Organ specific apoptosis following polymicrobial intraperitoneal infection.

BACKGROUND: Leukocyte apoptosis allows safe removal of potentially harmful cells and facilitates resolution of inflammation. We hypothesized that the number of apoptotic cells changes in a disproportionate fashion in parenchymal organs in response to intra-abdominal infection. MATERIALS AND METHODS: The percentage of apoptotic cells in the liver, spleen, lung, and peripheral blood was evaluated following cecal ligation and puncture (CLP) in mice. Tissue myeloperoxidase (MPO) levels were measured as an index of neutrophil extravasation. RESULTS: Liver & spleen MPO continually increased, while lung MPO remained low after CLP. In parallel to the increase in MPO, liver & spleen apoptosis continually increased throughout the 9-day follow-up period, whereas lung apoptosis remained unchanged. CONCLUSIONS: The distribution of apoptotic cells during intraperitoneal infection occurs in an organ specific manner, with significant increases in the spleen and liver. This distribution likely reflected the clearance of apoptotic cells as the inflammatory focus became contained.

Immediate and delayed leukocyte apoptosis in two models of peritonitis.

Leukocyte apoptosis is an energy-dependent process that facilitates resolution of the cellular inflammatory response. Levels of apoptosis can be accelerated or inhibited after exposure to various stimuli. To compare apoptosis in transmigrated leukocytes, two models of peritonitis in mice were used that both cause leukocyte influx into the peritoneal cavity: (1) intraperitoneal thioglycollate administration producing a sterile peritonitis and (2) cecal ligation and puncture (CLP) producing a polymicrobial bacterial peritonitis. Samples of blood and peritoneal exudate cells (PEC) were collected at multiple time points after induction of peritonitis. Leukocytes were either fixed immediately to determine an immediate apoptosis level or cultured for 24 h to determine a delayed apoptosis level. Apoptosis was assessed using terminal uridine-triphosphate nick-end labeling (TUNEL) assay, flow cytometry, and confocal microscopy. Leukocyte influx into the peritoneal cavity was confirmed in both models. At all time points, and in both models, there was increased immediate apoptosis in PEC compared with unmanipulated controls and this increase was maximal in CLP after 18 h, although it appeared to remain at a stable level in the sterile peritonitis model by 3 h. There was also an increase in PEC delayed apoptosis at early time points in both models, again maximal at 18 h for CLP, with the levels being significantly higher than the thioglycollate model at 6 h and 18 h. The mice had a relative peripheral neutropenia at 6 h after CLP, but not post thioglycollate injection, and this persisted until 42 h. Lung and liver MPO levels were elevated in CLP but did not increase after thioglycollate. There was no increase in immediate peripheral leukocyte apoptosis in either model, but an increase in delayed peripheral leukocyte apoptosis was observed by 18 h in both models. Peripheral leukocyte CD1lb expression, which is a marker of activation, was also persistently elevated in the CLP model, but not in sterile peritonitis. In conclusion, CLP is a more potent stimulus for apoptosis of leukocytes than their migration to the site of inflammation alone, as occurs in the thioglycollate model. Blood leukocyte apoptosis also appears not to be dependent on CD11b expression, and therefore activation status.

Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin.

Heparin-binding protein decreases apoptosis in human and murine neutrophils.

BACKGROUND: Heparin-binding protein (HBP), a serine protease without any known proteolytic activity, is found in human polymorphonuclear leukocyte (PMN) granules, but not in mice. HBP potentiates the endotoxin-induced release of tumor necrosis factor (TNF) alpha, interleukin (IL)-1, and IL-6 from isolated monocytes. HBP has also been shown to increase the survival of cultured monocytes and protect them from oxidative stress. However, whether HBP affects PMNs themselves is not known. MATERIALS AND METHODS: Based on our work with cultured monocytes and the survival benefit noted in experimental peritonitis, we hypothesized that HBP would have a beneficial effect on the survival of neutrophils. We evaluated the effect of HBP on apoptosis in murine peritoneal exudative cells elicited by intraperitoneal thioglycollate administration and in normal human neutrophils from volunteers. Leukocytes were isolated from the peritoneal cavity and blood of mice that underwent intraperitoneal thioglycollate instillation. The mouse peritoneal exudate cells and normal human neutrophils isolated from peripheral blood were used to study the effect of HBP on survival and apoptosis. RESULTS: HBP decreased percentage apoptosis of mouse cells in both serum-enriched (from 24.8 to 4.5%) and serum-deprived (from 23.1 to 8.2%) cultures. In human PMNs, the protective effect of HBP was seen only in the serum-deprived group, with a decrease in percentage apoptosis from 69.1 to 43.3%. CONCLUSIONS: For the first time, we have shown that HBP, in addition to its known augmentation of the proinflammatory response of monocytes, also acts as a prosurvival protein for neutrophils themselves, and thereby enhances local host defense.

In vitro assembly of the mouse U14 snoRNP core complex and identification of a 65-kDa box C/D-binding protein.

The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) that have been categorized into two major families based on evolutionarily conserved sequence elements. U14 snoRNA is a member of the larger, box C/D snoRNA family and possesses nucleotide box C and D consensus sequences. In previous studies, we have defined a U14 box C/D core motif that is essential for intronic U14 snoRNA processing. These studies also revealed that nuclear proteins that recognize boxes C/D are required. We have now established an in vitro U14 snoRNP assembly system to characterize protein binding. Electrophoretic mobility-shift analysis demonstrated that all the sequences and structures of the box C/D core motif required for U14 processing are also necessary for protein binding and snoRNP assembly. These required elements include a base paired 5',3' terminal stem and the phylogenetically conserved nucleotides of boxes C and D. The ability of other box C/D snoRNAs to compete for protein binding demonstrated that the box C/D core motif-binding proteins are common to this family of snoRNAs. UV crosslinking of nuclear proteins bound to the U14 core motif identified a 65-kDa mouse snoRNP protein that requires boxes C and D for binding. Two additional core motif proteins of 55 and 50 kDa were also identified by biochemical fractionation of the in vitro-assembled U14 snoRNP complex. Thus, the U14 snoRNP core complex is a multiprotein particle whose assembly requires nucleotide boxes C and D.