英语国际会议PPT
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4
Background:
•Patients with atopic dermatitis (AD) and psoriasis are frequently colonized with Staphylococcus aureus (S. aureus). one-third of them producing α -toxin, which is cor-related with the severity of eczema in AD. • Distinct expression of chemokine (C-C motif) ligand (配体CCL) and chemokine (C-X-C motif) ligand (配体CXCL) chemokines has been documented in both diseases.
Chemotaxis assay
The chemotactic activity of supernatants from a-toxin stimulated macrophages on lymphocytes (CD4+ T cells) was determined using a Boyden chamber technique
15
a-Toxin-stimulated macrophages could induce the migration of human CD4+ T cells via CXCR3
Both 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.
14
Figure 3 Punch biopsies (3 mm) from healthy individuals were left either unstimulated (A) or stimulated with 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.
• the effects of a-toxin on human macrophages in patients with AD and psoriasis?
5
Present study
Recent 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) ligand (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 .
Macrophages from patients with atopic dermatitis show a reduced CXCL10 expression in response to staphylococcal a-toxin.
1
Introduction
Materials and Methods
17
Low effect of a-toxin on CXCL10 induction (Th1-related chemokine) in macrophages 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.
flow cytometry Boyden chamber technique
9
Hale Waihona Puke Baiduatients
Cell isolation and culture
healthy donors and patients with AD or psoriasis
Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density-gradient centrifugation from healthy donors and from patients with AD and psoriasis
IFN-c-induced protein of 10-kDa (IP10)/CXCL10 qRT–PCR or ELISA macrophage-derived chemokine (MDC)/CCL22
Cell surface markers’ expression and chemotaxis
10
Western blot
macrophages were prestimulated with a-toxin (100 ng/ml), IFN-c (10 ng/ml), TNF-a (10 ng/ml) or LPS. Cell extracts were subjected to Western blot analysis Cell-free culture supernatants were harvested and analysed for CXCL10 or CCL22 , using a commercially available enzyme-linked immunosorbent assay (ELISA) system .
11
Cytokine assessment by ELISA
Results
One Induction of IP10/CXCL10 by staphylococcal a-toxin in human macrophages Two a-Toxin-stimulated macrophages could induce the migration of human CD4+ T cells via CXCR3 Four Effect of a-toxin on MDC production (Th2-related chemokine) in macrophages from patients with chronic inflammatory skin diseases
Microarray analysis revealed that CXCL10 was the most strongly upregulated 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 .
12
Three Low effect of a-toxin on CXCL10 induction (Th1related chemokine) in macrophages from patients with AD
Induction of IP-10/CXCL10 by staphylococcal a-toxin in human macrophages
16
Figure 4 Chemotactic 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.
6
Purpose
The effects of α-toxin on human macrophages still remain unclear. We show here for the first time that α-toxin induces the Th1related chemokine CXCL10 in human macrophages. we studied the effects of α-toxin on Th1- and Th2-related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.
13
Figure 1 Induction of chemokine (C-X-C motif) ligand (CXCL)10 following 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.
7
Materials and Methods
Materials and Methods Patients Cytokine assessment by ELISA Western blot
Cell isolation and culture
Statistical analysis
8
Methods
Results
Discussion
2
Introduction
keywords background present study purpose
3
Keywords
• atopic dermatitis; (特应性皮炎) • chemokines; （趋化因子） • human macrophages; （人巨噬细胞） • IP-10/CXCL10; • MDC/CCL22; • psoriasis; （牛皮癣；银屑病） • Staphylococcus aureus; （金黄色葡萄球菌） • α -toxin.（α -毒素）
Background:
•Patients with atopic dermatitis (AD) and psoriasis are frequently colonized with Staphylococcus aureus (S. aureus). one-third of them producing α -toxin, which is cor-related with the severity of eczema in AD. • Distinct expression of chemokine (C-C motif) ligand (配体CCL) and chemokine (C-X-C motif) ligand (配体CXCL) chemokines has been documented in both diseases.
Chemotaxis assay
The chemotactic activity of supernatants from a-toxin stimulated macrophages on lymphocytes (CD4+ T cells) was determined using a Boyden chamber technique
15
a-Toxin-stimulated macrophages could induce the migration of human CD4+ T cells via CXCR3
Both 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.
14
Figure 3 Punch biopsies (3 mm) from healthy individuals were left either unstimulated (A) or stimulated with 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.
• the effects of a-toxin on human macrophages in patients with AD and psoriasis?
5
Present study
Recent 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) ligand (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 .
Macrophages from patients with atopic dermatitis show a reduced CXCL10 expression in response to staphylococcal a-toxin.
1
Introduction
Materials and Methods
17
Low effect of a-toxin on CXCL10 induction (Th1-related chemokine) in macrophages 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.
flow cytometry Boyden chamber technique
9
Hale Waihona Puke Baiduatients
Cell isolation and culture
healthy donors and patients with AD or psoriasis
Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep density-gradient centrifugation from healthy donors and from patients with AD and psoriasis
IFN-c-induced protein of 10-kDa (IP10)/CXCL10 qRT–PCR or ELISA macrophage-derived chemokine (MDC)/CCL22
Cell surface markers’ expression and chemotaxis
10
Western blot
macrophages were prestimulated with a-toxin (100 ng/ml), IFN-c (10 ng/ml), TNF-a (10 ng/ml) or LPS. Cell extracts were subjected to Western blot analysis Cell-free culture supernatants were harvested and analysed for CXCL10 or CCL22 , using a commercially available enzyme-linked immunosorbent assay (ELISA) system .
11
Cytokine assessment by ELISA
Results
One Induction of IP10/CXCL10 by staphylococcal a-toxin in human macrophages Two a-Toxin-stimulated macrophages could induce the migration of human CD4+ T cells via CXCR3 Four Effect of a-toxin on MDC production (Th2-related chemokine) in macrophages from patients with chronic inflammatory skin diseases
Microarray analysis revealed that CXCL10 was the most strongly upregulated 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 .
12
Three Low effect of a-toxin on CXCL10 induction (Th1related chemokine) in macrophages from patients with AD
Induction of IP-10/CXCL10 by staphylococcal a-toxin in human macrophages
16
Figure 4 Chemotactic 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.
6
Purpose
The effects of α-toxin on human macrophages still remain unclear. We show here for the first time that α-toxin induces the Th1related chemokine CXCL10 in human macrophages. we studied the effects of α-toxin on Th1- and Th2-related chemokines in macrophages from patients with AD and psoriasis where the intrinsic abnormal and different chemokines production profile is well defined.
13
Figure 1 Induction of chemokine (C-X-C motif) ligand (CXCL)10 following 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.
7
Materials and Methods
Materials and Methods Patients Cytokine assessment by ELISA Western blot
Cell isolation and culture
Statistical analysis
8
Methods
Results
Discussion
2
Introduction
keywords background present study purpose
3
Keywords
• atopic dermatitis; (特应性皮炎) • chemokines; （趋化因子） • human macrophages; （人巨噬细胞） • IP-10/CXCL10; • MDC/CCL22; • psoriasis; （牛皮癣；银屑病） • Staphylococcus aureus; （金黄色葡萄球菌） • α -toxin.（α -毒素）