Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Role of histamine receptors (HRs) in mast cells (MCs) in acute inflammation-induced mesenteric perilymphatic mast cell-mediated changes of tissue histamine. A: a significant proportion of mesenteric MCs are located near mesenteric lymphatic vessels [MLVs; A.1, bright-field, MLV identified by its intraluminal valve, MCs stained with avidin conjugate (A.2) and with toluidine blue (TB; A.3–A.4)]. Scale bars, 80 μm. B: illustration of surgical isolation of mesenteric perilymphatic tissue segments (B.2) from intestinal loops (B.1). B.3 shows a live single MLV from isolated segment presented in B.2. C: effects of various experimental treatments on histamine concentration in mesenteric perilymphatic tissues (untreated control, treatment with LPS; Crmln + LPS, pretreatment with cromolyn sodium before and during treatment with LPS; MC H1R active + LPS, pretreatment with HR2–4 antagonists before and during treatment with LPS; MC H2R active + LPS, pretreatment with HR1 and -3/4 antagonists before and during treatment with LPS; MC H3R + H4R + LPS, pretreatment with HR1 and -2 antagonists before and during treatment with LPS [n = 6 rats, normalized to untreated control; *significant differences (P < 0.05) between certain treatments]. D: illustration of web-based STRING platform analysis of predicted protein-protein interactions between subtypes of HRs and the histamine-producing enzyme histidine decarboxylase (HDC): H1R-HDC (E.1), H2R-HDC (E.2). E and F: effects of various experimental treatments on expression of HDC in mesenteric perilymphatic MCs (untreated control, 48/80, treatment with compound 48/80; LPS, treatment with LPS; Crmln + LPS, pretreatment with cromolyn sodium before and during treatment with LPS; H1B + LPS or H2B + LPS, pretreatment with HR1 antagonist or HR2 antagonist before and during treatment with LPS). E: mean fluorescence intensity (MFI) of HDC measured in mesenteric perilymphatic MCs after various experimental treatments [n = 4 rats, normalized to untreated control; *significant differences (P < 0.05) between certain treatments]. F: representative images of mesenteric segments containing MCs stained by Alexa fluor 488-conjugated avidin, in green), additionally labeled for HDC (in red), and DAPI (in blue) after various experimental treatments. Scale bars, 50 μm.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Staining, Avidin-Biotin Assay, Isolation, Concentration Assay, Expressing, Fluorescence, Labeling

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Relative roles of histamine receptors (HRs) 1 and 2 in acute inflammation-induced mast cell (MC) degranulation. A: representative images of all variants of MC degranulation status. MCs stained with avidin or toluidine blue (T.Blue). B: quantitative analysis of variants of MC degranulation status under different experimental conditions (untreated control; LPS, treatment with LPS; pretreatment with HR1 antagonist before and during treatment with LPS (H1B + LPS); and pretreatment with HR2 antagonist before and during treatment with LPS (H2B + LPS); n = 4 rats, average mean values presented. C: representative images of MCs under each experimental condition stained with either avidin or T.Blue. Scale bars, 65 μm.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Staining, Avidin-Biotin Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Presence and functionality of histamine receptors (HRs) 1 and 2 in perilymphatic mast cells (MCs). A: representative confocal images of mesenteric perilymphatic tissue segments stained in red for HR1 (A.1) and HR2 (A.2) together with labeling of the same segment by Alexa fluor 488-conjugated avidin (in green) and DAPI (in blue). Scale bars, 20 μm. B: mean fluorescence intensity (MFI) of fluorescently labeled histamine measured in mesenteric perilymphatic MCs after various experimental treatments: H1B, pretreatment with H1R antagonist; H2B, pretreatment with H2R antagonist; H1H2B, pretreatment with both HR1 and HR2 antagonists [n = 3 rats, normalized to untreated control; *significant differences (P < 0.05) between treatment groups and control]. C: representative images of mesenteric segments containing MCs (stained by Texas Red-conjugated avidin, in red) bound with fluorescently labeled histamine (FLU-His, in green) after various experimental treatments. Scale bars, 50 μm.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Staining, Labeling, Avidin-Biotin Assay, Fluorescence

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Histamine itself is able to induce activation of perilymphatic mast cells (MCs). A: representative images of MCs stained with both ruthenium red (R.Red) and toluidine blue (T.Blue) under different experimental conditions [untreated control (A.1 and A.2); 48/80 (treatment with compound 48/80, A.3 and A.4); treatment with histamine (A.5 and A.6); pretreatment by cromolyn before and during treatment with histamine (Crmln + Histamine, A.7 and A.8); pretreatment by histamine receptor 1 (HR1) antagonist before and during treatment with histamine (H1B + Histamine, A.9 and A.10); and pretreatment by HR2 antagonist before and during treatment with histamine (H2B + Histamine, A.11 and A.12)]. Insets from each picture demonstrate detailed structure of MCs under experimental conditions. Scale bars, 200 μm. B: quantitative analysis of mesenteric perilymphatic MC activation under various experimental conditions [n = 3 rats; *significant differences (P < 0.05) between corresponding experimental conditions]. C: schema of the MC-histamine autocrine regulatory loop. HDC, histidine decarboxylase; TLR4. Toll-like receptor 4.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Activation Assay, Staining

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Mast cell (MC)-histamine autocrine regulatory loop: functional implications in mesenteric perilymphatic tissue compartments. A: expression of phosphorylated (p)NF-κB in mesenteric perilymphatic tissue segments under different experimental treatments [(untreated control; LPS-treated; Crmln + LPS, pretreatment with cromolyn sodium before and during treatment with LPS; H1B + LPS, pretreatment with histamine receptor 1 (HR1) antagonist before and during treatment with LPS; H2B + LPS, pretreatment with HR2 antagonist before and during treatment with LPS; H3/4B + LPS, pretreatment with HR3/4 antagonist before and during treatment with LPS (n = 4 rats, normalized to untreated control; *significant differences (P < 0.05) between certain treatments)]. Bottom: representative images of mesenteric perilymphatic tissue segments following various treatments. Scale bars, 80 μm. B: numerous cell types beyond MCs express HR1 and -2 in mesenteric perilymphatic tissues. Representative images showing immunohistochemical staining for HR1 or -2 (in red) and cell nuclei (DAPI, in blue) along with avidin conjugate to indicate MCs (in green). Scale bars, 50 μm. C: almost all CD11b/c-positive cells (in green, together with DAPI staining, in blue) express both HR1 and HR2 (in red): representative images. Scale bars, 20 μm. D and E: activation of MCs significantly increased the number of MCs that appeared physically associated with CD11b/c-positive cells. D: results of quantitative analysis [n = 4 rats; *significant differences (P < 0.05) between certain treatments: untreated control; 48/80, treatment with compound 48/80, LPS, treatment with LPS]. E: representative images (fluorescent labeling similar to 5B). Scale bars, 50 μm.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Functional Assay, Expressing, Immunohistochemical staining, Staining, Avidin-Biotin Assay, Activation Assay, Labeling

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Histamine-mediated autocrine signaling in mesenteric perilymphatic mast cells

doi: 10.1152/ajpregu.00255.2019

Figure Lengend Snippet: Roles of mast cell (MC) activation and histamine receptors (HRs) 1 and 2 in trafficking of CD11b/c-positive cells toward mesenteric lymphatic vessels (MLVs). A: representative images of trafficking of CD11b/c-positive cells (in green) toward MLVs under different experimental treatments [untreated control; 48/80, treatment with compound 48/80; LPS, treatment with LPS; Crmln + LPS, pretreatment with cromolyn sodium before and during treatment with LPS; H1B + LPS, pretreatment with HR1 antagonist before and during treatment with LPS; H2B + LPS, pretreatment with HR2 antagonist before and during treatment with LPS]. Scale bar, 100 μm. B: results of quantitative analysis [n = 4 rats; *significant differences (P < 0.05) between certain treatments]. C: schematic presentation of involvement of perilymphatic MCs in regulation of trafficking of CD11b/c-positive cells toward MLVs in response to LPS-induced acute inflammation. LEC, lymphatic endothelial cell; TLR4, Toll-like receptor 4; VCAM1, vascular cell adhesion molecule 1.

Article Snippet: Antibodies were obtained from the following sources: phospho-NF-κB p65 from Cell Signaling Technology, (cat. no. 3033S; Boston, MA), histidine decarboxylase (HDC) from Abcam (cat. no. ab37291; Boston, MA), HR1 (cat. no. AHR-001) and HR2 (cat. no. AHR-002) from Almone Laboratories, Jerusalem, Israel; HR2 from Novus Biologicals (cat. no. NLS1175), and CD11b/c from Biolegend (cat. no. 201801; San Diego, CA).

Techniques: Activation Assay