Responsible for Susceptibility in the Genetic Complement of Resistant Mice

Based on the previous observation that a single major autosomal gene controls susceptibility to Friend leukemia virus in mice, an attempt was made to place the gene for susceptibility, Fv(s), from susceptible DDD mice into the genetic complement of resistant C57BL/6 mice. The backcross system was adopted for this purpose, the heterozygotes being selected by progeny test at each generation. During successive backcrosses, the effect of gene Fv(s) was not diluted out, and progeny were almost always obtained as expected from the single-gene hypothesis, with respect to both genotype and phenotype. With the eighth backcross generation, brother-sister mating was done between the heterozygotes, and it produced mice homozygous for gene Fv(s). These susceptible homozygotes and their progeny produced by incross could be assumed congenic with C57BL/6 mice except for susceptibility to Friend leukemia virus. The results indicate that the appearance of early splenomegaly in Friend virus-infected mice is under the control of a single major autosomal gene.     

Effect of activated lymphocytes on the regulation of hematopoiesis: suppression of in vitro granulopoiesis by OKT8+Ia+T cells induced by alloantigen stimulation

We studied the effects of alloantigen-stimulated lymphocytes in the regulation of hematopoiesis. Alloantigen-stimulated lymphocytes were harvested on days 2 to 3, days 6 to 7, or days 9 to 10 of MLC and were tested for their effects on granulocyte/macrophage progenitor cells (CFU-C). Dose-dependent suppression of CFU-C was observed when alloantigen-stimulated lymphocytes from days 6 to 7 and days 9 to 10 MLC were added to the cultures of autologous or allogeneic bone marrow cells for CFU-C assays. Suppressive activity was detected in the T cell fraction but not in the non-T cell fraction. For further characterization of these CFU-C/suppressor cells, alloantigen-stimulated lymphocytes were treated with radiation (2000 rad) or with monoclonal antibodies against T cell subsets and complement (C) before culture. Suppressive activity was completely abolished by treatment with OKT8 or OKIa1 antibodies and C whereas suppression was retained after radiation treatment. These observations suggest that CFU-C/suppressor cells can be induced by alloantigen stimulation in MLC and that they are radioresistant OKT8+ and Ia+ T cells.     

Skeletal isotope microprofiles of growth perturbations in<Emphasis Type="Italic"> Porites</Emphasis> corals during the 1997–1998 mass bleaching event

Severe coral bleaching occurred throughout the tropics in 1997/98. We report high-resolution skeletal oxygen isotope (¹⁸O) and carbon isotope (¹³C) microprofiles for bleached corals from Pandora Reef, Great Barrier Reef, and Ishigaki Island, Japan, in order to examine the ability of Porites corals to record clear signals of bleaching. Analysis of the annual cycle in ¹⁸O revealed abrupt reductions in skeletal extension immediately after the 1997–98 summer temperature maximum, indicating that bleaching inhibits coral calcification. Skeletal ¹³C in the Ishigaki corals showed lower values during bleaching, indicating depressed coral metabolism associated with a reduction in calcification. In contrast, microprofiles of skeletal ¹³C from the shaded sides of Pandora Reef corals exhibited little change, possibly because algal photosynthesis was already slow prior to bleaching, thus subduing the ¹³C-response to bleaching. Comparison of ¹⁸O microprofiles from bleached corals with instrumental temperature records showed that Porites corals can recover following 5 months with little skeletogenesis. The results indicate that isotopic microprofiling may be the key to identifying gaps in coral growth that are diagnostic of past bleaching events. We have tested this hypothesis using blue UV fluorescent bands to guide us to coral skeleton where isotope microprofiling identifies bleaching events in 1986, 1989, and 1990. These events, detected by proxy, suggest that coral bleaching may have occurred more commonly on Ishigaki Island than previously recorded.     

Functional expression of nitrile hydratase in Escherichia coli: requirement of a nitrile hydratase activator and post-translational modification of a ligand cysteine

The nitrile hydratase (NHase) from Rhodococcus sp. N-771 is a photoreactive enzyme that is inactivated on nitrosylation of the non-heme iron center and activated on photo-dissociation of nitric oxide (NO). The nitrile hydratase operon consists of six genes encoding NHase regulator 2, NHase regulator 1, amidase, NHase alpha subunit, NHase beta subunit and NHase activator. We overproduced the NHase in Escherichia coli using a T7 expression system. The NHase was functionally expressed in E. coli only when the NHase activator encoded downstream of the beta subunit gene was co-expressed and the transformant was grown at 30 degrees C or less. A ligand cysteine, alphaCys112, of the recombinant NHase was also post-translationally modified to a cysteine-sulfinic acid similar to for the native NHase. Although another modification of alphaCys114 could not be identified because of the instability under acidic conditions, the recombinant NHase could be reversibly inactivated by nitric oxide.     

Specific and nontoxic silencing in mammalian cells with expressed long dsRNAs

A number of groups have developed libraries of siRNAs to identify genes through functional genomics. While these studies have validated the approach of making functional RNAi libraries to understand fundamental cellular mechanisms, they require information and knowledge of existing sequences since the RNAi sequences are generated synthetically. An alternative strategy would be to create an RNAi library from cDNA. Unfortunately, the complexity of such a library of siRNAs would make screening difficult. To reduce the complexity, longer dsRNAs could be used; however, concerns of induction of the interferon response and off-target effects of long dsRNAs have prevented their use. As a first step in creating such libraries, long dsRNA was expressed in mammalian cells. The 250 nt dsRNAs were capable of efficiently silencing a luciferase reporter gene that was stably transfected in MDA-MB-231 cells without inducing the interferon response or off-target effects any more than reported for siRNAs. In addition, a long dsRNA expressed in the same cell line was capable of silencing endogenous c-met expression and inhibited cell migration, whereas the dsRNA against luciferase had no effect on c-met or cell migration. The studies suggest that large dsRNA libraries are feasible and that functional selection of genes will be possible.