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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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Inhibitory effect of Slit2 on fMLP-induced human neutrophil and HL-60 migrationPercentage of A) human neutrophils and B) HL-60 cells that migrate towards or away from fMLP with or without the presence of Slit2 (N=4 independent experiments). For both cell types Slit2 significantly inhibits fMLP-induced migration (p<0.01 and p<0.001 respectively, Student's t test). C) Migratory persistence of human neutrophils is not affected by Slit2 (fMLP BD-Index = 0.89, fMLP+Slit2 BD-Index = 0.84). Directional persistence of migrating HL-60 cells is decreased by Slit2 (fMLP BD-Index = 0.63, fMLP+Slit2 BD-Index=0.39; p<0.001, Mann Whitney test). Bars represent mean and standard deviation of Bi-Directional Index. Speed of E) neutrophils and F) HL-60 migrating towards fMLP with or without Slit2 (arrows indicate the mean speed. Slit2 decreases migratory speed of HL-60 cells from 20.7±7.1μm min−1 to 12.8±5.9μm min−1 (p<0.01, Student's t test). Colors are: grey, fMLP alone; black, fMLP and Slit2; white, Slit2 alone. Bars represent mean ± SD. Combined data of N=4 (neutrophils) and N=3 (HL-60) independent experiments is shown. For neutrophils and fMLP, a total of 672 migrating out of 828 total cells were analyzed, fMLP+Slit (176 migrating/669 total), HL-60 (fMLP 530/1020), fMLP+Slit (221/1571).
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Figure 5: Inhibitory effect of Slit2 on fMLP-induced human neutrophil and HL-60 migrationPercentage of A) human neutrophils and B) HL-60 cells that migrate towards or away from fMLP with or without the presence of Slit2 (N=4 independent experiments). For both cell types Slit2 significantly inhibits fMLP-induced migration (p<0.01 and p<0.001 respectively, Student's t test). C) Migratory persistence of human neutrophils is not affected by Slit2 (fMLP BD-Index = 0.89, fMLP+Slit2 BD-Index = 0.84). Directional persistence of migrating HL-60 cells is decreased by Slit2 (fMLP BD-Index = 0.63, fMLP+Slit2 BD-Index=0.39; p<0.001, Mann Whitney test). Bars represent mean and standard deviation of Bi-Directional Index. Speed of E) neutrophils and F) HL-60 migrating towards fMLP with or without Slit2 (arrows indicate the mean speed. Slit2 decreases migratory speed of HL-60 cells from 20.7±7.1μm min−1 to 12.8±5.9μm min−1 (p<0.01, Student's t test). Colors are: grey, fMLP alone; black, fMLP and Slit2; white, Slit2 alone. Bars represent mean ± SD. Combined data of N=4 (neutrophils) and N=3 (HL-60) independent experiments is shown. For neutrophils and fMLP, a total of 672 migrating out of 828 total cells were analyzed, fMLP+Slit (176 migrating/669 total), HL-60 (fMLP 530/1020), fMLP+Slit (221/1571).

Mentions: In a final series of experiments, we wished to validate our microfluidic device for the analysis of anti-inflammatory agents. In these assays, we used Slit2 as a prototype inhibitor of neutrophil migratory responses36-38. As illustrated in Fig. 5A, Slit2 (5 μg mL−1) reduced the fraction of human neutrophils migrating towards fMLP (100 nM) by ~3 fold (from 74% of cells migrating to 23%, p<0.001). Similarly, we evaluated the effect of Slit2 on the migration of the clonal HL-60 cell line and found that its inhibitory effect was concentration-dependent (Fig.5). Lower numbers of cells migrated at higher concentrations of Slit2 (p<0.05). Slit2 (1.5 μg mL−1) reduced the number of HL-60 cells that migrate towards fMLP (100 nM) by ~3 fold (from 40% to 11%, p<0.01, Fig. 5B). Slit2 did not reduce the persistence of migrating neutrophils, but it slightly decreased the bi-directional index of migrating HL-60 cells from 0.63±0.57 to 0.39±0.68 (p<0.001, Fig. 5 C, D). In control experiments, where Slit-2 was present in media alone, a migratory response was evident only in 0.8±0.7 % of human neutrophils and 12.1±3.4% of HL-60 cells.


Microfluidic platform for the quantitative analysis of leukocyte migration signatures.

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

Inhibitory effect of Slit2 on fMLP-induced human neutrophil and HL-60 migrationPercentage of A) human neutrophils and B) HL-60 cells that migrate towards or away from fMLP with or without the presence of Slit2 (N=4 independent experiments). For both cell types Slit2 significantly inhibits fMLP-induced migration (p<0.01 and p<0.001 respectively, Student's t test). C) Migratory persistence of human neutrophils is not affected by Slit2 (fMLP BD-Index = 0.89, fMLP+Slit2 BD-Index = 0.84). Directional persistence of migrating HL-60 cells is decreased by Slit2 (fMLP BD-Index = 0.63, fMLP+Slit2 BD-Index=0.39; p<0.001, Mann Whitney test). Bars represent mean and standard deviation of Bi-Directional Index. Speed of E) neutrophils and F) HL-60 migrating towards fMLP with or without Slit2 (arrows indicate the mean speed. Slit2 decreases migratory speed of HL-60 cells from 20.7±7.1μm min−1 to 12.8±5.9μm min−1 (p<0.01, Student's t test). Colors are: grey, fMLP alone; black, fMLP and Slit2; white, Slit2 alone. Bars represent mean ± SD. Combined data of N=4 (neutrophils) and N=3 (HL-60) independent experiments is shown. For neutrophils and fMLP, a total of 672 migrating out of 828 total cells were analyzed, fMLP+Slit (176 migrating/669 total), HL-60 (fMLP 530/1020), fMLP+Slit (221/1571).
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Related In: Results  -  Collection

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Figure 5: Inhibitory effect of Slit2 on fMLP-induced human neutrophil and HL-60 migrationPercentage of A) human neutrophils and B) HL-60 cells that migrate towards or away from fMLP with or without the presence of Slit2 (N=4 independent experiments). For both cell types Slit2 significantly inhibits fMLP-induced migration (p<0.01 and p<0.001 respectively, Student's t test). C) Migratory persistence of human neutrophils is not affected by Slit2 (fMLP BD-Index = 0.89, fMLP+Slit2 BD-Index = 0.84). Directional persistence of migrating HL-60 cells is decreased by Slit2 (fMLP BD-Index = 0.63, fMLP+Slit2 BD-Index=0.39; p<0.001, Mann Whitney test). Bars represent mean and standard deviation of Bi-Directional Index. Speed of E) neutrophils and F) HL-60 migrating towards fMLP with or without Slit2 (arrows indicate the mean speed. Slit2 decreases migratory speed of HL-60 cells from 20.7±7.1μm min−1 to 12.8±5.9μm min−1 (p<0.01, Student's t test). Colors are: grey, fMLP alone; black, fMLP and Slit2; white, Slit2 alone. Bars represent mean ± SD. Combined data of N=4 (neutrophils) and N=3 (HL-60) independent experiments is shown. For neutrophils and fMLP, a total of 672 migrating out of 828 total cells were analyzed, fMLP+Slit (176 migrating/669 total), HL-60 (fMLP 530/1020), fMLP+Slit (221/1571).
Mentions: In a final series of experiments, we wished to validate our microfluidic device for the analysis of anti-inflammatory agents. In these assays, we used Slit2 as a prototype inhibitor of neutrophil migratory responses36-38. As illustrated in Fig. 5A, Slit2 (5 μg mL−1) reduced the fraction of human neutrophils migrating towards fMLP (100 nM) by ~3 fold (from 74% of cells migrating to 23%, p<0.001). Similarly, we evaluated the effect of Slit2 on the migration of the clonal HL-60 cell line and found that its inhibitory effect was concentration-dependent (Fig.5). Lower numbers of cells migrated at higher concentrations of Slit2 (p<0.05). Slit2 (1.5 μg mL−1) reduced the number of HL-60 cells that migrate towards fMLP (100 nM) by ~3 fold (from 40% to 11%, p<0.01, Fig. 5B). Slit2 did not reduce the persistence of migrating neutrophils, but it slightly decreased the bi-directional index of migrating HL-60 cells from 0.63±0.57 to 0.39±0.68 (p<0.001, Fig. 5 C, D). In control experiments, where Slit-2 was present in media alone, a migratory response was evident only in 0.8±0.7 % of human neutrophils and 12.1±3.4% of HL-60 cells.

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