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Trends Pharmacol Sci 27 1 —7. Abrogation of CC chemokine receptor 9 ameliorates collagen-induced arthritis of mice. Arthritis Res Ther 16 5 Lab Invest 86 9 — Panzer U, Uguccioni M. Prostaglandin E2 modulates the functional responsiveness of human monocytes to chemokines. Eur J Immunol 34 12 —9. Blood 2 — Blood 97 7 —4.

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Eotaxin-3 is a natural antagonist for CCR2 and exerts a repulsive effect on human monocytes. A human chemokine with a regulatory role. J Biol Chem 22 — J Leukoc Biol 76 3 —8. The unique property of the CC chemokine regakine-1 to synergize with other plasma-derived inflammatory mediators in neutrophil chemotaxis does not reside in its NH2-terminal structure. Mol Pharmacol 62 1 — J Immunol 11 — J Exp Med 5 — Eur J Immunol 41 4 — Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events. Mol Pharmacol 74 2 — Chemokine receptor homo- or heterodimerization activates distinct signaling pathways.

EMBO J 20 10 — Chemokine interaction with synergy-inducing molecules: fine tuning modulation of cell trafficking. A rich chemokine environment strongly enhances leukocyte migration and activities. Blood 9 — CCLinduced responses are powerfully enhanced by synergy inducing chemokines via CCR4: evidence for the involvement of first beta-strand of chemokine. Eur J Immunol 35 3 — Synergy-inducing chemokines enhance CCR2 ligand activities on monocytes. Eur J Immunol 39 4 — Int J Cancer 10 — Exp Cell Res 2 — Possible mechanisms involved in chemokine synergy fine tuning the inflammatory response.

Immunol Lett 1—2 —4. Disrupting functional interactions between platelet chemokines inhibits atherosclerosis in hyperlipidemic mice. Nat Med 15 1 — Weber C, Koenen RR. Trends Immunol 27 6 — Chapter 4. Interactions of chemokines with glycosaminoglycans. Methods Enzymol — Glycosaminoglycans interact selectively with chemokines and modulate receptor binding and cellular responses.

Biochemistry 38 39 — Rot A. Eur J Immunol 23 —6. Glycosaminoglycan binding and oligomerization are essential for the in vivo activity of certain chemokines. The monomer-dimer equilibrium and glycosaminoglycan interactions of chemokine CXCL8 regulate tissue-specific neutrophil recruitment.

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J Leukoc Biol 91 2 — Kinetics of chemokine-glycosaminoglycan interactions control neutrophil migration into the airspaces of the lungs. J Immunol 5 — Chemokine cooperativity is caused by competitive glycosaminoglycan binding. J Immunol 8 — J Exp Med 10 — CCL18 exhibits a regulatory role through inhibition of receptor and glycosaminoglycan binding.

PLoS One 8 8 :e Targeting glycosaminoglycans in the lung by an engineered CXCL8 as a novel therapeutic approach to lung inflammation. Eur J Pharmacol — Oligomerization of CXCL10 is necessary for endothelial cell presentation and in vivo activity. J Immunol 10 —8. Monomeric and dimeric CXCL8 are both essential for in vivo neutrophil recruitment.

Enzymology

PLoS One 5 7 :e Neutrophil activation by monomeric interleukin Science —2. The dependence of chemokine-glycosaminoglycan interactions on chemokine oligomerization. Glycobiology 26 3 — C dashed lines with numerical simulations continuous lines and the experimental measurements circles. The time when the chemoattractant front reaches the sample increases with the distance from the membrane. In the time frame of interest to our work i.


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It is worth mentioning that the value of D obtained by fitting the numerical simulation with the experimental data 1. Cell trajectories were reconstructed by means of a semi-automated image analysis macro [43] , [44] based on standard software libraries Image Pro Plus. The macro allows the user to identify each cell on the corresponding best focus layer at each time step. All the cells that were in focus in each layer were tracked.

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Cell position arrays were then processed by a Matlab script, in order to characterize quantitatively the effect of chemotaxis in terms of changes in motility parameters, i. The latter is used to quantitatively assess directional cell movement for observation times that are sufficiently long greater than the cell persistence time , and is defined as the ratio between the net movement in the direction of the gradient and the total curvilinear length of the cell trajectory.

The value of I at a given time is calculated by considering the motion of each cell from the beginning of the experiment cumulative chemotactic index , and is determined by averaging the values for all the cells analysed, weighted on the curvilinear length of each cell trajectory. Data from about 70 cells were averaged in order to calculate motility parameters. In order to determine the percentage of cells moving randomly, we calculated the velocity modules and components of every cell, grouping the values in 5 minutes intervals, and running the t-test over each of the grouped distributions.

In order to validate our chemotaxis assay, several experiments were performed on human neutrophils freshly isolated from healthy donors. The proposed assay allows the observation and analysis of the locomotory behaviour of cells in the absence and presence of external pro-migratory chemotactic factors during the same experiment.

The samples were imaged every minute for minutes and for each time point 70 cells were individually tracked by manually overlaying each cell contour. Here, we show representative results obtained from two different healthy donors referred to as A and B , as an experimental validation of the proposed experimental technique. The response of neutrophils to IL-8 was first analysed qualitatively through the reconstruction of cell trajectories.

Since collagen fibrils can become aligned contact guidance near the surface as the gel forms or within the gel as it compacts due to traction exerted by the entrapped cells, the possible contributions of these effects to directional cell migration and orientation need to be accounted for. Contact guidance is the phenomenon by which the extracellular matrix provides directional cues to the cells and directs the motility response via anisotropy in the microenvironment [45] — [47].

For instance, it has been shown that contact guidance from the alignment of collagen fibres promotes 3D migration of fibroblasts along the axis of collagen orientation [45] , [46]. Recent studies [48] show that density and spatial alignments of 3D collagen architecture, collagen concentration and the polymerization conditions, including pH and temperature, have considerable influence on scaffold structure and porosity and secondary impacts on cell morphology and behaviour including migration efficiency [49] , [50].

Furthermore, we used low cell densities in order to prevent any significant gel compaction during the observation period. Figure 3. A shows the trajectories of 54 cells, tracked for minutes, labelled with symbols having different shapes and grey intensities, in isotropic conditions. Each trajectory is referred to the same initial position that coincides with the origin of the coordinate system. In this control experiment where no chemoattractant was added, cell trajectories were uniformly distributed in space see Figure 3A , thus showing that cells were moving in a random orientation i.

Such random pattern confirms the absence of any matrix-mediated contact guidance effect.

CYTOKINES : ILs, INFs, TNFs, CSFs and Chemokines (FL-Immuno/04)

In the presence of the IL-8 concentration gradient, cell trajectories were instead directed towards the chemoattractant source as shown in Figure 3. B with results from 76 cells. Most of the cell paths have a preferential orientation toward the negative y axis, which is the direction of the concentration gradient. A: Random motion in absence of any chemical stimulus. Visual inspection of the time-lapse movie provided as movie S1 , shows that only a fraction of the cells did actually move during the entire experiment.

In Figure 4 the z-projection of 5 consecutive images from a z-stack acquired within the cell seeded collagen gel is reported, showing the neutrophils at time 0 Figure 4. A and at time minutes Figure 4. The circles indicate the position of cells in focus at the two times, respectively. Only the cells enclosed in black circles exhibited a significant movement, while the white ones showed no appreciable change in their positions over the entire experiment.

The complete trajectories described by the motile cells are drawn in Figure 4. The circles indicate the position of cells at two different times. Only the cells enclosed in a black circle move, while the white ones do not significantly change their position over the entire experiment. The complete trajectories described by motile cells are shown.

A cell was considered motile in a given time interval if its total displacement exceeded its own diameter.


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This criterion was used to exclude values representing minor cellular displacements that might be caused by either the repositioning error of the microscope motorized stage or by external perturbations of the system. The fraction of mobile cells was calculated for a time interval of 10 minutes in presence and absence of IL-8 for donors A and B, and is reported in Figure 5 A and B , respectively; the vertical line at 30 minutes from the start of the experiment shows the time IL-8 solution was added.

A different percentage of motile cells was clearly visible in unstimulated conditions Pre IL-8 for the two samples with donor B showing a higher motility. However, in both donors the stimulation of the cells by IL-8 induced a significant increase in the number of motile cells as detected in the Post time stages. The evolution over time of the fraction of motile cells suggests that at short times cells are not affected by the presence of chemoattractant, but the motile fraction progressively increases, until a maximum is reached which is then followed by a decrease at later times when the influence of the chemoattractant starts to vanish.

The vertical line indicates the moment when the IL-8 solution was added to the chemoattractant reservoir. To further characterize the effect of chemokine gradient on neutrophils motility, the average velocity of cells was calculated for each time point and averaged along 5 minute intervals. In basal conditions, i.

B , respectively. This value increased after the addition of IL-8 in both donors, in agreement with the increment of the fraction of motile cells, thus confirming that the chemoattractant gradient elicits greater cell motility. Once again, the vertical line on the graphs in Figure 6 corresponds to the time point when the IL-8 solution was added.

In Figure 6 the X and Y velocity components are also reported for both donors. The Y-component is the one showing the higher change after the IL-8 addition, evolving towards negative values, i. It is worth mentioning that for both donors the Y component of the average cell velocity showed a progressive decrease down to a negative peak, then slowly recovered towards basal values, a trend which confirms the presence of a transient peak in the chemotactic response. The statistical significance of these results was investigated by calculating the T-test for the velocity module and the X and Y velocity components.

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Chapter 4 Interactions of Chemokines with Glycosaminoglycans - Dimensions

The percentage of cells that rejected the null hypothesis i. This parameter shows a transient peak for the Y component of the velocity after the addition of the IL-8 solution as compared to the random conditions Pre IL-8 , whereas the same peak is absent in the velocity module and the X component.

This result provides further evidence that the effect of the IL-8 concentration gradient is to enhance cell movement mainly along the Y direction. A: Average cell velocity components and modulus as a function of time for neutrophils from donor A. C: Average cell velocity components and modulus as a function of time for neutrophils from donor B. As a further statistical analysis we report in Figure 7 the average velocity module V calculated over the entire Pre IL-8 and Post IL-8 periods for each of the 63 cells from donor A.

The continuous line refers to the average value, while the standard deviation is reported as the error bar. In the absence of IL-8 stimulation, i.