Journal: Journal of Neuroinflammation

Article Title: An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE

doi: 10.1186/s12974-018-1237-y

Figure Lengend Snippet: CD45 + CD11b + myeloid cells are a primary source of CXCL2 in the brainstem during aEAE. a Neutrophils (CD45 + CD11b + Ly6G + ), monocytes/macrophages (CD45 hi CD11b + Ly6G − ), microglia (CD45 lo CD11b + Ly6G − ), and non-myeloid immune cells (CD45 + CD11b − ) were FACS sorted from the brainstems of IFNγRKO mice at the onset of aEAE. CXCL2 mRNA was quantified by qPCR and normalized to GAPDH ( n = 15 mice pooled from two experiments). b Brainstem-infiltrating cells isolated at the onset of aEAE were stained on the cell surface with antibodies specific for myeloid cell markers and intracellularly with αCXCL2 prior to flow cytometric analysis ( n = 6 mice from two experiments)

Article Snippet: For immunofluorescent histology, primary antibodies included rabbit α-glial fibrillary acidic protein (GFAP) (Gibco), rat α-mouse CD45 (IBL-5/15, Millipore), goat α-mouse CXCL2 (R&D Systems), goat α-mouse CXCL1 (R&D Systems), rat α-mouse Ly6G (IA8, BD Biosciences), and hamster α-mouse CD3ε (BD Biosciences).

Techniques: Isolation, Staining

Journal: Journal of Neuroinflammation

Article Title: An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE

doi: 10.1186/s12974-018-1237-y

Figure Lengend Snippet: CXCL2 is produced by CD45 + cells in the brainstem infiltrates during aEAE. a Brainstem tissue was harvested from an IFNγRKO host at aEAE onset (clinical score 1). Sections were subjected to in situ hybridization to detect CXCL2 mRNA (red) combined with a DAPI (blue) counter stain. b Brainstem sections from a mouse with aEAE (clinical score 3) were stained with monoclonal antibodies specific for CD45 (green), GFAP (white), and CXCL2 (red) and counterstained with DAPI (blue). Arrows indicate CXCL2 + CD45 + cells. Arrow heads indicate GFAP + astrocytes

Article Snippet: For immunofluorescent histology, primary antibodies included rabbit α-glial fibrillary acidic protein (GFAP) (Gibco), rat α-mouse CD45 (IBL-5/15, Millipore), goat α-mouse CXCL2 (R&D Systems), goat α-mouse CXCL1 (R&D Systems), rat α-mouse Ly6G (IA8, BD Biosciences), and hamster α-mouse CD3ε (BD Biosciences).

Techniques: Produced, In Situ Hybridization, Staining, Bioprocessing

Journal: Journal of Neuroinflammation

Article Title: An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE

doi: 10.1186/s12974-018-1237-y

Figure Lengend Snippet: IFNγ signaling directly regulates CXCR2 expression by neutrophils. a IFNγR-sufficient and IFNγRKO neutrophils (left panels) and monocytes/macrophages (right panels) were FACS sorted from the brainstems (upper panels), spinal cords (middle panels), and bone marrow (lower panels) of mixed bone marrow chimeric mice. CXCL2 transcript levels were measured by qPCR and normalized to GAPDH ( n = 13 chimeric mice from two experiments). b CD45 + CD11b + Ly6G + neutrophils were FACS sorted from the spinal cord, brainstem, and bone marrow of WT mice at the onset of cEAE, and IFNγRKO mice at the onset of aEAE. Bone marrow cells from naïve mice were used as controls. CXCR2 mRNA levels were quantified by qPCR and normalized to GAPDH ( n = 10 WT mice and 15 IFNγRKO mice with EAE, pooled from two experiments; n = 4 WT naive mice and 5 IFNγRKO naïve mice). c IFNγR-sufficient and IFNγRKO neutrophils (defined as CD45 + CD11b + Ly6G + ) were FACS sorted from the bone marrow, spinal cord, and brainstem of mixed bone marrow chimeric mice at the onset of EAE. CXCR2 transcript levels were normalized to GAPDH ( n = 13 mice from two experiments)

Article Snippet: For immunofluorescent histology, primary antibodies included rabbit α-glial fibrillary acidic protein (GFAP) (Gibco), rat α-mouse CD45 (IBL-5/15, Millipore), goat α-mouse CXCL2 (R&D Systems), goat α-mouse CXCL1 (R&D Systems), rat α-mouse Ly6G (IA8, BD Biosciences), and hamster α-mouse CD3ε (BD Biosciences).

Techniques: Expressing

Journal: Journal of Neuroinflammation

Article Title: An IFNγ/CXCL2 regulatory pathway determines lesion localization during EAE

doi: 10.1186/s12974-018-1237-y

Figure Lengend Snippet: IFNγ suppresses CXCL2 expression in neutrophils and monocytes stimulated with CXCR2 or TLR ligands, respectively. a , b Bone marrow neutrophils were cultured in media alone, or with recombinant CXCL2, CXCL2 plus IL-1β, CXCL1, or G-CSF, in the presence or absence of IFNγ. RNA was extracted 1 h later to measure CXCL2 transcript levels via qPCR. Fold induction of CXCL2 in the stimulated neutrophils over unstimulated controls was calculated as 2 −ΔΔCt , using GAPDH as an internal control for normalization. Each symbol represents a neutrophil culture derived from an individual mouse. ( a n = 8 mice, with data pooled from three experiments. b Representative of two experiments with n = 3 mice each). c Bone marrow monocytes were stimulated with LPS in the presence or absence of IFNγ for 4 h. Intracellular CXCL2 protein expression was assessed by flow cytometry (Representative of three experiments with n = 3 mice each)

Article Snippet: For immunofluorescent histology, primary antibodies included rabbit α-glial fibrillary acidic protein (GFAP) (Gibco), rat α-mouse CD45 (IBL-5/15, Millipore), goat α-mouse CXCL2 (R&D Systems), goat α-mouse CXCL1 (R&D Systems), rat α-mouse Ly6G (IA8, BD Biosciences), and hamster α-mouse CD3ε (BD Biosciences).

Techniques: Expressing, Cell Culture, Recombinant, Control, Derivative Assay, Flow Cytometry

Journal: Cell Biology and Toxicology

Article Title: ATG5-dependent autophagy in Sertoli cells protects against cadmium-disrupted blood-testis barrier via perturbing CXCL2/CXCR2 axis

doi: 10.1007/s10565-025-10089-5

Figure Lengend Snippet: Cd exposure disrupts the blood-testis barrier by activating the CXCL2/CXCR2 axis in mice . A - D The male mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 0, 2, 8, 24, 48, and 72 h after Cd treatment. A The mRNA expression of T-bet , Gata3 , Rorc , Foxp3 , Nos2 , Tnf-α , Il-10 , Tgf-β , Pd-1 , Pd-L1 , Gas6 , Acvr1 , Cxcl2 , Il-17a , Il-17f , Tlr4 , Il-23 , and IL-22 were measured by RT-qPCR. B - D The expression of CXCL2 and CXCR2 was detected by western blotting. E - L The male mice were injected (i.p.) with 10.0 mg/kg SB225002 or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 72 h after Cd treatment. E Experimental design flowchart. F The representative images of biotin tracer permeability assay. Biotin was combined with Streptavidin-Alexa Fluor 488 (green), and nuclei were stained with DAPI (blue). The diffusion distance was denoted with red Line, and the basal part of seminiferous tubules was denoted with red circles. Scale bar: 50 μm. G Quantification of biotin tracer permeability. The diffusion distance was defined as D signal , and the seminiferous tubules'radius was defined as D radius . H-J The expression of Connexin 43 and Occludin was detected by western blotting. K, L Representative images and quantification of mIHC assay. White arrows denoted the Connexin 43 puncta surrounding WT1. Scale bar: 50 μm. M, N The male mice were injected (i.p.) with 10.0 mg/kg SB225002 or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 8 h after Cd treatment. The expression of CXCR2 was detected by western blotting. Data were displayed as mean ± SEM . n = 3–6. * P < 0.05, ** P < 0.01 vs Ctrl group; # P < 0.05, ## P < 0.01 vs Cd group

Article Snippet: Antibodies against GAPDH (60004–1-IG), CXCL2 (26791–1-AP), Occludin (27260–1-AP), and Desmocollin 2 (13876–1-AP) were gained from Proteintech Group.

Techniques: Injection, Expressing, Quantitative RT-PCR, Western Blot, Permeability, Staining, Diffusion-based Assay

Journal: Cell Biology and Toxicology

Article Title: ATG5-dependent autophagy in Sertoli cells protects against cadmium-disrupted blood-testis barrier via perturbing CXCL2/CXCR2 axis

doi: 10.1007/s10565-025-10089-5

Figure Lengend Snippet: Autophagy inhibition aggravates Cd-induced testicular CXCL2 expression upregulation and blood-testis barrier disruption in mice . A - F The male mice were injected (i.p.) with 20.0 mg/kg 3-MA or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 72 h after Cd treatment. A Experimental design flowchart. B The representative images of biotin tracer permeability assay. Biotin was combined with Streptavidin-Alexa Fluor 488 (green), and nuclei were stained with DAPI (blue). The diffusion distance was denoted with red Line, and the basal part of seminiferous tubules was denoted with red circles. Scale bar: 50 μm. C Quantification of biotin tracer permeability. The diffusion distance was defined as D signal , and the seminiferous tubules'radius was defined as D radius . D - F The expression of Connexin 43 and Occludin were detected by western blotting. G - K The male mice were injected (i.p.) with 20.0 mg/kg 3-MA or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 8 h after Cd treatment. G - I The expression of CXCL2 and CXCR2 was detected by western blotting. J , K Representative images and quantification of mIHC assay. White arrows denoted the LC3B puncta surrounding WT1. Scale bar: 50 μm. Data were displayed as mean ± SEM . n = 3–6. * P < 0.05, ** P < 0.01 vs Ctrl group; # P < 0.05, ## P < 0.01 vs Cd group

Article Snippet: Antibodies against GAPDH (60004–1-IG), CXCL2 (26791–1-AP), Occludin (27260–1-AP), and Desmocollin 2 (13876–1-AP) were gained from Proteintech Group.

Techniques: Inhibition, Expressing, Disruption, Injection, Permeability, Staining, Diffusion-based Assay, Western Blot

Journal: Cell Biology and Toxicology

Article Title: ATG5-dependent autophagy in Sertoli cells protects against cadmium-disrupted blood-testis barrier via perturbing CXCL2/CXCR2 axis

doi: 10.1007/s10565-025-10089-5

Figure Lengend Snippet: Autophagy activation protects against Cd-induced testicular CXCL2 expression upregulation and blood-testis barrier disruption in mice. A - F The male mice were injected (i.p.) with 1.0 mg/kg Rap or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 72 h after Cd treatment. A Experimental design flowchart. B The representative images of biotin tracer permeability assay. Biotin was combined with Streptavidin-Alexa Fluor 488 (green), and nuclei were stained with DAPI (blue). The diffusion distance was denoted with red Line, and the basal part of seminiferous tubules was denoted with red circles. Scale bar: 50 μm. C Quantification of biotin tracer permeability. The diffusion distance was defined as D signal , and the seminiferous tubules'radius was defined as D radius . D - F The expression of Connexin 43 and Occludin were detected by western blotting. G - K The male mice were injected (i.p.) with 1.0 mg/kg Rap or vehicle, respectively. After 1 h, mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 8 h after Cd treatment. G - I The expression of CXCL2 was detected by western blotting. J , K Representative images and quantification of mIHC assay. White arrows denoted the LC3B puncta surrounding WT1. Scale bar: 50 μm. Data were displayed as mean ± SEM . n = 3–6. *P < 0.05, **P < 0.01 vs Ctrl group; # P < 0.05, ## P < 0.01 vs Cd group

Article Snippet: Antibodies against GAPDH (60004–1-IG), CXCL2 (26791–1-AP), Occludin (27260–1-AP), and Desmocollin 2 (13876–1-AP) were gained from Proteintech Group.

Techniques: Activation Assay, Expressing, Disruption, Injection, Permeability, Staining, Diffusion-based Assay, Western Blot

Journal: Cell Biology and Toxicology

Article Title: ATG5-dependent autophagy in Sertoli cells protects against cadmium-disrupted blood-testis barrier via perturbing CXCL2/CXCR2 axis

doi: 10.1007/s10565-025-10089-5

Figure Lengend Snippet: Knockout of Atg5 in Sertoli cells aggravates Cd-induced testicular CXCL2 expression upregulation and blood-testis barrier disruption in mice . A - H The WT and Atg5 −/− male mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 72 h after Cd treatment. A Experimental design flowchart. B The expression of ATG5 and WT1 in primary Sertoli cells was detected by western blotting. C The representative images of biotin tracer permeability assay. Biotin was combined with Streptavidin-Alexa Fluor 488 (green), and nuclei were stained with DAPI (blue). The diffusion distance was denoted with red Line, and the basal part of seminiferous tubules was denoted with red circles. Scale bar: 50 μm. D Quantification of biotin tracer permeability. The diffusion distance was defined as D signal , and the seminiferous tubules'radius was defined as D radius . E – H The expression of Desmocollin 2, Connexin 43, and Occludin were detected by western blotting. I-M The WT and Atg5 −/− male mice were injected (i.p.) with 2.0 mg/kg CdCl 2 or vehicle, respectively. Mouse testes were collected at 8 h after Cd treatment. I - K The expression of CXCL2 and CXCR2 was detected by western blotting. L, M Representative images and quantification of mIHC assay. White arrows denoted the LC3B puncta surrounding WT1. Scale bar: 50 μm. Data were displayed as mean ± SEM . n = 3–6. *P < 0.05, **P < 0.01 vs Ctrl-treated WT group; # P < 0.05, ## P < 0.01 vs Cd-treated WT group

Article Snippet: Antibodies against GAPDH (60004–1-IG), CXCL2 (26791–1-AP), Occludin (27260–1-AP), and Desmocollin 2 (13876–1-AP) were gained from Proteintech Group.

Techniques: Knock-Out, Expressing, Disruption, Injection, Western Blot, Permeability, Staining, Diffusion-based Assay

Journal:

Article Title: Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies

doi: 10.1128/IAI.69.5.2988-2995.2001

Figure Lengend Snippet: MIP-2 ELISA of Chinese hamster corneas infected with Acanthamoeba. Corneas were removed from three hamsters at the indicated times postinfection. Corneal lysates were individually prepared and assayed for MIP-2. The bars show means ± SE of three hamster corneas per time point. ∗∗∗, significantly different from uninfected control corneas (P < 0.001).

Article Snippet: Goat anti-mouse MIP-2 antibody and mouse rMIP-2 were purchased from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Infection

Journal:

Article Title: Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies

doi: 10.1128/IAI.69.5.2988-2995.2001

Figure Lengend Snippet: MPO assay of Chinese hamster corneas infected with Acanthamoeba. Hamster corneas were injected with rMIP-2, anti-MIP-2, or control IgG and infected with Acanthamoeba trophozoites. Corneas were removed on days 2 and 6, and corneal lysates were individually prepared and assayed for MPO activity. Each bar represents the mean ± SE of three corneas per treatment on both days. ∗∗ and ∗∗∗, significantly different from IgG-treated corneas (P < 0.010 and P < 0.001, respectively).

Article Snippet: Goat anti-mouse MIP-2 antibody and mouse rMIP-2 were purchased from R&D Systems.

Techniques: MPO Assay, Infection, Injection, Activity Assay

Journal:

Article Title: Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies

doi: 10.1128/IAI.69.5.2988-2995.2001

Figure Lengend Snippet: In vivo effect of anti-MIP-2 treatment on the severity of Acanthamoeba keratitis in Chinese hamsters. Animals were subconjunctivally administered either anti-MIP-2 antibody or control IgG 4 h before infection. Identical concentrations of anti-MIP-2 and control IgG were administered topically on days 1 to 3 postinfection. Infected lenses were removed after 3 days, and corneas were scored for clinical severity for the period indicated. On day 18 (∗), five of six anti-MIP-2-treated animals but only one of six control animals still demonstrated evidence of disease. Each graph line represents the mean severity of six animals for the observed times. The results shown are representative of three separate experiments.

Article Snippet: Goat anti-mouse MIP-2 antibody and mouse rMIP-2 were purchased from R&D Systems.

Techniques: In Vivo, Infection

Journal:

Article Title: Exacerbation of Acanthamoeba Keratitis in Animals Treated with Anti-Macrophage Inflammatory Protein 2 or Antineutrophil Antibodies

doi: 10.1128/IAI.69.5.2988-2995.2001

Figure Lengend Snippet: Photomicrographs of corneas from Chinese hamsters untreated, treated with anti-MIP-2 antibody or rMIP-2, and challenged with amoeba-laden contact lenses. Eyes were removed from untreated (A, C, and E), anti-MIP-2-treated (B, D, F, and J), and rMIP-2-treated (G to I) animals on days 3, 6, and 9 postinfection. Untreated corneas displayed mild corneal swelling and few PMN at day 3 postinfection (A). On day 6, extensive corneal swelling and neutrophils were present in the stroma (C). By day 9, the corneal edema and swelling had subsided (E). Anti-MIP-2-treated corneas on day 3 displayed significant corneal swelling, edema, and plasma cells, with few macrophages and PMN in the stroma (B). At day 6, the extensive corneal swelling persisted while general stromal inflammation increased (B). At higher magnification, the cornea reveals a high preponderance of mononuclear cells (J). Note the persistence of stromal swelling and edema on day 9 postinfection (F). rMIP-2-treated animals displayed the greatest stromal swelling and PMN infiltration at day 3 in both the stroma and anterior chamber (G). By day 6, PMN infiltration and corneal swelling were significantly reduced (H). The stromal swelling and edema had subsided by day 9 postinfection (I). A to I, bars = 70 μm; J, bar = 25 μm.

Article Snippet: Goat anti-mouse MIP-2 antibody and mouse rMIP-2 were purchased from R&D Systems.

Techniques: