Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators

Objective: Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1α (Ccl3/MIP-1α) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Methods and Results: Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1α induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B4 (LTB4) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB4, but not Ccl2/JE/MCP-1 and Ccl3/MIP-1α, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/ MCP-1- and Ccl3/MIP-1α-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. Conclusions: Ccl2/JE/MCP-1 and Ccl3/MIP-1α mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage

Intraoperative Near-Infrared Autofluorescence and Indocyanine Green Imaging to Identify Parathyroid Glands: A Comparison

Objective. To investigate the feasibility of near-infrared autofluorescence (AF) and indocyanine green (ICG) fluorescence to identify parathyroid glands intraoperatively. Methods. Fluorescence imaging was carried out during open parathyroid and thyroid surgery. After visual identification, parathyroid glands were exposed to near-infrared (NIR) light with a wavelength between 690 and 770 nm. The camera of the Storz (R) NIR/ICG endoscopic system used detects NIR light as a blue signal. Therefore, parathyroid AF was expected to be displayed in the blue color channel in contrast to the surrounding tissue. Following AF imaging, a bolus of 5 mg ICG was applied intravenously. ICG fluorescence was detected using the same NIR/ICG imaging system. Well-vascularized parathyroid glands were expected to show a strong fluorescence in contrast to surrounding lymphatic and adipose tissue. Results. We investigated 78 parathyroid glands from 50 patients. 64 parathyroid glands (82%) displayed AF showing the typical bluish violet color. 63 parathyroid glands (81%) showed a strong and persistent fluorescence after application of ICG. The sensitivity of identifying a parathyroid gland by AF was 82% (64 true positive and 14 false negative results), while ICG imaging showed a sensitivity of 81% (63 true positive and 15 false negative results). The Fisher exact test revealed no significant difference between both groups at p < 0.05. Neither lymph nodes nor adipose tissue revealed substantial AF or ICG fluorescence. Conclusion. AF and ICG fluorescence reveal a high degree of sensitivity in identifying parathyroid glands. Further, ICG imaging facilitates the assessment of parathyroid perfusion. However, in the current setting both techniques are not suitable as screening tools to identify parathyroid glands at an early stage of the operation

Urokinase-Type Plasminogen Activator Promotes Paracellular Transmigration of Neutrophils Via Mac-1, But Independently of Urokinase-Type Plasminogen Activator Receptor

Background: Urokinase-type plasminogen activator (uPA) has recently been implicated in the pathogenesis of ischemia-reperfusion (I/R) injury. The underlying mechanisms remain largely unclear. Methods and Results: Using in vivo microscopy on the mouse cremaster muscle, I/R-elicited firm adherence and transmigration of neutrophils were found to be significantly diminished in uPA-deficient mice and in mice treated with the uPA inhibitor WX-340, but not in uPA receptor (uPAR)–deficient mice. Interestingly, postischemic leukocyte responses were significantly reduced on blockade of the integrin CD11b/Mac-1, which also serves as uPAR receptor. Using a cell transfer technique, postischemic adherence and transmigration of wild-type leukocytes were significantly decreased in uPA-deficient animals, whereas uPA-deficient leukocytes exhibited a selectively reduced transmigration in wild-type animals. On I/R or stimulation with recombinant uPA, >90% of firmly adherent leukocytes colocalized with CD31-immunoreactive endothelial junctions as detected by in vivo fluorescence microscopy. In a model of hepatic I/R, treatment with WX-340 significantly attenuated postischemic neutrophil infiltration and tissue injury. Conclusions: Our data suggest that endothelial uPA promotes intravascular adherence, whereas leukocyte uPA facilitates the subsequent paracellular transmigration of neutrophils during I/R. This process is regulated via CD11b/Mac-1, and does not require uPAR. Pharmacological blockade of uPA interferes with these events and effectively attenuates postischemic tissue injury

Plasmin Inhibitors Prevent Leukocyte Accumulation and Remodeling Events in the Postischemic Microvasculature

Clinical trials revealed beneficial effects of the broad-spectrum serine protease inhibitor aprotinin on the prevention of ischemia-reperfusion (I/R) injury. The underlying mechanisms remained largely unclear. Using in vivo microscopy on the cremaster muscle of male C57BL/6 mice, aprotinin as well as inhibitors of the serine protease plasmin including tranexamic acid and ε-aminocaproic acid were found to significantly diminish I/R-elicited intravascular firm adherence and (subsequent) transmigration of neutrophils. Remodeling of collagen IV within the postischemic perivenular basement membrane was almost completely abrogated in animals treated with plasmin inhibitors or aprotinin. In separate experiments, incubation with plasmin did not directly activate neutrophils. Extravascular, but not intravascular administration of plasmin caused a dose-dependent increase in numbers of firmly adherent and transmigrated neutrophils. Blockade of mast cell activation as well as inhibition of leukotriene synthesis or antagonism of the platelet-activating-factor receptor significantly reduced plasmin-dependent neutrophil responses. In conclusion, our data suggest that extravasated plasmin(ogen) mediates neutrophil recruitment in vivo via activation of perivascular mast cells and secondary generation of lipid mediators. Aprotinin as well as the plasmin inhibitors tranexamic acid and ε-aminocaproic acid interfere with this inflammatory cascade and effectively prevent postischemic neutrophil responses as well as remodeling events within the vessel wall

Ccl2 and Ccl3 Mediate Neutrophil Recruitment via Induction of Protein Synthesis and Generation of Lipid Mediators

Objective: Although the chemokines monocyte chemoattractant protein-1 (Ccl2/JE/MCP-1) and macrophage inflammatory protein-1α (Ccl3/MIP-1α) have recently been implicated in neutrophil migration, the underlying mechanisms remain largely unclear. Methods and Results: Stimulation of the mouse cremaster muscle with Ccl2/JE/MCP-1 or Ccl3/MIP-1α induced a significant increase in numbers of firmly adherent and transmigrated leukocytes (>70% neutrophils) as observed by in vivo microscopy. This increase was significantly attenuated in mice receiving an inhibitor of RNA transcription (actinomycin D) or antagonists of platelet activating factor (PAF; BN 52021) and leukotrienes (MK-886; AA-861). In contrast, leukocyte responses elicited by PAF and leukotriene-B4 (LTB4) themselves were not affected by actinomycin D, BN 52021, MK-886, or AA-861. Conversely, PAF and LTB4, but not Ccl2/JE/MCP-1 and Ccl3/MIP-1α, directly activated neutrophils as indicated by shedding of CD62L and marked upregulation of CD11b. Moreover, Ccl2/JE/ MCP-1- and Ccl3/MIP-1α-elicited leakage of fluorescein isothiocyanate dextran as well as collagen IV remodeling within the venular basement membrane were completely absent in neutrophil-depleted mice. Conclusions: Ccl2/JE/MCP-1 and Ccl3/MIP-1α mediate firm adherence and (subsequent) transmigration of neutrophils via protein synthesis and secondary generation of leukotrienes and PAF, which in turn directly activate neutrophils. Thereby, neutrophils facilitate basement membrane remodeling and promote microvascular leakage

Effect of plasmin on neutrophil activation.

<p>Fluorescence histograms for expression of CD11b/Mac-1 and CD62L/L-selectin on murine neutrophils undergoing stimulation with plasmin, PMA (open histograms, broken lines), or vehicle (open histograms, solid lines) as compared to control IgG (solid histograms).</p

Systemic leukocyte counts as well as microhemodynamic parameters, including inner vessel diameter, blood flow velocity, and wall shear rate were obtained as detailed in <i>Material and Methods</i> (mean±SEM for <i>n</i> = 3–6 per group).

<p>Systemic leukocyte counts as well as microhemodynamic parameters, including inner vessel diameter, blood flow velocity, and wall shear rate were obtained as detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017229#s4" target="_blank"><i>Material and Methods</i></a> (mean±SEM for <i>n</i> = 3–6 per group).</p

Effect of plasmin on leukocyte rolling, firm adherence, and transmigration.

<p>Leukocyte responses in the cremaster muscle after intraarterial (A, C, E) or intrascrotal (B, D, F) injection of plasmin (mean±SEM for <i>n</i> = 3 per group; *p<0.05, vs. control).</p

Role of mast cells and lipid mediators for plasmin-dependent leukocyte responses.

<p>The number of ruthenium red-positive cells in the cremaster muscle of mice treated with cromolyn, MK-886, or BN52021 undergoing stimulation with plasmin (A). Leukocyte responses in the cremaster muscle of mast cell-depleted mice as well as of mice treated with cromolyn (B, D), MK-886, BN52021 (C, E), or vehicle undergoing stimulation with plasmin (mean±SEM for <i>n</i> = 4–6 per group; #p<0.05, vs. unstimulated; *p<0.05, vs. vehicle; §p<0.05, vs. MK-886; °p<0.05, vs. cromolyn).</p

Effect of TXA, EACA, and aprotinin on postischemic leukocyte responses.

<p>Postcapillary venules in the postischemic cremaster muscle treated with ε-aminocaproic acid (EACA) or vehicle (A; scale bar 25 µm). Leukocyte rolling (B), firm adherence (C), and transmigration (D) in mice treated with tranexamic acid (TXA), EACA, or aprotinin undergoing I/R (mean±SEM; n = 6 per group; #p<0.05, vs. sham; *p<0.05, vs. vehicle).</p