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Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

Boneschansker L, Yan J, Wong E, Briscoe DM, Irimia D - Nat Commun (2014)

Bottom Line: We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells.These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression.Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent.

View Article: PubMed Central - PubMed

Affiliation: 1] Transplant Research Program and The Division of Nephrology, Department of Medicine, Boston Children's Hospital, Boston, Massachusetts 02115, USA [2] The Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Surgery, BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, Massachusetts 02129, USA [4].

ABSTRACT
Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

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Migratory patterns of neutrophil subpopulationsA) Human neutrophils were fluorescence-activated cell sorted in two groups: highest and lowest quartile expressing IL-8R (CXCR1, CD181). B) Subpopulations of neutrophils expressing high levels of CD177 (CD177hi) were sorted and compared to CD177lo cells. The percentage of CD177hi cells ranged from 20-80% of total neutrophils. C) IL-8Rhi and IL-8Rlo expressing neutrophils show bi-directional migration patterns, but greater numbers of IL-8Rhi cells migrate away than towards IL-8 (p<0.05, Student's t test). D) Both CD177hi and CD177lo migrate in a bi-directional manner in response to C5a or IL-8. Greater numbers of CD177hi cells migrate in response to C5a vs. CD177lo cells (ns p=0.06). Higher numbers of CD177hi cells migrate away vs. towards C5a (p<0.01, Student's t test). E) Migratory patterns of IL-8Rhi and IL-8Rlo neutrophils in an IL-8 gradient; no difference was found in directional persistence. Bars represent mean and standard deviation of Bi-Directional Index. F) Migratory persistence of neutrophil subpopulations towards and away from a gradient of IL-8 and C5a. There is no difference in migration patterns of the subpopulations. Bars represent mean ± SD. Data from N=3 independent experiments. Number of cells analyzed: IL-8Rhi 580 migrating cells; IL-8Rlo 543 cells; CD177hi C5a 220 cells; CD177lo C5a 308 cells; CD177hi IL-8 165 cells; CD177lo IL-8 69 cells.
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Figure 3: Migratory patterns of neutrophil subpopulationsA) Human neutrophils were fluorescence-activated cell sorted in two groups: highest and lowest quartile expressing IL-8R (CXCR1, CD181). B) Subpopulations of neutrophils expressing high levels of CD177 (CD177hi) were sorted and compared to CD177lo cells. The percentage of CD177hi cells ranged from 20-80% of total neutrophils. C) IL-8Rhi and IL-8Rlo expressing neutrophils show bi-directional migration patterns, but greater numbers of IL-8Rhi cells migrate away than towards IL-8 (p<0.05, Student's t test). D) Both CD177hi and CD177lo migrate in a bi-directional manner in response to C5a or IL-8. Greater numbers of CD177hi cells migrate in response to C5a vs. CD177lo cells (ns p=0.06). Higher numbers of CD177hi cells migrate away vs. towards C5a (p<0.01, Student's t test). E) Migratory patterns of IL-8Rhi and IL-8Rlo neutrophils in an IL-8 gradient; no difference was found in directional persistence. Bars represent mean and standard deviation of Bi-Directional Index. F) Migratory persistence of neutrophil subpopulations towards and away from a gradient of IL-8 and C5a. There is no difference in migration patterns of the subpopulations. Bars represent mean ± SD. Data from N=3 independent experiments. Number of cells analyzed: IL-8Rhi 580 migrating cells; IL-8Rlo 543 cells; CD177hi C5a 220 cells; CD177lo C5a 308 cells; CD177hi IL-8 165 cells; CD177lo IL-8 69 cells.

Mentions: We also wished to determine if the chemoattraction and chemorepulsion response(s) of neutrophils in IL-8 and C5a gradients are associated with cellular phenotype. We FACS-sorted IL-8Rhi-expressing (CXCR1/CD181) (highest 25%ile) and IL-8Rlo (lowest 25%ile) cells and evaluated their respective migratory response to IL-8 (Fig. 3, Table 3). We found that both groups responded with a similar pattern and displayed bi-directional migratory behavior. Similar numbers of IL-8hi and IL-8Rlo cells responded to IL-8 (~35% vs. ~31% respectively). Nevertheless, the IL-8Rhi neutrophils showed a significant bias for migrating away from IL-8 (20.8±2.1% away vs. 14.5±1.6% towards, p<0.05). IL-8Rlo neutrophils migrated in equal proportions in both directions (15.1±1.1% towards vs. 16.2±1.1% away from IL-8). We did not find significant differences in directional persistence and speed between the two groups (Supplementary Fig. 4A, B).


Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

Boneschansker L, Yan J, Wong E, Briscoe DM, Irimia D - Nat Commun (2014)

Migratory patterns of neutrophil subpopulationsA) Human neutrophils were fluorescence-activated cell sorted in two groups: highest and lowest quartile expressing IL-8R (CXCR1, CD181). B) Subpopulations of neutrophils expressing high levels of CD177 (CD177hi) were sorted and compared to CD177lo cells. The percentage of CD177hi cells ranged from 20-80% of total neutrophils. C) IL-8Rhi and IL-8Rlo expressing neutrophils show bi-directional migration patterns, but greater numbers of IL-8Rhi cells migrate away than towards IL-8 (p<0.05, Student's t test). D) Both CD177hi and CD177lo migrate in a bi-directional manner in response to C5a or IL-8. Greater numbers of CD177hi cells migrate in response to C5a vs. CD177lo cells (ns p=0.06). Higher numbers of CD177hi cells migrate away vs. towards C5a (p<0.01, Student's t test). E) Migratory patterns of IL-8Rhi and IL-8Rlo neutrophils in an IL-8 gradient; no difference was found in directional persistence. Bars represent mean and standard deviation of Bi-Directional Index. F) Migratory persistence of neutrophil subpopulations towards and away from a gradient of IL-8 and C5a. There is no difference in migration patterns of the subpopulations. Bars represent mean ± SD. Data from N=3 independent experiments. Number of cells analyzed: IL-8Rhi 580 migrating cells; IL-8Rlo 543 cells; CD177hi C5a 220 cells; CD177lo C5a 308 cells; CD177hi IL-8 165 cells; CD177lo IL-8 69 cells.
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Related In: Results  -  Collection

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Figure 3: Migratory patterns of neutrophil subpopulationsA) Human neutrophils were fluorescence-activated cell sorted in two groups: highest and lowest quartile expressing IL-8R (CXCR1, CD181). B) Subpopulations of neutrophils expressing high levels of CD177 (CD177hi) were sorted and compared to CD177lo cells. The percentage of CD177hi cells ranged from 20-80% of total neutrophils. C) IL-8Rhi and IL-8Rlo expressing neutrophils show bi-directional migration patterns, but greater numbers of IL-8Rhi cells migrate away than towards IL-8 (p<0.05, Student's t test). D) Both CD177hi and CD177lo migrate in a bi-directional manner in response to C5a or IL-8. Greater numbers of CD177hi cells migrate in response to C5a vs. CD177lo cells (ns p=0.06). Higher numbers of CD177hi cells migrate away vs. towards C5a (p<0.01, Student's t test). E) Migratory patterns of IL-8Rhi and IL-8Rlo neutrophils in an IL-8 gradient; no difference was found in directional persistence. Bars represent mean and standard deviation of Bi-Directional Index. F) Migratory persistence of neutrophil subpopulations towards and away from a gradient of IL-8 and C5a. There is no difference in migration patterns of the subpopulations. Bars represent mean ± SD. Data from N=3 independent experiments. Number of cells analyzed: IL-8Rhi 580 migrating cells; IL-8Rlo 543 cells; CD177hi C5a 220 cells; CD177lo C5a 308 cells; CD177hi IL-8 165 cells; CD177lo IL-8 69 cells.
Mentions: We also wished to determine if the chemoattraction and chemorepulsion response(s) of neutrophils in IL-8 and C5a gradients are associated with cellular phenotype. We FACS-sorted IL-8Rhi-expressing (CXCR1/CD181) (highest 25%ile) and IL-8Rlo (lowest 25%ile) cells and evaluated their respective migratory response to IL-8 (Fig. 3, Table 3). We found that both groups responded with a similar pattern and displayed bi-directional migratory behavior. Similar numbers of IL-8hi and IL-8Rlo cells responded to IL-8 (~35% vs. ~31% respectively). Nevertheless, the IL-8Rhi neutrophils showed a significant bias for migrating away from IL-8 (20.8±2.1% away vs. 14.5±1.6% towards, p<0.05). IL-8Rlo neutrophils migrated in equal proportions in both directions (15.1±1.1% towards vs. 16.2±1.1% away from IL-8). We did not find significant differences in directional persistence and speed between the two groups (Supplementary Fig. 4A, B).

Bottom Line: We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells.These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression.Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent.

View Article: PubMed Central - PubMed

Affiliation: 1] Transplant Research Program and The Division of Nephrology, Department of Medicine, Boston Children's Hospital, Boston, Massachusetts 02115, USA [2] The Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA [3] Department of Surgery, BioMEMS Resource Center, Massachusetts General Hospital, Harvard Medical School, Shriners Hospitals for Children, Boston, Massachusetts 02129, USA [4].

ABSTRACT
Leukocyte migration into tissues is characteristic of inflammation. It is usually measured in vitro as the average displacement of populations of cells towards a chemokine gradient, not acknowledging other patterns of cell migration. Here, we designed and validated a microfluidic migration platform to simultaneously analyse four qualitative migration patterns: chemoattraction, -repulsion, -kinesis and -inhibition, using single-cell quantitative metrics of direction, speed, persistence and fraction of cells responding. We find that established chemokines, complement component 5a and IL-8 induce chemoattraction and repulsion in equal proportions, resulting in the dispersal of cells. These migration signatures are characterized by high persistence and speed and are independent of the chemokine dose or receptor expression. Furthermore, we find that twice as many T lymphocytes migrate away than towards stromal cell-derived factor 1 and their directional migration patterns are not persistent. Overall, our platform helps discover migratory signature responses and uncovers an avenue for precise characterization of leukocyte migration and therapeutic modulators.

Show MeSH
Related in: MedlinePlus