The Effect of Heterozygous Mutation of Adenylate Kinase 2 Gene on Neutrophil Differentiation.

Mitochondrial ATP production plays an important role in most cellular activities, including growth and differentiation. Previously we reported that Adenylate kinase 2 (AK2) is the main ADP supplier in the mitochondrial intermembrane space in hematopoietic cells, especially in the bone marrow. AK2 is crucial for the production of neutrophils and T cells, and its deficiency causes reticular dysgenesis. However, the relationship between ADP supply by AK2 and neutrophil differentiation remains unclear. In this study, we used CRISPR/Cas9 technology to establish two heterozygous AK2 knock-out HL-60 clones as models for reticular dysgenesis. Their AK2 activities were about half that in the wild-type (WT). Furthermore, neutrophil differentiation was impaired in one of the clones. In silico analysis predicted that the obtained mutations might cause a structural change in AK2. Time course microarray analysis of the WT and mutants revealed that similar gene clusters responded to all-trans retinoic acid treatment, but their expression was lower in the mutants than in WT. Application of fructose partially restored neutrophil differentiation in the heterozygous knock-out HL-60 clone after all-trans retinoic acid treatment. Collectively, our study suggests that the mutation of N-terminal region in AK2 might play a role in AK2-dependent neutrophil differentiation and fructose could be used to treat AK2 deficiency.

Elemental Diet Regulates Intestinal Permeability and Antibody Production in Indomethacin-Induced Intestinal Injury Rats.

Crohn's disease is a type of inflammatory bowel disease of unknown etiology. Administration of indomethacin (Indo) to rats induces acute mucosal lesions similar to those observed in Crohn's disease patients, but the damage can be prevented by feeding the animals an elemental diet (ED). In this study, we examined changes in intestinal macroscopic appearance, permeability, and immunoglobulin production after administration of Indo to male Sprague-Dawley rats fed normal lab chow or an ED. Intestinal damage was induced by subcutaneous injection of Indo on two successive days. Mucosal permeability, as measured by urinary excretion of phenolsulfonphthalein, peaked on day 2 after Indo injection, whereas the most severe intestinal damage, as scored by macroscopic inflammatory changes, was observed on day 3. Flow cytometric analysis of mesenteric lymph node cells revealed that the proportion of CD45RA+ cells was increased after Indo treatment. Furthermore, in vitro-cultured mesenteric lymph node and spleen lymphocytes from Indo-treated rats produced higher levels of IgA and IgG than did cells from vehicle-treated rats. In contrast, IgG and albumin concentrations in plasma were significantly decreased by Indo administration. Notably, none of the Indo-induced changes was observed in ED-fed rats. These findings suggest that an ED may prevent the appearance of Indo-induced mucosal lesions, at least in part, by modulating intestinal permeability and antibody production.

Glutamine and arginine affect Caco-2 cell proliferation by promotion of nucleotide synthesis.

OBJECTIVE: We tested our hypothesis that 1) the major effect of Gln is as a nitrogen donor, not an energy source, for nucleotides (NT) and 2) the supplementation of culture medium with arginine (Arg) decreases the flux of glutamine (Gln) for conversion to Arg, thus accelerating NT synthesis. METHODS: Various concentrations of nucleosides (NS+NT) Gln, and glutamate (Glu) in culture were tested for their effect on Caco-2 cell proliferation. (Arg was tested in media with and without Gln to evaluate the Gln pathway. The incorporation of (15)N from L-[5-(15)N]-Gln into NTs of DNA was measured under different NS + NT and Arg concentrations.) RESULTS: The proliferation of Caco-2 cells was increased by NS + NT and Gln supplementation, but not by Glu. The effective concentration of NS + NT was 100-fold smaller than that of Gln. An Arg effect was observed only in the presence of Gln. The NT synthesis from Gln, as indicated by (15)N incorporation from L-[5-(15)N]-Gln, was increased by Arg supplementation and decreased by NS + NT supplementation. CONCLUSION: These results support our hypothesis that the effects of Gln and Arg on Caco-2 cell proliferation are by the promotion of NT synthesis and that the major role of Gln is not energy supply.

Higher concentrations of interferon-gamma enhances uptake and transport of dietary antigens by human intestinal cells: a study using cultured Caco-2 cells.

Besides functioning as a mucosal barrier and transporting nutrients, intestinal epithelial cells (IECs) also serve as antigen-presenting cells (APCs). Modification of protein antigens by proteolysis is one of the principal steps in antigen presentation to Th cells. We used a Caco-2 intestinal epithelial cell line to investigate the transepithelial transport of the dietary antigen, ovalbumin (OVA). We also examined the effects of the proinflammatory cytokine interferon-gamma (IFN-gamma) on the antigen transport process in Caco-2 cell layers. Caco-2 cell layers transferred both intact and degraded OVA from the mucosal to the serosal side. IFN-gamma stimulated OVA transport and most of the transported OVA in such cells were degraded. We also examined OVA uptake by Caco-2 cells using immunohistochemical means. Caco-2 cells incorporated OVA in a time-dependent manner and IFN-gamma significantly enhanced antigen internalization. Flow cytometry also demonstrated that IFN-gamma elevated the internalization of FITC-OVA. We also determined the effects of low and high concentrations of IFN-gamma on mucosal permeability and internalization of FITC-OVA. Although both 10 and 50 ng/mL IFN-gamma stimulated mucosal permeability to the same extent, more FITC-OVA was internalized by Caco-2 cells incubated with 50 than with 10 ng/mL IFN-gamma. These results suggest that the effects of IFN-gamma on mucosal permeability and the internalization of antigens by intestinal epithelial cells are brought about by different mechanisms. Therefore, higher concentrations of IFN-gamma stimulate the uptake, processing, and transport of dietary antigens by IECs.

Immunoglobulin secretions in the mesenteric lymph node in stressed rats.

To study the effects of different types of stressors on intestinal immune function, the lymphocyte subsets and associated immunoglobulin production in stressed rats were observed. Physical (electric foot shock) or psychological (non foot-shock) stress respectively were induced using a communication box. Rats were exposed to stress for 2 h per day, and the treatment was maintained for 14 consecutive days. Lymphocytes were isolated from the mesenteric lymph node (MLN) and spleen using Lympholyte-Rat. There was no change the lymphocyte subsets in MLN or spleen in either group. Foot-shock stress increased immunoglobulin secretions in MLN lymphocytes. These results demonstrated that intestinal immune functions were adaptively regulated under conditions of moderate stress.

An elemental diet controls inflammation in indomethacin-induced small bowel disease in rats: the role of low dietary fat and the elimination of dietary proteins.

Elemental diets (EDs) are effective in treating Crohn's disease. We hypothesize that low dietary fat and amino acids used as the sole nitrogen source are the major contributors for the success of EDs. We examined the influences of the addition of dietary fat and protein to an ED using an indomethacin-induced inflammation model in rat small intestine. In the ED-fed rats, the intestinal damage score was decreased compared with that in the standard chow group with decreasing intestinal permeability. By supplementing an ED with soybean oil (SO), intestinal permeability was increased to a level similar to that of the standard chow group. For this group, the intestinal damage score also increased compared with that of the ED group but did not reach the levels observed in the standard chow group. The addition of dietary proteins (using heat-denatured pancreatin) resulted in intestinal damage scores that were significantly higher than those of the ED+SO-fed group. The dietary protein increased the intestinal damage score. These results suggest that EDs control inflammation by decreasing intestinal permeability and the elimination of dietary proteins.