Asymmetric Distribution Of Cytokines In The Cortical Astrocytes Cultured In Vitro

Preliminary data indicate that the distribution of brain interleukin-6 is asymmetrical both in cortex and hippocampus. cytokines in the cortex of the asymmetric distribution of cytology source is only clear from glial cells at present. The glial cells on the immune function of inflammatory injury in the bilateral hemispheres is the asymmetric distribution . Well, cytokines come from which kind of cells in the asymmetric distribution of cortical glial cells?With this question, we studied astrocytes shouldered most of glial cells function with largest number and the most widely distributed. We examined the IL-6,TNF-αand IL-1-βsecreting abilities of the astrocytes both the left and right neocortex treated with LPS cultured in vitro .Two groups of astrocytes cultured in vitro from the two cerebral cortex of the neonatal BALB/c mice were selected and the levels of IL-6,TNF-αand IL-1-βwere measured after an lipopolysaccharide-treated and untreated 24h. Generally, left cortical astrocytes had higher IL-6 levels than right examined from lipopolysaccharide-treated astrocytes cultured supernatants. To confirm the gene array data on secreting of IL-6 by left and right cortical astrocytes, semiquantitative RT-PCR was conducted. RT-PCR statistical data from eight experiments indicate that the left cortical astrocytes significantly increased IL-6 mRNA level (relative to right and control; p<0.05 compared with left cortical astrocytes) .The results showed asymmetrical distribution of brain IL-6 releasing by cerebral cortical astrocytes to the inflammatory insults both in protein and mRNA levels. It is believed that TLR4, CD14 molecule and MD-2 constituted a "recognition of LPS receptor complex". The results showed that left cortical astrocytes had higher IL-6 levels than right examined from lipopolysaccharide-treated astrocytes. Then left and right cortical astrocytes LPS recognition of the TLR4 receptor, CD14 and MD-2 molecule is there asymmetry? With this question,we examined the expression of CD14 and MD-2 molecules on the left and right cortical astrocytes treated with LPS. The results showed that CD14 moleculeup-regulate and TLR4 / MD-2 molecule down-regulate. In addition, use of influenza virus H1N1 and H5N1 infected human fetal glial cells and examine the expression of IL-6, TNF-a and IL-1βby RT-PCR. The results showed that LPS stimulation and influenza virus H1N1 and H5N1 could induce the expression of inflammatory cytokines such as IL-6, TNF-a and IL-1β. Materials and methodsAstrocyte isolation. Astrocytes were prepared from neonatal (<24 hr) BABL/c mouse brains. Briefly, BALB/c mouse Left and right cortex were dissected free of meninges, The primary Left and right glial cell cultures were maintained in Dulbecco modified Eagle medium-Ham F12 (1:1) supplemented with 10% fetal calf serum in a humidified atmosphere of 5% CO2/95% air at 37°C, and the growth medium was replaced every 2 to 3 days. Remove the complete medium and washed three times with DMEM without fetal calf serum , Both left and right cortical astrocytes were treated with 10μg/ml Lipopolysaccharide in the DMEM-F12(1:1) absence of serum.Commercial ELISA (R&D Systems) kits were used to determine culture supernatant protein levels of the cytokines IL-6,TNF-αand IL-1-βaccording to the manufacturer's protocol. Total cellular RNA was obtained from astrocytes using TRIzol (Invitrogen) reagent according to the manufacturer's protocol. First-strand cDNA was generated from 1μg of total RNA from the astrocytes by using oligo(dT)15 primer and a protocol for RT for PCR Kit (Promega). Astrocytes labeled with FITC-Conjugated Anti-mouse CD14 and PE-Conjugated Tol-like receptor 4/ MD-2 were detected by Flow cytometry. H1N1 and H5N1 infect human fetal cortical glial cells. Total cellular RNA was obtained from astrocytes using TRIzol reagent according to the manufacturer's protocol. First-strand cDNA was generated from 1μg of total RNA from the astrocytes by using oligo(dT)15 primer and a protocol for RT for PCR Kit.Results1.The morphology of cultured glia cells and the identification of astocytesThe primary glial cell cultures from the left and right cortex were maintained after removing the adherent fibroblast. Glial cells were flat, thinly spread out, tripolar and polygonally shaped when plated either in plastic tissue culture flasks or on glass coverslips. The expression of GFAP in cultured cells was detected by immunocytochemistry. The positive immunostaining was detected in the cytoplasm of more than 98% cells.2.IL-6,TNF-αand IL-1-βlevels in cortical astrocytesAfter astrocytes treated with LPS 24h , differential levels of IL-6 were observed between left and right cortical astrocytes .Post hoc test showed that IL-6 levels were higher secreted from the left cortical astrocytes culture supernatants as compared with the right and control group(p<0.05).Control group were also detected an lower amount levels of IL-6 untreated with LPS. TNF-αlevels were higher secreted from the left cortical astrocytes compared with the left control (p<0.01),and the right cortical astrocytes compared with the right control (p<0.05). IL-1-βlevels levels were higher secreted from the left and right cortical astrocytes compared with the control,but no differences are found between them.3.IL-6 mRNA levels in cortical astrocytesTo confirm the gene array data on secreting of IL-6 by left and right cortical astrocytes, semiquantitative RT-PCR was conducted.Both left and right cortical astrocytes were treated and untreated with LPS(10 ug/ml). Astrocytes stimulated for 24 h with LPS dramatically up-regulated the levels of IL-6 mRNA both in left and right cortex. RT-PCR statistical data from six independent experiments indicate that the left cortical astrocytes significantly increased IL-6 mRNA level (relative to right and control;p<0.05compared with left cortical astrocytes) When astrocytes were stimulated with LPS.4. TLR4 / MD-2 expression on cortical astrocytesTLR4 / MD-2 molecule down-regulate both on left and right cortical astrocytes treated with LPS.5. CD14 expression on cortical astrocytesCD14 moleculeup-regulate on right cortical astrocytes as compared with the left.6. Influenza virus H1N1 and H5N1 infection of human fetal glial cellsExpression of inflammatory cytokines IL-6, TNF-a and IL-1βon human fetal glial cells infected by H1N1 and H5N1 and induced with LPS are promoted.Conclusion1. Astrocytes could be purified by an orbital shaker which were agitated on for 2 hr at 200 rpm at 37°C for more than 4 times.2. After astrocytes treated with LPS 24h ,IL-6 levels were higher secreted from the left cortical astrocytes culture supernatants as compared with the right and control group(p<0.05).3. Astrocytes stimulated for 24 h with LPS dramatically up-regulated the levels of IL-6 mRNA both in left and right cortex. The left cortical astrocytes significantly increased IL-6 mRNA level compared with left cortical astrocytes. 4. Control group were also detected an lower amount of IL-6 both in protein and mRNA levels untreated with LPS.5. TLR4 / MD-2 molecule down-regulate both on left and right cortical astrocytes treated with LPS. CD14 molecule up-regulate on right cortical astrocytes as compared with the left.6. Expression of inflammatory cytokines IL-6, TNF-a and IL-1βon glial cells infected by H1N1 and H5N1 and induced with LPS are promoted.