| ObjectiveHuman natural killer cells are cytotoxic lymphocytes that are responsible for inducing rapid apoptosis of virus infected cells or tumors.They play cytolytic activity through several distinct pathways,including Fas-L,TRAIL,and perforin-granzymes effector molecules.Among them,the secretion of cytotoxic granules is principal event and dependent on perforin-granzymes pathway.After conjugate with target cells,NK cells impair target cells by secreting cytotoxic granules.This function involves the expression of several granzyme gene products,including granzyme A,B,H,K and M, which initiate distinct apoptotic pathways.Granzymes are closely related to one another structurally,and their genes are clustered in several loci on distinct chromosomes,especially,GZM-H has a very high amino acid identity(90%)with many portions of the GZM-B sequence.GZM-B is the most well studied granzyme and is responsible for the rapid induction of caspase-dependent apoptosis pathway.Human GZM-B exerts its function is mainly through mitochondria pathway.Granzyme A-mediated apoptosis is mainly characterized by generation of single stranded DNA nicks.Moreover,GZM-A does not result in activation of caspases which is different from that of GZM-B.GZM- H and GZM-K are called orphan enzymes because their substrates have not been identified.GZM-H and K induced cell death is independent of caspase activation too. GZM-M induced cell apoptosis rapidly and in a caspase-independent pathway.Previous studies have demonstrated that several cytokines such as IL-2,IL-15, IL- 12 and IFN-αcan enhance cytotoxic activity of NK cells.It has been reported that the abilities of these cytokines in enhancing target cell recognition,killing and at a little times,granzymes proteins are similar,but the precise events in granzymes gene regulation by these different cytokines are lacking.To investigate the functional characterizations of IL-2,IL-15,IL-12 and IFNαon regulating the transcription of the members of granzyme family,including GZM-A,B,H,K and M genes in human NK cells,we used real-time PCR to examine the changes of granzyme mRNA levels after NK cells were stimulated with these cytokines and analyzed the differences between each other.This study illustrated that these cytokines are not entirely similar and they may preferentially induce discrete NK cell granzyme profiles.Methods1.Real-time PCR:real-time PCR was used to analysis the expression of granzyme genes after IL-2,IL-12,IL-15 or IFNαtreatment.2.Western blot:Western blot was used to analysis GZM-B protein expression after IL-2 or IFNαtreatment.3.MACS:MACS was performed to isolate and purify human peripheral blood NK cells.4.FACS:FACS was used to determine the purity of human peripheral blood NK cells by MACS technique.5.siRNA transfection:siRNA was used to silence GZM-B gene expression.6.MTT:MTr was used to determine the cytotoxity of NK-92 cells after treatment by IL-2 or IFNα.Results1.IL-2 up-regulated GZM-A and GZM-B gene transcription in human NK-92 cells2.Gene transcription of GZM-A,B were up-regulated by IL-15,while GZM-H was down regulated in human NK-92 cells3.GZM-A and GZM-B were up-regulated in human NK-92 cells by IL-124.The effects of IFNαon gene expression of granzymes in NK-92 cells.5.The features of IL-2,IL-12,IL-15 and IFNαon granzymes gene regulation were different.6.IL-2 or IFNαup-regulated GZM-B protein level in human NK-92 cells 7.IL-2 or IFNαcan enhance cytotoxicity of NK-92 cells against K562 cells partly dependent on GZM-B.8.IL-2 or IFNαup-regulated GZM-A and B gene level in human peripheral blood NK cells.ConclusionGZM-B is the most sensitive one to all four cytokines when compared to other granzyme members,especially in response to IL-2 stimulation,and IL-2 seems to be the most efficient stimulator of GZM-B expression.IL-15 mainly stimulates both GZM-A and GZM-B expression.At the same time,the expression of GZM-H was dramatically down-regulated in response to IL-15,while IL-2,IL-12 and IFNαdidn't show detectable effect on GZM-H.Compared with IL-15,the responses of both GZM-A and GZM-B to IL-12 were quicker and the augment levels were similar. Different from interleukins(IL-2,IL-12 and IL-15),IFNαalso up regulate GZM-K mRNA transcripts in a 4-fold increase in addition to GZM-A and GZM-B up-regulation.IL-2 or IFNαcan enhance cytotoxicity of NK92 against K562 cells partly dependent on GZM-B.IL-2 or IFNαup-regulated GZM-A and B gene levels in human peripheral blood NK cells.In summary,though the four cytokines(IL-2,IL-12,IL-15 and IFNα)have overlapping roles in promoting the gene expression of granzymes in NK-92 cells, each cytokine appeared to be distinct from each other in aspects of their regulatory effects.The display of these unique features of these cytokines on gramzymes gene expression will help to explain their distinct functions on NK cells.
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