Effects Of Immunosuppressants And Ultraviolets On Secretion Of Chemokines By Keratinocytes

IntroductionIFN-inducible T cellαchemoattractant(CXCL11/I-TAC)and monokine induced by gamma-interferon(CXCL9/Mig)are type 1 chemokines which specifically chemoattracts CXCR3-expressing Th1/Tc1 cells.Macrophage-derived chemokine (CCL22/MDC)is a type 2 chemokine which specifically chemoattracts CCR4-expressing Th2/Tc2 cells.In recent years,there have been increasing evidences that CXCL11/I-TAC and CXCL9/Mig are overexpressed in Th1-dominated skin disorders such as lichen planus and that CCL22/MDC is highly expressed in a Th2-dominated skin disorder,atopic dermatitis(AD).Th1 cytokine IFN-γand TNF-αare overexpressed and play important roles in both Th1-dominated skin diseases and chronic phase of AD lesions.In previous studies,the two cytokines have been shown to be strong inducers of CXCL11/I-TAC,CXCL9/Mig or CCL22/MDC in keratinocytes.Immunosuppressants and phototherapy have been effective in treating both Th1-and Th2-dominated skin diseases.However,the effects of immunosuppressants or ultraviolets on chemokine secrection by keratinocytes has not been investigated.ObjectiveTo investigate the effects of three immunosuppressants,dexamethasone, methotrexate,triptolide as well as ultraviolet A(UVA)and narrow-band ultraviolet B (NB-UVB)on CXCL11/I-TAC,CXCL9/Mig or CCL22/MDC secretion by a human keratinocyte cell line HaCaT.Materials and MethodsCell culture HaCaT cells were cultured at 37℃,5%CO₂ in RPMI 1640 containing 10%fetal bovine serum(FBS)and 100 U/ml penicillin-streptomycin(Hyclone,USA).When confluence was achieved,the cells were trypsinized,washed,resuspended in RPMI 1640 with 10%FBS at 1×10⁶ cells/ml,and 1 ml was added to each well of the six-well plates.When the cells reached confluence,the medium was completely removed,washed twice,and 1 ml RPMI 1640 without FBS was added to each well. Then,cytokines or drugs were added immediately.Recombinant IFN-γand TNF-αwere from PeproTech(London,UK).Dexamethasone and methotrexate were from Sigma(Saint Louis,MI).Triptolide was from Institute of Dermatology,Chinese Academy of Medical Sciences(Nanjing,China).Ultraviolet irradiationFor UV irradiations,we used a SS-03AB UV radiator(Xigema,Shanghai,China). UVA was at a peak of 365 nm in wavelength and irradiated by TLK 40W/10R lamps (Philips,Holland).NB-UVB was 311 nm±1 nm in wavelength and irradiated by TL 20W/01RS lamps(Philips,Holland).Reverse transeription-polymerase chain reactionTotal RNA was isolated using a TRIzol Reagent(Invitrogen,Carlsbad,CA) according to the manufacturer's instructions.Reverse transcription and polymerase chain reaction(RT-PCR)was performed using a RNA PCR Kit Ver 2.1(Takara,Japan). I-TAC upstream primer:5'-GCT ATA GCC TTG GCT GTG ATA TTG TG-3';I-TAC downstream primer:5'-CTG CCA CTT TCA CTG CTT TTA CC-3'.The mixture was predenaturated for 2 rain at 94℃and then subjected to 28 cycles:94℃0.5 min,60℃0.5 min,72℃1 min.Amplified DNA fragments were judged from 2.5%agarose gel electrophoresis.Humanβ-actin was used as control primer.β-actin upstream primer: 5'-ACA CTG TGC CCA TCT ACG AGG GG-3';β-actin downstream primer:5'- ATG ATG GAG TTG AAG GTA GTT TCG TGG AT-3'.The expected fragment length was 218 bp for I-TAC and 340 bp forβ-actin.Enzyme-linked immunosorbent assay(ELISA)Supernatant chemokine levels were detected using human CXCL11/I-TAC, CXCL9/Mig or CCL22/MDC ELISA kits(R&D Systems,Minneapolis,MN)according to the manufacturer' protocol.Optical density was measured at 450 nm with a microplate ELISA reader(Model 550,Bio-Rad Laboratories Inc.,Hercules,CA).Cell viabilityAfter culture for 24 hrs,supematants were collected,centrifuged and stored at -70℃.Cells adhering to the bottom of the plates were trypsinized,collected.Cell viability was evaluated by trypan blue dye exclusion.Each of the experiments was repeated at least three times.The cells were tested in duplicate in each independent experiment.Statistical analysisData were analyzed using the Student's t-test.A P value less than 0.05 was considered to be statistically significant.ResultsI-TAC mRNA in HaCaT cells was upregulated by IFN-γor TNF-αReverse transcription-polymerase chain reaction(RT-PCR)revealed that CXCL11/I-TAC mRNA expression was not seen when HaCaT cells were cultured without any stimulations.When cultured with IFN-γ(10 ng/ml)or TNF-α(10 ng/ml), CXCL11/I-TAC mRNA expression was upregulated.However,when cultured with dexamethasone(10 ng/ml),methotrexate(10 ng/ml),triptolide(1 ng/ml),UVA(4 J/cm²),NB-UVB(0.01 J/cm²)or NB-UVB(0.1 J/cm²),I-TAC mRNA expression was not induced.Effect of dexamethasone,methotrexate,triptolide,UVA and NB-UVB on chemokine secretion by HaCaT cellsWithin certain dose ranges,dexamethasone,methotrexate,triptolide or UVA inhibited the IFN-γand TNF-αenhanced CXCL11/I-TAC,CXCL9/Mig or CCL22/MDC secretion(P＜0.05).NB-UVB showed either upregulatory or downregulatory effects on the chemokine secretion at different tested doses.Cell viability At the above dexamethasone,methotrexate,triptolide,UVA or NB-UVB doses,24 h cultured HaCaT cells showed no significant changes in cell viabilities.The percentages of viable cells,based on three counts per plate,and two plates per treatment,were not significantly different among the unstimulated controls,the IFN-γand TNF-αtreated HaCaT cells and the dexamethasone,methotrexate,triptolide,UVA or NB-UVB treated HaCaT cells.ConclusionsThis is the first report describing the regulation of CXCL11/I-TAC,CXCL9/Mig or CCL22/MDC secretion from HaCaT cells by immunosuppressants and ultraviolets. The therapeutic mechanism of immunosuppressants and ultraviolets in Th1/Tc1-or Th2/Tc2-dominated inflammatory skin diseases may be associated with their effects on keratinocytes through inhibiting the secretion of type 1 or type 2 chemokines, consequently inhibiting Th1/Tc1 or Th2/Tc2 cells entering the skin.