Macrophage activation and differentiation signals regulate schlafen-4 gene expression: evidence for Schlafen-4 as a modulator of myelopoiesis

Background

The ten mouse and six human members of the Schlafen (Slfn) gene family all contain an AAA domain. Little is known of their function, but previous studies suggest roles in immune cell development. In this report, we assessed Slfn regulation and function in macrophages, which are key cellular regulators of innate immunity.

Methodology/Principal Findings

Multiple members of the Slfn family were up-regulated in mouse bone marrow-derived macrophages (BMM) by the Toll-like Receptor (TLR)4 agonist lipopolysaccharide (LPS), the TLR3 agonist Poly(I∶C), and in disease-affected joints in the collagen-induced model of rheumatoid arthritis. Of these, the most inducible was Slfn4. TLR agonists that signal exclusively through the MyD88 adaptor protein had more modest effects on Slfn4 mRNA levels, thus implicating MyD88-independent signalling and autocrine interferon (IFN)-β in inducible expression. This was supported by the substantial reduction in basal and LPS-induced Slfn4 mRNA expression in IFNAR-1^−/− BMM. LPS causes growth arrest in macrophages, and other Slfn family genes have been implicated in growth control. Slfn4 mRNA levels were repressed during macrophage colony-stimulating factor (CSF-1)-mediated differentiation of bone marrow progenitors into BMM. To determine the role of Slfn4 in vivo, we over-expressed the gene specifically in macrophages in mice using a csf1r promoter-driven binary expression system. Transgenic over-expression of Slfn4 in myeloid cells did not alter macrophage colony formation or proliferation in vitro. Monocyte numbers, as well as inflammatory macrophages recruited to the peritoneal cavity, were reduced in transgenic mice that specifically over-expressed Slfn4, while macrophage numbers and hematopoietic activity were increased in the livers and spleens.

Conclusions

Slfn4 mRNA levels were up-regulated during macrophage activation but down-regulated during differentiation. Constitutive Slfn4 expression in the myeloid lineage in vivo perturbs myelopoiesis. We hypothesise that the down-regulation of Slfn4 gene expression during macrophage differentiation is a necessary step in development of this lineage.     

A rapid and sensitive method for measuring cell adhesion

We have adapted the CyQuant® assay to provide a simple, rapid, sensitive and highly reproducible method for measuring cell adhesion. The modified CyQuant® assay eliminates the requirement for labour intensive fluorescent labelling protocols prior to experimentation and has the sensitivity to measure small numbers (>1000) of adherent cells.     

Neuroprotection against CCl4 induced brain damage with crocin in Wistar rats

Owing to its lipophilic property, carbon tetrachloride (CCl₄) is rapidly absorbed by both the liver and brain. We investigated the protective effects of crocin against brain damage caused by CCl₄. Fifty rats were divided into five groups of ten: control, corn oil, crocin, CCl₄ and CCl₄ + crocin. CCl₄ administration decreased glutathione (GSH) and total antioxidant status (TAS) levels, and catalase (CAT) activity, while significant increases were observed in malondialdehyde (MDA) and total oxidant status (TOS) levels and superoxide dismutase (SOD) activity. The cerebral cortex nuclear lamina developed a spongy appearance, neuronal degeneration was observed in the hippocampus, and heterochromatic and pyknotic neurons with increased cytoplasmic eosinophilia were observed in the hippocampus after CCl₄ treatment. Because crocin exhibits strong antioxidant properties, crocin treatment increased GSH and TAS levels and CAT activities, and decreased MDA and TOS levels and SOD activity; significant improvements also were observed in histologic architecture. We found that crocin administration nearly eliminated CCl₄ induced brain damage by preventing oxidative stress.     

Sexual behavior and serum concentrations of reproductive hormones in normal stallions

Recently, it has been reported that impotence in the stallion has a physiological basis that involves decreased serum concentrations of luteinizing hormone (LH) and estradiol-17beta, but not testosterone. We have found such a hormonal profile in two of nine stallions studied during an ongoing investigation of the endocrinology of the normal stallion. Nevertheless, both of these stallions possessed vigorous libido and normal seminal characteristics. We conclude that the hormonal profile of low LH, low estradiol and normal testosterone, although it may accompany impotence in the stallion, is not predictive of, or causally related to, abnormalities in sexual behavior.     

Molecular species analysis of 1,2-diacylglycerols and phosphatidic acid formed during bombesin stimulation of Swiss 3T3 cells

Swiss 3T3 cells were labelled with [3H]glycerol and stimulated with bombesin over a time course of 20 min. The individual 1,2-diacylglycerols produced were quantified by acetylation followed by analysis by HPLC and argentation chromatography. The major phospholipids and phosphatidic acid were acetolysed and then analysed in the same manner. The data show that even at an early time of stimulation (30 s), stimulated diacylglycerol formation comes from at least two sources--phosphoinositides and phosphatidylcholine.     

Non-destructive measurement of stem water content by time domain reflectometry using short probes

Time Domain Reflectometry (TDR) has previously been used todetermine the water content of soils. Here, TDR is assessedas a method of tracking the seasonal change in water contentof the stems of mature trees (Pinus sylvestris L.). The longerprobes used for soil were replaced by 50 mm probes, insertedradially into the stems at 1 m above the ground. Tests on blocksof wood in the laboratory suggested that the probe is influencedby the water content several centimetres around it, and thesensitivity of the system enables volumetric water content (W)in the physiological range to be measured to a resolution greaterthan 0.01 m³ (H₂0) m^–3. The use of very short probes preventsthe development of a universal calibration between measureddielectric constant and W. Calibrations for both 50 mm and 20mm long probes are reported. The effect of temperature on thecalibration was negligible. The system was used successfullyon mature trees in 1995, and no evidence of wound reaction aroundthe permanently installed probes was apparent in the measurements. Key words: TDR, dielectnc constant, water content, Pinus sylvestris, xylem embolism