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<p>Autoantibody production and autoantibody-mediated inflammation are hallmarks of a number of autoimmune diseases. The K/BxN serum-transfer arthritis is one of the most widely used models of the effector phase of autoantibody-induced pathology. Several hematopoietic lineages including neutrophils, platelets, and mast cells have been proposed to contribute to inflammation and tissue damage in this model. We have previously shown that the Syk tyrosine kinase is critically involved in the development in K/BxN serum-transfer arthritis and bone marrow chimeric experiments indicated that Syk is likely involved in one or more hematopoietic lineages during the disease course. The aim of the present study was to further define the lineage(s) in which Syk expression is required for autoantibody-induced arthritis. To this end, K/BxN serum-transfer arthritis was tested in conditional mutant mice in which Syk was deleted in a lineage-specific manner from neutrophils, platelets, or mast cells. Combination of the MRP8-Cre, PF4-Cre, or Mcpt5-Cre transgene with floxed Syk alleles allowed efficient and selective deletion of Syk from neutrophils, platelets, or mast cells, respectively. This has also been confirmed by defective Syk-dependent in vitro functional responses of the respective cell types. In vivo studies revealed nearly complete defect of the development of K/BxN serum-transfer arthritis upon neutrophil-specific deletion of Syk. By contrast, Syk deletion from platelets or mast cells did not affect the development of K/BxN serum-transfer arthritis. Our results indicate that autoantibody-induced arthritis requires Syk expression in neutrophils, whereas, contrary to prior assumptions, Syk expression in platelets or mast cells is dispensable for disease development in this model.</p

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<p>Autoantibody production and autoantibody-mediated inflammation are hallmarks of a number of autoimmune diseases. The K/BxN serum-transfer arthritis is one of the most widely used models of the effector phase of autoantibody-induced pathology. Several hematopoietic lineages including neutrophils, platelets, and mast cells have been proposed to contribute to inflammation and tissue damage in this model. We have previously shown that the Syk tyrosine kinase is critically involved in the development in K/BxN serum-transfer arthritis and bone marrow chimeric experiments indicated that Syk is likely involved in one or more hematopoietic lineages during the disease course. The aim of the present study was to further define the lineage(s) in which Syk expression is required for autoantibody-induced arthritis. To this end, K/BxN serum-transfer arthritis was tested in conditional mutant mice in which Syk was deleted in a lineage-specific manner from neutrophils, platelets, or mast cells. Combination of the MRP8-Cre, PF4-Cre, or Mcpt5-Cre transgene with floxed Syk alleles allowed efficient and selective deletion of Syk from neutrophils, platelets, or mast cells, respectively. This has also been confirmed by defective Syk-dependent in vitro functional responses of the respective cell types. In vivo studies revealed nearly complete defect of the development of K/BxN serum-transfer arthritis upon neutrophil-specific deletion of Syk. By contrast, Syk deletion from platelets or mast cells did not affect the development of K/BxN serum-transfer arthritis. Our results indicate that autoantibody-induced arthritis requires Syk expression in neutrophils, whereas, contrary to prior assumptions, Syk expression in platelets or mast cells is dispensable for disease development in this model.</p

Dynorphin A (DYN-A) (1–13) and (1–17) induce strong contraction of rat cerebral arteries.

<p>Effects of cumulative concentrations of DYN-A (1–8), (1–13), (1–17) and Dynorphin B (DYN-B) on the resting tension of rat basilar (A) and middle cerebral (B) arteries. DYN-A (1–13) and DYN-A (1–17) induce strong, dose-dependent vasoconstriction in both vessels, whereas DYN-A (1–8) and DYN-B have no significant effect. Values are expressed as mean±SEM percentage of the reference contraction induced by 124 mmol/L K⁺ Krebs solution, n = 6–37. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 vs. DYN-A (1–8); ^##<i>P</i><0.01, ^###<i>P</i><0.001 vs. DYN-B.</p

Dynorphin A (DYN-A) induced cerebral vasoconstriction is partly mediated by heterotrimeric G_i/o-proteins.

<p>Effect of the inhibition of G_i/o-signaling with pertussis toxin (PTX, applied as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037798#s2" target="_blank">Methods</a>”) on the responsiveness of rat basilar (A) and middle cerebral (B) arteries to DYN-A (1–13). In control vessels the slightly weaker reactions to DYN-A (as compared to the previous figures) were probably the consequence of overnight incubation in the Krebs solution containing the vehicle of PTX. PTX inhibited the vasoconstrictor effects of DYN-A. Values are expressed as mean±SEM percentage of the reference contraction induced by 124 mmol/L K⁺ Krebs solution, n = 6–8. Asterisks indicate significant (*<i>P</i><0.05, **<i>P</i><0.01) differences between PTX-treated and vehicle-treated control vessels.</p

Dynorphin A (DYN-A) is present in perivascular nerves of rat and human intraparenchymal arteries.

<p>Oblique sections of rat (A and B) as well as longitudinal sections of rat (C and D) and human (E and F) cerebral arteries are shown. Merged images of DYN-A (1–13) immunoreactivity (green fluorescence) and transmitted-light (grayscale) indicate the perivascular localization of DYN-A. Broken line shows the surface of the brain on panels A and B, asterisks indicate red blood cell clots in the lumen of the artery on panel F. Bar = 50 µm on each panel.</p

Dynorphin A (DYN-A) (2–13) induces contraction of rat cerebral arteries independently of κ-opiate receptors.

<p>Effects of DYN-A (2–13) on the tone of rat basilar (A) and middle cerebral (B) arteries before and after treatment with the κ-opiate receptor antagonist <i>nor</i>-Binaltorphimine dihydrochloride (NORBI, 50 µM). DYN-A (2–13), which is reportedly inactive at opiate receptors, evoked weaker [in comparison to DYN-A (1–13) shown in previous figures] but significant contractions in both vessels, and these effects were resistant to NORBI. Values are expressed as mean±SEM percentage of the reference contraction induced by 124 mmol/L K⁺ Krebs solution, n = 6–9.</p

Dynorphin A (DYN-A) is present in perivascular nerves of the rat basilar artery.

<p>Localization of DYN-A in perivascular nerve fibers of rat basilar artery by immunofluorescent confocal microscopy. Anti-synaptophysin staining (red fluorescence) indicates the synaptic vesicles of perivascular nerves on the surface of the basilar artery (A). DYN-A (1–13) (green fluorescence) is abundantly found in the adventitia (B) and co-localized with synaptophysin (C). Bar = 100 µm.</p

Dynorphin A (DYN-A) induced cerebral vasoconstriction is partly mediated by κ-opiate receptors.

<p>Effect of the κ-opiate receptor antagonist <i>nor</i>-Binaltorphimine dihydrochloride (NORBI, 50 µM) on the responsiveness of rat basilar (A) and middle cerebral (B) arteries to DYN-A (1–13). NORBI was able to reduce the vasoconstrictor effect of DYN-A in both vessels, whereas its vehicle (saline) was without any effect (C and D). Values are expressed as mean±SEM percentage of the reference contraction induced by 124 mmol/L K⁺ Krebs solution, n = 8–10. Asterisks indicate significant (**<i>P</i><0.01, ***<i>P</i><0.001) differences between values before and after NORBI treatment.</p

Weak activation of thromboxane receptors aggravates while inhibition of TXA₂ synthesis attenuates CoBF oscillations developed in the absence of NO.

<p><b>A–C</b>: Original recordings of the cerebrocortical laser-Doppler flux <i>in vivo</i> before (left panels) and after (right panels) administration of the TP-receptor agonist U-46619 (A), the thromboxane synthase inhibitor ozagrel (C) or their vehicle (saline) (B) in rats pretreated by the NO synthase inhibitor L-NAME. <b>D</b>: Quantitative analysis of slow wave oscillations with discrete Fourier transformation. The peak magnitudes of the power spectra are compared before and after treatments in the three experimental groups. Values are mean ± SEM (n = 20, 12 and 14 in Group IIa, IIb, and IIc, respectively) *p<0.05 vs. “Before Treatment”.</p

Activation of thromboxane or endothelin receptors induce Rho-kinase dependent vasomotion in NO synthase blocked MCAs.

<p>Quantitative analysis of slow wave oscillations with discrete Fourier transformation in L-NAME treated vessels before and after the administration of the TP-receptor agonist U-46619 (Panel A) or endothelin-1 (Panel B) followed by the Rho-kinase inhibitor Y-27632. Values are mean ± SEM fold changes of the peak magnitudes of the power spectra compared to the baseline. *p<0.05, ***p<0.001 vs. L-NAME, ##<0.01 vs. ET-1 and ###<0.001 vs. U-46619 (n = 10–20).</p