Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 1. Semiquantitative RT-PCR analysis for expression of CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples were prepared from salivary glands, colon, appendix, small intestine with- out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old BALB/c mice. RT-PCR was conducted as described in Materials and Methods. Representative results from three separate experiments are shown. Relative signal intensities obtained by normalization with G3PDH are shown in the lower panels as mean SD.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3B220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After 4 h at 37°C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3B220low cells; open bars, CD3B220 cells. The data are mean SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¨nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, 1000.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Expressing, Migration, Isolation, Incubation, Control, Labeling, Staining, Chemotaxis Assay, Cytometry, Clinical Proteomics, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- maldehyde-prefixed frozen sections of human submandibular gland (A–C, J, and K), mouse parotid gland (D and G), mouse submandibular gland (E and F), and mouse sublingual gland (F and I) were stained with goat anti- human CCL28 (A, B, and J), goat anti-mouse CCL28 (D–F), or normal goat IgG (C, G–I, and K). Immunohistochemistry (A–I): scale bar, 100 m (A–C) and 20 m (D–I); , acinus; , duct. Immunoelectron microscopy (J and K): scale bar, 1 m; arrow, immunoreactive granule; Lu, lumen.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant CCL28, parotid saliva samples (10 l), and mature milk samples (20 l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial peptides. MEC/CCL28 and CCL28-C are boxed.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Sequencing, Comparison, Binding Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells were stained with FITC-labeled annexin V for 10 min and were washed again. After addition of PI at 2 g/ml, cells were immediately analyzed on FACSCalibur. The representative results of three independent experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Staining, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning electron microscopy. C. albicans (A–F) was mock treated (A and D) or treated with 10 M CCL28 for 30 min (B and E) or 60 min (C and F). P. aeruginosa (G and H) and Streptococcus mutans (I and J) were mock treated (G and I) or treated with 10 M CCL28 for 2 h (H and J). Microbes were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A, 2 m; D, 200 nm; and G, 300 nm.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Electron Microscopy, Microscopy

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects of NaCl concentrations on the antimicrobial activity of CCL28 against C. albicans and P. aeruginosa were examined by using CFU assay. For de- tails, see Materials and Methods. All assays were done in triplicate. Ver- tical bars indicate SD. Representative results from two separate experi- ments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 2 Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 3 Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 6 CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 7 A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: CCR10-transfected HepG2 and LO2 cells were treated with either the CCR10 agonist-ligand CCL28 or the Akt inhibitor A6730. a Activation of the CCL28-CCR10 axis by CCL28 significantly increased Akt phosphorylation, PCNA protein expression, and b relative cell proliferation in both cell lines, while Akt inhibition produced the opposite effects. Relative cell proliferation is defined as the fold-change in proliferation relative to the untreated parent cell line. * P < 0.05 vs. CCR10 group. All values are reported as means ± standard errors of the mean (SEMs)

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Transfection, Activation Assay, Phospho-proteomics, Expressing, Inhibition, Produced

Journal: Cell Death & Disease

Article Title: The chemokine receptor CCR10 promotes inflammation-driven hepatocarcinogenesis via PI3K/Akt pathway activation

doi: 10.1038/s41419-018-0267-9

Figure Lengend Snippet: Following short-term DEN-induced inflammation (10 days after i.p. DEN injection), ( a ) Western blotting analysis showed significantly enhanced TNF protein expression, CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Knocking-out CCR10 significantly opposed these inflammation-induced effects but did not significantly affect TNF or PI3K protein expression.* P < 0.05 vs. vehicle WT group, † P < 0.05 vs. DEN-treated WT group. b Western blotting analysis of CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression in murine liver tissue 6 h after intraperitoneal (i.p.) injection of TNF, which produced significant increases in CCR10 protein expression, PI3K protein expression, Akt phosphorylation, and PCNA protein expression. Pretreatment with the CCR10 agonist-ligand CCL28 significantly increased Akt phosphorylation and PCNA expression levels, while pretreatment with the Akt inhibitor A6730 produced the opposite effects. Neither CCL28 nor A6730 had any significant effect upon CCR10 or PI3K expression. * P < 0.05 vs. vehicle group, † P < 0.05 vs. TNF group. All values are reported as means ± standard errors of the mean (SEMs). n = 12 mice in each group

Article Snippet: For some in vitro experiments, HepG2 and LO2 cell lines were pre-treated with the pro-inflammatory cytokine TNF (concentrations as indicated; R&D Systems, Minneapolis, MN, USA) for 4 h, recombinant human CC chemokine ligand 28 (CCL28) (400 nM; R&D Systems) for 2 h, or the allosteric Akt inhibitor A6730 (10 μM; Sigma, St. Louis, MO, USA) for 2 h . Human CCL28 is a natural ligand-agonist for human CCR10 .

Techniques: Injection, Western Blot, Expressing, Phospho-proteomics, Produced

Journal: Inflammatory Bowel Diseases

Article Title: B Cells Secrete Eotaxin-1 in Human Inflammatory Bowel Disease

doi: 10.1097/mib.0b013e3182802950

Figure Lengend Snippet: FIGURE 5. TLR4 ligands induce CCR9 and CCR10 expression by eosinophils. A, Histogram depicting levels of homing receptors in eosinophils. Gray fill, isotype control; thick black line, anti-CCR10; thin black line, anti-CCR9; gray line, anti-a4b7. Eosinophils from a patient with UC. B. CCR9 expression levels on peripheral blood eosinophils from healthy subjects (n ¼ 9), patients with CD (n ¼ 33), and patients with UC (n ¼ 16) and CCR10 expression levels on eosinophils from healthy subjects (n ¼ 9), patients with CD (n ¼ 22), and patients with UC (n ¼ 10), **P , 0.001, *P , 0.01, compared with healthy subjects. C, TLR4 expression by eosinophils correlates with CCR10 expression in patients with UC (n ¼ 22). D, Plasma concentrations of LPS correlate with percentages of eosinophils that express CCR10 in patients with UC (n ¼ 39). E, E. coli LPS increases CCR9 expression by purified eosinophils from patients with IBD. Gray fill, media alone; black line, 1 mg/mL E. coli LPS. Representative data from 5 blood samples (CD, n ¼ 3; UC, n ¼ 2). F, Eosinophils isolated from patients with IBD actively chemotaxis in response to CCL25 and CCL28. Treatment with E. coli LPS for 18 hours increased responses to CCL28 compared with untreated cells (P ¼ 0.015). Eosinophils from 2 patients with CD and 1 patient with UC.

Article Snippet: Bottom chambers contained recombinant human chemokines, CCL25 and CCL28 (R&D Systems), at 1000 ng/mL in R10.

Techniques: Expressing, Control, Clinical Proteomics, Isolation, Chemotaxis Assay

Journal: Inflammatory Bowel Diseases

Article Title: B Cells Secrete Eotaxin-1 in Human Inflammatory Bowel Disease

doi: 10.1097/mib.0b013e3182802950

Figure Lengend Snippet: FIGURE 6. Model depicting the possible effect of systemic LPS on eosinophil migration patterns in IBD. During decreased disease activity, hypo-acylated LPS induces eotaxin-1 release from TLR4+ B cells. Eotaxin-1 may direct circulating eosinophils to traffic to certain regions of the gastrointestinal tract or induce emigration of nascent eosinophils from the bone marrow. During increased disease activity, hexa-acylated LPS stimulates CCR9 and CCR10 expression by TLR4+ eosinophils. CCL25 and CCL28 are tissue expressed and may thus direct eosinophils to specific areas of the gastrointestinal tract other than those directed by eotaxin-1.

Article Snippet: Bottom chambers contained recombinant human chemokines, CCL25 and CCL28 (R&D Systems), at 1000 ng/mL in R10.

Techniques: Migration, Activity Assay, Expressing

Journal: Frontiers in medicine

Article Title: Activated Hepatic Stellate Cells Induce Infiltration and Formation of CD163 + Macrophages via CCL2/CCR2 Pathway.

doi: 10.3389/fmed.2021.627927

Figure Lengend Snippet: FIGURE 3 | The high level of CCL2 in aHSCs was associated with CD163+ macrophage infiltration and increased with liver fibrosis progression. (A) The primary aHSCs are typically fusiform and express the activation marker α-SMA, together with a high expression of CCL2 protein. (B) The aHSCs secrete high levels of CCL2 (Continued)

Article Snippet: When indicated, recombinant human CCL2 protein (rh CCL2, 2ng/ml, R&D Systems, Abingdon, UK) and INCB 3284 (100ng/ml, Tocris Bioscience, UK) were accordingly added, after which the macrophages were harvested, counted, and analyzed.

Techniques: Activation Assay, Marker, Expressing

Journal: Frontiers in medicine

Article Title: Activated Hepatic Stellate Cells Induce Infiltration and Formation of CD163 + Macrophages via CCL2/CCR2 Pathway.

doi: 10.3389/fmed.2021.627927

Figure Lengend Snippet: FIGURE 4 | CCL2 was responsible for macrophages infiltration and differentiation into M2 phenotype during liver fibrosis. (A) Representative images of macrophage infiltration under different chemotaxis treatments including aHSC, aHSC+INCB, Rh CCL2 and medium. (B) Statistical analysis of the number of macrophages (Continued)

Article Snippet: When indicated, recombinant human CCL2 protein (rh CCL2, 2ng/ml, R&D Systems, Abingdon, UK) and INCB 3284 (100ng/ml, Tocris Bioscience, UK) were accordingly added, after which the macrophages were harvested, counted, and analyzed.

Techniques: Chemotaxis Assay

Journal: Journal of reproductive immunology

Article Title: Detection of CCL25 and the correlation between CCL25, CCL28, IL-7, and TSLP in human breast milk.

doi: 10.1016/j.jri.2022.103783

Figure Lengend Snippet: Fig. 1. Comparison of chemokine and cytokine levels between colostrum and mature milk. The concentrations of chemokines and cytokines in colostrum and mature milk were analyzed: (a) CCL25, (b) CCL28, (c) IL-7, and (d) TSLP. The levels of each of the chemokines and cytokines were compared between colostrum and mature milk using Wilcoxon signed rank test. Medians of chemokine and cytokine levels in BM are shown with a bar. An asterisk indicates a significant difference at P < 0.05.

Article Snippet: CCL25, CCL28, IL-7, and TSLP levels in whey were measured using Human CCL25 DuoSet ELISA kit (R&D Systems), Human CCL28 DuoSet ELISA kit (R&D Systems), Human IL-7 DuoSet ELISA kit (R&D Systems), and TSLP Human Uncoated ELISA Kit (Invitrogen) according with the previous reports (Aihara et al., 2014; Hossny et al., 2020; Pallister et al., 2015).

Techniques: Comparison

Journal: Journal of reproductive immunology

Article Title: Detection of CCL25 and the correlation between CCL25, CCL28, IL-7, and TSLP in human breast milk.

doi: 10.1016/j.jri.2022.103783

Figure Lengend Snippet: Fig. 2. Comparison of chemokine and cytokine levels in BM between primiparous and multiparous women.CCL25, CCL28, TSLP, and IL-7 levels in BM were compared between primiparous and multiparous women: (a) CCL25, (b) CCL28, (c) TSLP, and (d) IL-7. Medians of chemokine and cytokine levels in BM are shown with a bar. Primi. and Multi. indicate primiparous and multiparous women, respectively. Cytokine levels in colostrum and mature milk are shown with triangles and circles, respectively.

Article Snippet: CCL25, CCL28, IL-7, and TSLP levels in whey were measured using Human CCL25 DuoSet ELISA kit (R&D Systems), Human CCL28 DuoSet ELISA kit (R&D Systems), Human IL-7 DuoSet ELISA kit (R&D Systems), and TSLP Human Uncoated ELISA Kit (Invitrogen) according with the previous reports (Aihara et al., 2014; Hossny et al., 2020; Pallister et al., 2015).

Techniques: Comparison

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Cervical lymphatic activation in mice after stroke: a Male C57BL6 mice were subjected to transient 60 min of focal ischemia. Note: the artery occlusion time was titrated to achieve equivalent levels of injury in mice and rats. Sixty min of transient middle cerebral artery occlusion in mice result in the same infarct volume as 100 min of occlusion in rats, i.e. approximately 30% of the ipsilateral hemisphere. Western blot demonstrated that LYVE-1 expression was significantly upregulated in CLNs. LYVE-1 upregulation was robust in superficial CLNs compared to deep CLNs after focal ischemia. There were no significant changes in axillary lymph nodes or spleen after focal ischemia (n = 6 biologically independent animals). * P < 0.05 vs Sham resulted from Fisher’s LSD test. b , c Immunohistochemistry using LYVE-1 antibody (Sham; n = 13, MCAO; n = 6 biologically independent animals) and Podoplanin antibody (Sham; n = 7, MCAO; n = 7 biologically independent animals) confirmed that lymphatic endothelial intensity was increased in superficial CLNs at 24 h after focal cerebral ischemia. Scale: 100 nm. * P < 0.05, ** P < 0.01, unpaired t-test. Images were taken in the area of subcapsular sinus. d Experimental design for microarray using lymphatic endothelium isolated from CLNs after focal ischemia. Male C57BL6 mice were subjected to transient focal ischemia. At 3 h after reperfusion, LYVE-1 positive lymphatic vessels were collected from CLNs pooled from 4 mice, and RNAs were extracted for microarray analysis. e Focal ischemia upregulated genes in lymphatic endothelium (red in heat maps, n = 3, total animal number = 12). f Gene Set Enrichment Analysis (GSEA) suggested that differentially expressed genes were largely related to matrix and transmembrane receptor protein tyrosine kinase pathways. g Volcano plot showed some upregulated genes after cerebral ischemia including Fkbp5, Hdc, Sesn1, Lyve1, Cyp2b10, Dccp2, Flt1, Ccl28, IL1b, Kdr , and Flt4 . All values are mean +/− SD.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: Activation Assay, Western Blot, Expressing, Immunohistochemistry, Microarray, Isolation

Journal: Nature Communications

Article Title: Brain-to-cervical lymph node signaling after stroke

doi: 10.1038/s41467-019-13324-w

Figure Lengend Snippet: Cervical lymphatic inflammation through VEGFR3 tyrosine kinase in mice after stroke: a Male C57BL6 mice were subjected to transient focal ischemia and, immediately after reperfusion the vehicle (PBS 10 µL) or MAZ51 (3 ng/10 µL) were injected into the nasal cavity. b MAZ51 treatment suppressed tyrosine phosphorylation in superficial CLN lymphatic endothelium at 72 h after focal ischemia (Sham; n = 4, MCAO; n = 9, MCAO + MAZ51; n = 8 biologically independent animals). * P < 0.05, one-way ANOVA followed by Fisher’s LSD test. c FACS analysis demonstrated that MAZ51 treatment significantly decreased pro-inflammatory TNF-α positive macrophages in superficial CLNs; no clear changes were noticed in TGF-β positive macrophages. * P < 0.05, one-way ANOVA followed by Fisher’s LSD test. d – f . Immunostaining revealed that CD169 positive macrophages increased TNF-α, while MAZ51 treatment decreased pro-inflammatory macrophages. Scale: 100 nm. g – h Confocal microscopy analysis demonstrated that TNF-α was highly co-localized with CD169 positive macrophages in the subcapsular sinus of superficial CLNs. MAZ51 treatment decreased the co-localization. TNF-α expression was not observed in ILN macrophages. Scale: 100 nm. i – l Western blot confirmed that TNF-α, IL-1β and CCL28 were significantly increased in post-stroke superficial CLNs. MAZ51 treatment reduced cytokine/chemokine expression ( n = 4 biologically independent animals). ILNs weakly responded to focal cerebral ischemia as to TNF-α, IL-1β or CCL28 expression ( n = 3 biologically independent animals). * P < 0.05, ** P < 0.01, one-way ANOVA followed by Fisher’s LSD test. All values are mean +/− SD.

Article Snippet: Anti-β-actin (1:1,000, A5441, Sigma-aldrich), anti-p-Tyr antibody (1:500, sc-7020, Santa Cruz), anti-VEGF-C antibody (1:500, sc-374628, Santa Cruz), anti-VEGFR3 antibody (1:500, sc-365748, Santa Cruz), anti-iNOS antibody (1:500, ab3523, Abcam), anti-IL-1β antibody (1:500, ab9722, Abcam), anti-TNF-α antibody (1:200, GTX110520, GeneTex), anti-TGF-β antibody (1:200, ab64715, Abcam), anti-CCL28 antibody (1:500, MAB717, R&D systems), anti-Podoplanin antibody (1:200, sc-166906, Santa Cruz), anti-CD31 antibody (1:200, 550274, BD biosciences), anti-LYVE-1 antibody (1:500, NB100–725B, NOVUS biologicals), anti-vWF antibody (1:100, A0082, Agilent), FITC anti-CD4 antibody (1:100, 561828, BD Biosciences), FITC Neutrophil antibody (1:100, ab53453, Abcam), PE anti-CD34 antibody (1:100, 551387, BD Biosciences), APC anti-F4/80 antibody (1:200, 123116, BioLegend), FITC anti-CD16/32 antibody (1:200, 101306, BioLegend), PE anti-CD11b antibody (1:200, 557397, BD biosciences), anti-CD45 antibody (1:400, 20103–1-AP, Proteintech).

Techniques: Injection, Phospho-proteomics, Immunostaining, Confocal Microscopy, Expressing, Western Blot

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A–B ) cDNA was isolated from a panel of human pancreatic cancer cell lines, Panc1, MiaPaCa2, Capan2, Capan1, HPAFII, and Hs766t, and screened by RT-PCR for expression of CCR (A), CXCR, CX 3 CR, or XCR (B) family of receptors. C–D ) The same panel of human cell lines was probed for expression of the known ligands for CCR10 (C) and CXCR6 (D). E ) RT-PCR analysis of patient-derived pancreatic cells (MCW PDAC cell lines) as well as a human pancreatic epithelial nestin-expressing (HPNE) confirmed CXCR6-CXCL16 and CCR10-CCL28 transcript expression. F ) Pancreatic tumor cells derived from the KPC murine model exhibited varying levels of the ligands and receptors. GAPDH and actin were analyzed as loading controls. Regions of the MBL and NRAMP genes were assessed as genomic DNA controls. Positive control was RNA from PBMCs.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Derivative Assay, Positive Control

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Normal pancreatic tissue was sectioned and processed for histopathologic analysis for CCL28, CCR10 and CK19. The arrowhead denotes the epithelial cell lining. B) Tissue from pancreatic tumor was sectioned and processed for immunohistochemical staining with antibodies specific for CCL28, CCR10, CK19, α-SMA as well as H&E, Masson’s trichrome (3C) or Movat’s pentachrome (5C). ‘T’ denotes the tumor cells and ‘S’ denotes the stromal compartments of the tissue section as defined by CK19, α-SMA and trichrome staining. C–D ) Representative stained tissue was scored by an investigator blinded to the tissue or antibody source for staining intensity. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Tissue staining shown in A and B representative of tissues sections from 14 normal, 12 PanIN, and 12 PDAC patients, based on clinical diagnosis and examination by a board-certified pathologist.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunohistochemical staining, Staining, Biomarker Discovery

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A) Sections of pancreatitis tissue were processed for immunostaining of CCR10 and CCL28, CXCR6, and CXCL16. B–C) . Scoring for the number of ductal (CK19+) or stromal (SMA+) regions for stain of CXCL16, CXCR6, CCL28 or CCR10. Gray line is the mean staining intensity of normal pancreatic tissue shown in and included as a reference. D ) Parallel tissue sections were processed for immunohistochemistry for CXCL12, CXCR7, CXCR4 and CK19. E ) Quantitative staining intensity of CXCL12, CXCR7 and CXCR4 in CK19+ cells. *** = P ≤ 0.001, **** = P ≤ 0.0001. Tissue staining shown in A and D are representative of tissue specimens from 5–9 individual patients clinically and pathologically diagnosed with pancreatitis.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunostaining, Staining, Immunohistochemistry

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: A ) Sandwich ELISA revealed that the panel of tissue culture pancreatic cancer cells secrete CCL28 when stimulated with 50 ng/mL IFNγ. Levels of CCL28 were undetectable in HPSCs treated with IFNγ. B ) Flow cytometric analysis of HPSCs revealed the cell surface expression of CCR10 (top panel). The mean fluorescence intensity (MFI) was determined for cells stained with the isotype control primary antibody (light gray) and anti-CCR10 antibody (dark gray). C ) CCL28 stimulates chemotaxis. HPSCs were plated in the top well of a transwell insert in serum free media. The bottom well contained media (serum free; SF or full growth; FG). Recombinant CCL28 [30 nM] was added to the bottom or top well as indicated. After 4h, inserts were swabbed, stained with DAPI and enumerated. D ) CCL28-mediated HPSC migration is dose dependent. HPSCs were plated in the top well of a transwell insert. The bottom well contained either full growth media (+; positive control) or serum-free media that either lacked stimulant (-; negative control), or increasing concentrations of recombinant CCL28. E ) HPSC migration is mitigated by an anti-CCL28 neutralizing antibody. The transwell migration setup as above was modified to include the following conditions: no stimulation (NS; negative control), 10 ng/mL TGF-β (positive control) or 30 nM recombinant CCL28. Neutralizing antibody to CCL28 was added to the bottom well for 30 minutes prior to incubation with the cells. F ) CCL28-mediated directional migration of HPSCs is through a G-coupled protein receptor pathway. HPSCs were pretreated with pertussis toxin (PTX) or vehicle, prior to CCL28 stimulation. G) Transcript expression of CCR10 in HPSC cells. RNA was isolated and used as a template for RT-PCR analysis using CCR10 or GAPDH as a loading control. H) CCL28 stimulates chemotactic migration of patient-derived HSPC2 cells. HPSC2 cells were added to a transwell insert and placed in a well containing either serum free medium in the absence (NS) or presence of CCL28 (CCL28) or full growth medium (FG) as a positive control (FG). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Sandwich ELISA, Expressing, Fluorescence, Staining, Control, Chemotaxis Assay, Recombinant, Migration, Positive Control, Negative Control, Modification, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Three independent HPSC lines were treated with 30 nM CCL28, left untreated (NS), treated with 10 ng/mL TGF-β (C+), treated with SB-431542 [25 µM], a TGF-β1R inhibitor (C-) or treated with vehicle controls (V). A) Representative immunofluorescence images show digital intensity “heat maps” of detected αSMA, corresponding to intensity color bar scale. Scale bars represent 50 µm. B) Semi-quantitative imaging analysis of the mean integrated intensity (Integ Int) of αSMA detection normalized for each HPSC line (HPSC, 2, 3) using the vehicle (V) set as 1 arbitrary unit. HPSC: #, P = 0.0179. ***, P ≤ 0.0002. HPSC2: #, P = 0.0144; ****, P <0.0001; HPSC3, *****, P < 0.0001. C) Representative images of HPSC-derived ECMs produced under experimental conditions as in A and analyzed using Orientation-J plugin of Image-J software. Color tones were normalized using hue values for common mode angle (cyan/green boarded color) visualization as represented on the orientation bar. D) Box and whisker plot of mean percent of fibers distributed within 15° angles from the mode corresponding to the indicated experimental conditions. HPSC: #, P = 0.0304; *, P = 0.028; HPSC2: *, P = 0.0117; HPSC3: C + versus Vs, *, P = 0.0101. No substantial ECM production was obtained from HPSC3 under TGF-β inhibition and was designated as not available (NA).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Immunofluorescence, Imaging, Derivative Assay, Produced, Software, Whisker Assay, Inhibition

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Schematic representation of the experimental plan ( A ). Briefly, PDAC epithelial cells were seeded to the bottom well of a transwell dish (1) then stimulated with 50 ng/mL IFNγ to elicit chemokine secretion. HPSCs were plated onto the top chamber of the transwell insert (2) while CCL28 neutralizing antibody was added to the bottom chamber. The insert with HPSCs was added (3) and incubated for 4h prior to (4) fixing, DAPI-staining and cell counting. Stimulation of Panc1 ( B–C ) and MiaPaCa2 ( D–E ) cells by IFNγ resulted in directional migration of HPSCs that was inhibited by the neutralizing CCL28 antibody. HPSC migrate towards PDAC tumor cells and not non-transformed epithelial HPNE cells ( F–G ). Representative images ( C, E ) of 5 independent biological replicates are presented. * = P ≤ 0.05. ** = P ≤ 0.01.

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Incubation, Staining, Cell Counting, Migration, Transformation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Cancer cell chemokines direct chemotaxis of activated stellate cells in pancreatic ductal adenocarcinoma

doi: 10.1038/labinvest.2016.146

Figure Lengend Snippet: Normal pancreatic duct epithelial cells and quiescent stromal fibroblasts almost no ligand CCL28 (purple circles) and basal levels of the receptor CCR10 (left panel). In inflammation, such as in pancreatitis, CCL28 expression is increased by cytokines such as IFNγ, which also participate in the activation of stellate cells into cancer-associated fibroblasts. CCL28 directs the migration of stellate cells toward the epithelium (middle panel). CCL28 produced by transformed ductal cells direct the sustained migration of activated stellate cells into the remodeling tumor microenvironment (right panel).

Article Snippet: Stimulation or inhibition of migration was mediated via incubation (all treatments were added to the bottom well) with recombinant CCL28 (717-VC, R&D Systems) or polyclonal neutralizing antibodies against CCL28 (AF717, R&D Systems).

Techniques: Expressing, Activation Assay, Migration, Produced, Transformation Assay