1、英语国际会议英语国际会议PPT2CONTENTSCONTENTSIntroductionResultsMaterials and MethodsDiscussion6Present studyRecent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines.Two main subfamilies,recently renamed chemokine(C-C motif)ligand(CCL)and chemokine(C-X-C motif)l
2、igand(CXCL)chemo-kines,have been distinguished.Several studies have suggested a crucial involvement of CCL chemokines in inflammation.Chemokines and their receptors are implicated in the development of symptoms of AD and psoriasis.CXCL10 plays a role in pathogenesis of psoriasis.7PurposeThe effects
3、of-toxin on human macrophages still remain unclear.We show here for the first time that -toxin induces the Th1-related chemokine CXCL10 in human macrophages.we studied the effects of-toxin on Th1-and Th2-related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnorm
4、al and different chemokines production profile is well defined.8Materials and MethodsMaterials and MethodsPatientsPatientsCell isolation and cultureStatistical analysisCytokine assessment by ELISAWestern blot9Methodsmacrophage-derived chemokine(MDC)/CCL22 Cell surface markers expression and chemotax
5、is IFN-c-induced protein of 10-kDa(IP-10)/CXCL10qRTPCR or ELISA flow cytometry Boyden chamber technique10Patients healthy donors and patients with AD or psoriasis Cell isolation and culturePeripheral blood mononuclear cells(PBMCs)were isolated by Lymphoprep density-gradient centrifugation from healt
6、hy donors and from patients with AD and psoriasisChemotaxis assay The chemotactic activity of supernatants from a-toxin stimulated macrophages on lymphocytes(CD4+T cells)was determined using a Boyden chamber technique 11Western blot macrophages were prestimulated with a-toxin(100 ng/ml),IFN-c(10 ng/
7、ml),TNF-a(10 ng/ml)or LPS.Cell extracts were subjected to Western blot analysis Cytokine assessment by ELISACell-free culture supernatants were harvested and analysed for CXCL10 or CCL22,using a commercially available enzyme-linked immunosorbent assay(ELISA)system.12ResultsResultsOneOne Induction of
8、 IP-10/CXCL10 by staphylococcal a-toxin in human macrophagesThreeThreeLow effect of a-toxin on CXCL10 induction(Th1-related chemokine)in macrophages from patients with ADTwoTwo a-Toxin-stimulated macrophages could induce the migration of human CD4+T cells via CXCR3FourFour Effect of a-toxin on MDC p
9、roduction(Th2-related chemokine)in macrophages from patients with chronic inflammatory skin diseases13Induction of IP-10/CXCL10 by Induction of IP-10/CXCL10 by staphylococcal a-toxin in human staphylococcal a-toxin in human macrophagesmacrophagesMicroarray analysis revealed that CXCL10 was the most
10、strongly up-regulated gene in macrophages.An increased expression was not observed for other chemokines.The mean ratio for all these chemokines was between 0.2 and 2 when comparing not stimulated with a-toxin-stimulated macrophages.14 Figure 1 Induction of chemokine(C-X-C motif)ligand(CXCL)10 follow
11、ing a-toxin stimulation in human macrophages at the mRNA level.Figure 2 Induction of chemokine(C-X-C motif)ligand(CXCL)10 following a-toxin stimulation in human macrophages at the protein level.15 Figure 3 Punch biopsies(3 mm)from healthy individuals were left either unstimulated(A)or stimulated wit
12、h a-toxin(100 ng/ml)(B)or IFN-c(100 ng/ml)(C)for 24 h at 37C.5-lm paraffin sections were stained for CXCL10 along with appropriate isotype as well as CD68.16 a-Toxin-stimulated macrophages could a-Toxin-stimulated macrophages could induce the migration of human CD4+T induce the migration of human CD
13、4+T cells via CXCR3cells via CXCR3Both inhibitors(anti-IP-10 antibody&anti-CXCR3 antibody)abrogated the migration induced by a-toxin-treated macrophage supernatants.a-Toxin in cell culture medium without macrophages did not directly induce the migration of human CD4+lymphocytes.17 Figure 4 Chemotact
14、ic activity of a-toxin-stimulated macrophages and inhibition of chemokine(C-X-C motif)ligand(CXCL)10-induced chemotaxis using a neutralizing anti-CXCL10 or anti-CXCR3 Ab.18Low effect of a-toxin on CXCL10 Low effect of a-toxin on CXCL10 induction(Th1-related chemokine)in induction(Th1-related chemoki
15、ne)in macrophages from patients with ADmacrophages from patients with AD a-toxin induced significantly lower levels of CXCL10 expression or secretion in patients with AD compared with healthy controls as well as psoriasis at all time points and doses tested.19 Figure 5 Macrophages from patients with
16、 atopic dermatitis(AD)and psoriasis(PSO)as well as from healthy controls(HC)were left either unstimulated(medium control)or stimulated with a-toxin as indicated or with IFN-c(10 ng/ml)or TNF-a(20 ng/ml),respectively,for the indicated periods of time.20 Effect of a-toxin on MDC production Effect of a
17、-toxin on MDC production(Th2-related chemokine)in(Th2-related chemokine)in macrophages from patients with chronic macrophages from patients with chronic infammatory skin diseasesinfammatory skin diseasesMacrophages from patients with AD as well as patients with psoriasis showed an intrinsically higher CCL22 production compared to healthy controls by trend.On the whole,a-toxin did not regulate CCL22 secretion.21Thank you
