Journal: International Journal of Molecular Sciences

Article Title: Nintedanib Regulates GRK2 and CXCR2 to Reduce Neutrophil Recruitment in Endotoxin-Induced Lung Injury

doi: 10.3390/ijms22189898

Figure Lengend Snippet: Nintedanib (Nin) administration restored the changes in chemokine (C-X-C motif) receptor 2 (CXCR2) and G protein-coupled receptor kinase 2 (GRK2) expression on circulating neutrophils and prevented pulmonary neutrophil accumulation in mice with LPS-induced ALI. LPS reduced the expression of GRK2 and induced the expression of CXCR2 on circulating neutrophils in mice with LPS-induced ALI, and nintedanib administration restored these changes. Data are means ± standard deviations. * p < 0.05 vs. control, # p < 0.05 vs. LPS; n = 6 per group. PBS, phosphate-buffered saline.

Article Snippet: Cells from BALF and blood were subjected to cytospinning, fixed, permeabilized, and stained with Ly6G (LS-C36561, 1:100; LifeSpan Biosciences), VLA-4 (1:100, ab202969; Abcam), GRK2 (GTX101682, 1:100; GeneTex, Hsinchu City, Taiwan), or Alexa 647-labeled CXCR2 (129101, 1:100; BioLegend, San Diego, CA, USA) antibodies as primary antibodies.

Techniques: Expressing, Control, Saline

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: The sections were then incubated overnight at 4°C with the following primary antibodies: GFAP antibody (1:1000, mouse; Millipore Bioscience Research Reagents), Cx43 antibody (1:1000, rabbit; Sigma), NeuN antibody (1:1000, mouse; Millipore Bioscience Research Reagents), CXCL1 (1: 200, rabbit; Boster), and CXCR2 antibody (1: 200, rabbit; Boster).

Techniques: Expressing, Activity Assay, Transmission Assay

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: The time course of changes in CXCR2, CXCL1, CXCL2, CXCL3 mRNAs (A–D) and proteins (E–H) in the spinal cord tissues on the 2nd, 7th, 14th, and 28th days after chronic constriction injury (CCI) in rats. The RT-qPCR and Western blot data are presented as the means ± SEM of 6–10 and 4–6 samples per group in each method, respectively. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, *** p < 0.001 indicate differences vs. naive rats. CCI, chronic constriction injury; N, naïve.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Quantitative RT-PCR, Western Blot

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: The spinal localization of CXCR2 and its ligand CXCL3 in naive and CCI-exposed rats. Immunofluorescent staining was performed on paraffin-embedded 7 μm (A,B) co-staining of CXCR2 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). (C,D) co-staining of CXCL3 (green) and neuronal marker NeuN (red; upper row); astroglial marker GFAP (red, middle row), and microglial marker IBA1 (red, bottom row). White arrows indicate representative CXCL3-positive cells that co-localize with IBA1-positive cells. Scale bar for all pictures: 25 μm.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Staining, Marker

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of single (A,B) (different doses: 10, 20, and 30 μg/5 μl) intrathecal NVP CXCR2 20 administration on mechanical ( A ; von Frey test) and thermal ( B ; cold plate test) hypersensitivity as measured 0.5, 1, 2, 4, 6, 24 h after NVP CXCR2 20 injection on day 7 in CCI-exposed rats. Effects of repeated (C,D) (one dose: 10 μg/5 μl i.t .; 16 h and 1 h before CCI and then once a day for 7 days) intrathecal NVP CXCR2 20 administration on mechanical ( C ; von Frey test) and thermal ( D ; cold plate test) hypersensitivity as on day 2 or 7 in CCI-exposed rats. Tactile and thermal hypersensitivity were assessed at 120 and 125 min after the last NVP CXCR2 20 injection, respectively. The horizontal dotted line shows the cut-off value. Data are presented as the means ± SEM of 10–18 rats after single administration and 8 rats after repeated administration per group. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test measured separately at each time point. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences between vs. naive rats. ## p < 0.01, ### p < 0.001 indicate differences between V- and NVP-treated, CCI-exposed rats. CCI, chronic constriction injury; N, naive; V, vehicle; NVP, NVP CXCR2 20. Additionally, the results presented on graphs A and B were additionally evaluated using two-way ANOVA to determine the time × drug interaction (please see results in chapter 3.4).

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Injection

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of the repeated administration of NVP CXCR2 20 (NVP; 10 μg/5 μl; i.t .; 16 h and 1 h before CCI and then once a day for 7 days) on the protein levels of CXCR2, IBA1, GFAP, CXCL1, CXCL2, and CXCL3 proteins (A–I) in the spinal cord (A–F) and DRG (G–I) on the 7th day after CCI in rats. The data are presented as the mean fold changes relative to the control ± SEM (5–6 samples per group). Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences vs. naive rats. # p < 0.05, indicate differences between V-treated and NVP-treated rats. CCI, chronic constriction injury; N, naive; V, vehicle; NVP, NVP CXCR2 20.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques:

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of single NVP CXCR2 20 administration on a single CXCL3 injection and nociceptive transmission in naive mice (B,C) . Single intrathecal administrations of vehicle (V) or NVP CXCR2 20 (60 μg/5 μl) were performed 120 min before a single intrathecal administration of V or CXCL3 (2 ng/5 μl). The effects of administrations on mechanical (von Frey test; B ) and thermal (cold plate test; C ) hypersensitivity were measured 3.5, 7 and 26 h after the NVP CXCR2 20 injection (1.5, 5, and 24 h after the CXCL3) (A) . Data are presented as the means ± SEM (6–8 mice per group). The results were evaluated using one-way ANOVA followed by Bonferroni's test for comparisons of selected pairs. ** p < 0.01, *** p < 0.001 indicate differences in comparison with V+V-treated animals at the indicated time points. ## p < 0.01, ### p < 0.001 indicate differences in comparison with V+CXCL3-treated animals at the indicated time points. V, vehicle; NVP, NVP CXCR2 20.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Injection, Transmission Assay

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Effects of on NVP CXCR2 20 levels of the CXCL1, CXCL2, and CXCL3 proteins (A–F) in primary rat microglial (A–C) and astroglial (D–F) cell cultures. Samples were analyzed 24 h after cells were stimulated with LPS. The data are presented as the fold change relative to the control and relative protein levels. Fold change relative to control : the Western blot data are presented as the means ± SEM and represent the normalized averages derived from analyses of 3–4 independent experiments. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001 indicate differences in comparison with the control group (vehicle-treated non-stimulated cells); # p < 0.05, ## p < 0.01, ### p < 0.001 indicate differences between vehicle-treated and NVP-treated LPS-stimulated cells. Relative protein level: Inter-group differences in relative protein level were analyzed using a t-test. ### p < 0.001 indicates differences compared to the vehicle-treated LPS-stimulated cells. LPS-, vehicle-treated non-stimulated cells; NVP-, NVP-treated non-stimulated cells, LPS+, vehicle-treated LPS-stimulated cells; NVP+, NVP-treated LPS-stimulated cells. In (D–F) the blots are cropped which was shown with a dotted line on the representation bands below the figures.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Western Blot, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Pharmacological Blockade of Spinal CXCL3/CXCR2 Signaling by NVP CXCR2 20, a Selective CXCR2 Antagonist, Reduces Neuropathic Pain Following Peripheral Nerve Injury

doi: 10.3389/fimmu.2019.02198

Figure Lengend Snippet: Scheme of drug co-administration (A) . Effects of single (B,C) administration of NVP CXCR2 20 (NVP; 10 μg/5 μl; single dose i.t .; on the 7th day post-CCI) (A) on pain-related behaviors (von Frey test A ; cold plate test B ) and the analgesic effects of morphine (M; 2.5 μg/5 μl; single dose i.t .; on the 7th day post-CCI, 4 h after NVP or V injection) and buprenorphine (B; 2.5 μg/5 μl; single dose i.t .; on the 7th day post-CCI, 4 h after NVP or V injection) on CCI-exposed rats. The data are presented as the means ± SEM of 6 rats per group. Intergroup differences were analyzed using ANOVA with Bonferroni's multiple comparisons test. ** p < 0.01, *** p < 0.001 indicate differences compared with naïve rats. # p < 0.05, ## p < 0.01, ### p < 0.001 indicate differences compared V+V-treated, CCI-exposed rats. B, buprenorphine; CCI, chronic constriction injury; M, morphine; N, naive; NVP, NVP CXCR2 20; V, vehicle.

Article Snippet: Membranes were blocked with 5% non-fat dry milk (Bio-Rad, Warsaw, Poland) in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at RT, washed with TBST, and incubated with the following commercially available primary antibodies (reactivity of rat and specified by the producer of the observed molecular weight) overnight at 4°C: rabbit anti-Iba-1 (1:1,000, Proteintech, 10904-1-AP), anti-CXCR2 (1:2,000, LSBio, LS-C388292), anti-CXCL1 (1:200, LSBio, LS-C104778), anti-CXCL2 (1:200, Novus, MAB525), anti-CXCL3 (1:250, Novus, AF516), anti-GFAP (1:10,000, Novus, NB300-141), and mouse anti-GAPDH (1:5,000, Millipore, MAB374).

Techniques: Injection