Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA, and CNS tissue was harvested at various time-points corresponding to milestones in disease development. (A) Gene expression analysis was performed by Agilent whole mouse genome array on laser capture microdissected tissues. The data are expressed as fold induction in immunized mice compared with naïve controls. The results indicate high CCL22 expression in CNS lesion and peri-lesion areas. (B) Real-time RT-PCR analysis of CCL22 expression was conducted on spinal cord homogenates corresponding to the indicated times after and associated with disease development using SYBR Green chemistry. The data are expressed as mean (±sd) fold induction from individual mice (n=3) calculated by the Δ comparative threshold method relative to the housekeeping gene G3PDH. (C) CCL22 protein expression in the spinal cord was determined by assessing CNS homogenates from PLP139–151-immunized mice at the indicated time-points after disease induction by ELISA. The data are expressed as mean (±sd) from individual mice (n=3) at each time-point. *P < 0.05 for all experiments where statistical analysis was performed. These results are representative of multiple experimental replicates with similar results.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Gene Expression, Expressing, Quantitative RT-PCR, SYBR Green Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA at Day 0 and treated with normal goat Ig or polyclonal anti-murine CCL22 at Days –2 and 0 relative to immunization. (A) Anti-CCL22 treatment significantly (*P<0.05) inhibited acute and relapsing clinical disease. The data represent the mean clinical disease score at each specific day postimmunization. (B) In the same experiment, mean cumulative clinical score (±sd) was calculated, and there was a significant (*P<0.05) reduction in the anti-CCL22-treated group. (C) The mean number of relapses (±sd) was calculated for each group, and there was a significant (*P<0.05) reduction in the anti-CCL22-treated mice. The clinical results are representative of three independent experiments. (D) Anti-CCL22 treatment at Day +10, relative to disease induction, did not significantly inhibit acute clinical disease. The data represent the mean clinical disease score at each specific day postimmunization.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques:

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Representative photomicrograph (original magnification, 100×) of the H&E-stained lumbar spinal cord section from a control Ig-treated mouse. The data indicate extensive mononuclear cell infiltration (arrows). (B) Representative photomicrograph (original magnification, 100×) of a H&E-stained lumbar spinal cord section from an anti-CCL22-treated mouse. The data indicate a lack of mononuclear cell infiltration. (C) Representative flow cytometric analysis of CNS-infiltrating mononuclear cells from a control Ig-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 38% lymphocytes, 40% macrophages/DCs, and 11% microglia. (D) Representative flow cytometric analysis of CNS-infiltrating mononuclear cells from an anti-CCL22-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 10% lymphocytes, 2% macrophages/DCs, and 83% microglia. (E) Representative flow cytometric analysis of CNS-infiltrating CD4 and CD8 T cells from a control Ig-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 40% CD4 T cells and 4% CD8 T cells. (F) Representative flow cytometric analysis of CNS-infiltrating CD4 and CD8 T cells from an anti-CCL22-treated mouse. The dot plots are derived from a combined forward- and side-scatter and CD45+ gate. The percentages of cells in each quadrant are indicated: 8% CD4 T cells and 3% CD8 T cells. (G) Quantification of the total number of CNS-infiltrating CD4 and CD8 T cells and macrophages. Individual mice (n=5) in control- and anti-CCL22-treated groups were assessed for the presence of mononuclear cells in the spinal cord at the time of peak clinical disease in the control-treated group by flow cytometry. The data are expressed as the mean cell number (±sd) and demonstrate significantly (*P<0.05) fewer CD4 and CD8 T cells and macrophages in the anti-CCL22-treated mice. The results are representative of three independent histologic experiments and two independent flow cytometric analyses.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Staining, Control, Derivative Assay, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) CNS-infiltrating CD11b+Ly6Chi macrophages from acute EAE express CCR4. Mice were immunized to develop EAE, and at the peak of acute clinical disease, the CNS mononuclear cells were harvested and assessed for CCR4 expression by flow cytometry. The results are shown for a representative animal (out of a total of five), the data are derived from a CD45hiCD11b+ gate, and the results show CCR4 expression as a function of Ly6Chi expression. (B) CNS-infiltrating CD11b+Ly6Chi macrophages from relapsing EAE express CCR4. Mice were immunized to develop EAE, and at the peak of relapsing clinical disease, the CNS mononuclear cells were harvested and assessed for CCR4 expression by flow cytometry. The results are shown for a representative animal (out of the remaining five), the data are derived from a CD45hiCD11b+ gate, and the results show CCR4 expression as a function of Ly6Chi expression. (C) The presence of Ly6ChiCD11b+ macrophages was assessed in the CNS of control Ig-treated mice at peak clinical disease using flow cytometry. The results are displayed from a representative animal (out of a total of three) as the percentage of total CD11b+ macrophages out of the CD45+ gate (R1=37%) and the percentage of only Ly6Chi cells from the CD45+ gate (R2=18%). (D) The presence of Ly6ChiCD11b+ macrophages was assessed in the CNS of anti-CCL22-treated mice at the time controls were showing peak clinical disease using flow cytometry. The results are displayed from a representative animal (out of a total of three) as the percentage of total CD11b+ macrophages out of the CD45+ gate (R3=22%) and the percentage of only Ly6Chi cells from the CD45+ gate (R4=5%). The results are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Expressing, Flow Cytometry, Derivative Assay, Control

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: Mice were immunized with PLP139–151 in CFA and treated with control Ig or anti-CCL22 at Days –2 and 0 relative to antigen priming. At the indicated time-points, three mice/group/time were assessed for proliferative and cytokine responses. (A) Splenic PLP139–151-specific T cell-proliferative responses were determined from control- or anti-CCL22-treated mice at Day 10 postimmunization and at times corresponding to peak clinical disease, remission, and peak-relapsing disease. The data are displayed as mean cpm (±sd), and there are no significant differences at any time-point measured. (B) Splenic PLP139–151-specific IFN-γ T cell responses were determined using ELISPOT analysis of cells from each group of mice at 10 days postimmunization. The data are displayed as mean number of cytokine-secreting cells (±sd) at each dose of peptide (0, 5, 50, and 100 nM). There are no significant differences between the two groups at any peptide dose measured. (C) Splenic, PLP139–151-specific IL-17 T cell responses were determined using ELISPOT analysis of cells from each group of mice at 10 days postimmunization. The data are displayed as mean number of cytokine-secreting cells (±sd) at each dose of peptide (0, 5, 50, and 100 μM). There are no significant differences between the two groups at any peptide dose measured. (D) The presence of Tregs in the LN and spleen was assessed by flow cytometric analysis of Foxp3 expression by CD4+CD25+-gated lymphocytes from control- or anti-CCL22-treated mice at the time when control mice showed clinical disease. The data are expressed as mean percentage of Foxp3+ cells from the CD4+ T cell population (±sd) in the LN and spleen of control (n=3)- and anti-CCL22-treated mice (n=3). There are no significant differences between the two groups in either organ analyzed. These results are representative of at least two independent experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Control, Enzyme-linked Immunospot, Expressing

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Two groups of recipient mice (n=10) received i.v. injections of 5 × 106 PLP139–151-primed and reactivated lymphocyte blasts and were treated with control or polyclonal anti-murine CCL22 at Days 4 and 6 relative to T cell transfer. The data represent the mean clinical disease score at each specific day post-cell transfer and significantly demonstrate (*P<0.05) disease development in the anti-CCL22-treated group. (B) The CNS of control- or anti-CCL22-treated mice was evaluated by flow cytometry for leukocyte infiltration. The results show a decreased percentage of lymphocytes (CD45hiCD11b–) and macrophages (CD45hiCD11b+) in a representative anti-CCL22-treated animal compared with control. (C) The CNS of control- or anti-CCL22-treated mice was evaluated by flow cytometry for inflammatory macrophage infiltration. The results show a decreased percentage of inflammatory macrophages (CD45hiF4/80+Ly6Chi) in a representative anti-CCL22-treated animal compared with control. (D) The number of CNS-infiltrating leukocytes was determined in control- and anti-CCL22-treated mice (n=5, each group). The results show a significantly (*P<0.05) decreased number of total leukocytes, macrophages, and Ly6Chi inflammatory macrophages in the CNS of anti-CCL22-treated mice. (E) CNS-infiltrating Th1 and Th17 cells from mice with EAE do not express CCR4. The results shown are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Control, Flow Cytometry

Journal: Journal of Leukocyte Biology

Article Title: CCL22 regulates experimental autoimmune encephalomyelitis by controlling inflammatory macrophage accumulation and effector function

doi: 10.1189/jlb.0810442

Figure Lengend Snippet: (A) Cytokine expression patterns from CD11b+Ly6Chi macrophages from control- or anti-CCL22-treated mice were determined by ELISA from cells sorted from control- or anti-CCL22-treated mice. The data are displayed as mean cytokine production (±sd) and indicate a significantly (*P<0.05) decreased TNF level and a significantly (P<0.05) increased IL-10 level by CD11b+Ly6Chi macrophages from anti-CCL22- compared with control-treated mice. (B) Stimulation of Ly6Chi macrophages with CCL22 and LPS together results in significantly increased TNF and significantly (*P<0.05) decreased IL-10 compared with macrophages. These results are representative of two similar experimental replicates.

Article Snippet: Biotinylated goat anti-mouse CCL22 (5 ng/well; R&D Systems) was added, and plates were incubated for 1 h at 37°C.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: Methods in Molecular Biology

Article Title: Allergy

doi: 10.1007/978-1-4939-9591-2

Figure Lengend Snippet: Fig. 1 Different types of immunoelectrophoresis of cat hair extract (Fel d) using a polyclonal rabbit antibody raised against the same extract (a Fel d). (a) CIE of Fel d extract, (b) FRIE following a purification (immunoab- sorption) of the major allergen Fel d 1, (c) CIE of purified Fel d 1 against a Fel d, (d) CLIE with Fel d 1 in inter- mediate gel for identification of the allergen, (e) TCIE with Fel d extract and Fel d 1 showing a double peak, (f) CIIE with monospecific Fel d 1 in intermediate gel

Article Snippet: Detection: Biotinylated chicken anti-human MDC polyclonal (R&D Systems #BAF336 or PeproTech).

Techniques: Immunoelectrophoresis, Purification

Journal: The Journal of Experimental Medicine

Article Title: Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

doi: 10.1084/jem.20082901

Figure Lengend Snippet: Peptide immunotherapy inhibits levels of Th2 cytokines, Th2 chemokines, and serum IgE levels. Mice were sacrificed at day 61. Serum was obtained by cardiac puncture, BAL was performed to obtain BAL fluid, and single lung lobes were harvested and enzymatically digested. (a) BAL fluid and (b) lung digest homogenate cytokines IL-4, IL-5, IL-13, and IFN-γ were measured by ELISA according to manufacturer’s protocols. Th2 chemokines CCL11/eotaxin, CCL17/TARC, and CCL22/MDC in BAL fluid (c) and lung digest homogenates (d) were measured by ELISA according to the manufacturer’s protocols. Total IgE in serum samples (e) was quantified by ELISA using paired antibodies according to the manufacturer’s protocol. Levels of Fel d 1–specific IgE (f) were measured in serum by coating plates with recombinant Fel d 1 (5 µg/ml), incubating with serum samples, and detecting bound IgE with chromogen-labeled anti-IgE. Data are expressed as mean ± SEM. n = 5 mice/group, and are representative of three independent experiments. *, P < 0.05 and **, P < 0.01 for sensitized/HA (306–318) compared with sensitized/DR1 Feld1 -treated mice.

Article Snippet: CCL22 levels were measured using a sandwich ELISA generated by coating ELISA plates with anti–mouse MDC (a gift from ICOS Corp.) and detecting bound antibody with biotinylated anti–mouse MDC (R&D Systems) against a standard curve generated using recombinant MDC (R&D Systems) as previously described ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Labeling

Journal: The Journal of Experimental Medicine

Article Title: Peptide immunotherapy in allergic asthma generates IL-10–dependent immunological tolerance associated with linked epitope suppression

doi: 10.1084/jem.20082901

Figure Lengend Snippet: Peptide immunotherapy inhibits levels of Th2 cytokines, Th2 chemokines, and serum IgE levels. Mice were sacrificed at day 61. Serum was obtained by cardiac puncture, BAL was performed to obtain BAL fluid, and single lung lobes were harvested and enzymatically digested. (a) BAL fluid and (b) lung digest homogenate cytokines IL-4, IL-5, IL-13, and IFN-γ were measured by ELISA according to manufacturer’s protocols. Th2 chemokines CCL11/eotaxin, CCL17/TARC, and CCL22/MDC in BAL fluid (c) and lung digest homogenates (d) were measured by ELISA according to the manufacturer’s protocols. Total IgE in serum samples (e) was quantified by ELISA using paired antibodies according to the manufacturer’s protocol. Levels of Fel d 1–specific IgE (f) were measured in serum by coating plates with recombinant Fel d 1 (5 µg/ml), incubating with serum samples, and detecting bound IgE with chromogen-labeled anti-IgE. Data are expressed as mean ± SEM. n = 5 mice/group, and are representative of three independent experiments. *, P < 0.05 and **, P < 0.01 for sensitized/HA (306–318) compared with sensitized/DR1 Feld1 -treated mice.

Article Snippet: CCL22 levels were measured using a sandwich ELISA generated by coating ELISA plates with anti–mouse MDC (a gift from ICOS Corp.) and detecting bound antibody with biotinylated anti–mouse MDC (R&D Systems) against a standard curve generated using recombinant MDC (R&D Systems) as previously described ( ).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Labeling