Delay of migrating leukocytes by the basement membrane deposited by endothelial cells in long-term culture

Abstract

We investigated the migration of human leukocytes through endothelial cells (EC), and particularly their underlying basement membrane (BM). EC were cultured for 20days on 3μm-pore filters or collagen gels to form a distinct BM, and then treated with tumour necrosis factor-α, interleukin-1β or interferon-γ. Neutrophil migration through the cytokine-treated EC and BM was delayed for 20-day compared to 4-day cultures. The BM alone obstructed chemotaxis of neutrophils, and if fresh EC were briefly cultured on stripped BM, there was again a hold-up in migration. In studies with lymphocytes and monocytes, we could detect little hold-up of migration for 20-day versus 4-day cultures, in either the filter- or gel-based models. Direct microscopic observations showed that BM also held-up neutrophil migration under conditions of flow. Treatment of upper and/or lower compartments of filters with antibodies against integrins, showed that neutrophil migration through the endothelial monolayer was dependent on β(2)-integrins, but not β1- or β(3)-integrins. Migration from the subendothelial compartment was supported by β1- and β(2)-integrins for all cultures, but blockade of β(3)-integrin only inhibited migration effectively for 20-day cultures. Flow cytometry indicated that there was no net increase in expression of β1- or β3-integrins during neutrophil migration, and that their specific subendothelial function was likely dependent on turnover of integrins during migration. These studies show that BM is a distinct barrier to migration of human neutrophils, and that β(3)-integrins are particularly important in crossing this barrier. The lesser effect of BM on lymphocytes and monocytes supports the concept that crossing the BM is a separate, leukocyte-specific, regulated step in migration.

Supplementary Fig. 1

Phase-contrast photomicrographs taken as one focuses down from the endothelial surface, through the filter and onto the coverslip below. Neutrophils on the endothelial surface appear phase-bright with round or distorted outline, while those underneath are phase-dark and spread (circle). Neutrophil on either of the two lower surfaces are phase-bright, some round, some with distorted shape (circled). They do not spread and appear phase-dark like those immediately under the endothelial monolayer.

Fig. 1

Comparison of transmigration of neutrophils through HUVEC which had been cultured for 4 () or 20 () days and then treated with TNF or IL-1. A,B. Scanning electron micrographs of matrix deposited by EC on filters after culture for 4 days or 20 days. C. Transmigration after 2 h through HUVEC treated with different doses of TNF. D. Time course of transmigration through HUVEC treated with 100 U/ml TNF. E. Comparison of transmigration through HUVEC treated with TNF (100 U/ml) or IL-1 (50 pg/ml). F. Effect of adding IL-8 to the bottom well on transmigration through HUVEC treated with TNF (100 U/ml). Data are the mean ± SEM from 3 to 7 independent experiments. In C, ANOVA showed a significant effect of TNF dose (p < 0.01) and culture duration (p < 0.01). In D, ANOVA showed a significant effect of culture duration (p < 0.05) and assay time (p < 0.01). In E and F, ANOVA showed significant effects of culture duration (p < 0.01). *p < 0.05, **p < 0.01 compared to 4-day by paired t-test.

Fig. 2

Comparison of transmigration of neutrophils through HUVEC cultured for different durations undisturbed or re-seeded on matrix deposited over different periods. HUVEC were cultured for 4 or 20 days, and then stripped from the underlying matrix. Fresh HUVEC were added to the matrix and cultured for 2 days. Control HUVEC were cultured undisturbed in parallel for 6 or 22 days. The final ‘age’ of matrices was therefore 6 or 22 days. All HUVEC were treated with 100 U/ml TNF for 4 h before neutrophils were added to the upper chamber and allowed to transmigrate for 2 h. Data are the mean ± SEM from three (undisturbed) or five (re-seeded) experiments. ANOVA showed a significant effect of matrix age on transmigration (p < 0.01). *p < 0.05 compared to 6-day matrix by paired t-test.

Fig. 3

Comparison of transmigration of (A) mononuclear cells, (B), lymphocytes, (C), monocytes, through HUVEC which had been cultured for 4 () or 20 days () and then treated with TNF + IFNγ. Data are the mean ± SEM from 3 independent experiments in which migration was analysed after 2, 4 and 24 h.

Fig. 4

Comparison of transmigration of different leukocytes through HUVEC and into collagen gels after culture for 2 or 20 days and treatment with TNF for 4 h. The proportions of adherent leukocytes were counted below the endothelial monolayer or within the gel, for neutrophils (A,B), lymphocytes (C,D), mononuclear cells (E,F) or monocytes (G,H). Data are the mean ± SEM from 3 or more independent experiments with each type of cell. ANOVA showed a significant effect of culture duration on migration in A (p < 0.05), B (p < 0.01) and D (p < 0.01). *p < 0.05, **p < 0.01 for comparison between 4-day and 20-day cultures by paired t-test.

Fig. 5

Comparison of behaviour of neutrophils adhering from flow to TNF-treated HUVEC cultured for 4 or 20 days. A. Percentage of adherent neutrophils rolling, stationary adherent or transmigrated through the monolayer after 11 min. B. Percentage of adherent neutrophils that transmigrated through the filter over time. HUVEC were cultured for 4 or 20 days, treated with 100 U/ml TNF for 4 h and transferred to a flow chamber before perfusion of neutrophils for 4 min, followed by washout. Time zero was the start of neutrophil perfusion. Data are the mean ± SEM from 4 experiments. In B, ANOVA showed a significant effect of culture duration on migration (p < 0.01).

Fig. 6

Function and expression of β₂-integrins during transmigration of neutrophils through TNF-treated HUVEC cultured for 4 or 20 days. Effects of adding antibody against β₂-integrin to the upper chamber of Transwell filters (with neutrophils) or the lower chamber only, on: (A) adhesion to HUVEC; (B) transmigration; (C) surface expression of β₂-integrin on neutrophils retrieved from upper or lower chambers (i.e., non-adherent or transmigrated neutrophils respectively). Results for 4-day () and 20-day () cultures are shown, expressed relative to untreated control. In A, data are the mean ± SEM from 5 experiments. ANOVA showed a significant effect of treatment of the upper chamber; *p < 0.05 compared to control by paired t-test. In B, data are the mean ± SEM from 5 experiments. ANOVA showed a significant effect of treatment of either type; *p < 0.05 compared to control by paired t-test. In C, data are the mean ± SEM from 3 experiments. ANOVA showed a significant effect of treatment of either type, but there was no significant effect of treatment for the individual days.

Fig. 7

Function and expression of β₁-integrins during transmigration of neutrophils through TNF-treated HUVEC cultured for 4 or 20 days. Effects of adding antibody against β₁-integrin to the upper chamber of Transwell filters (with neutrophils) or the upper and lower chambers, on: (A) adhesion to HUVEC; (B) transmigration; (C) surface expression of β₁-integrin on neutrophils retrieved from upper or lower chambers (i.e., non-adherent or transmigrated neutrophils respectively). Results for 4-day () and 20-day () cultures are shown, expressed relative to untreated control. In A, data are the mean ± SEM from 5 experiments. In B, data are the mean ± SEM from 5 experiments. ANOVA showed a significant effect of treatment of both chambers; **p < 0.01 compared to control by paired t-test. In C, data are the mean ± SEM from 3 experiments.

Fig. 8

Function and expression of β₃-integrins during transmigration of neutrophils through TNF-treated HUVEC cultured for 4 or 20 days. Effects of adding antibody against β₃-integrin to the upper chamber of Transwell filters (with neutrophils) or the upper and lower chamber only, on: (A) adhesion to HUVEC; (B) transmigration; (C) surface expression of β₃-integrin on neutrophils retrieved from upper or lower chambers (i.e., non-adherent or transmigrated neutrophils respectively). Results are shown for 4-day () and 20-day () cultures, expressed relative to untreated control. In A, data are the mean ± SEM from 6 experiments. In B, data are the mean ± SEM from 6 experiments. ANOVA showed a significant effect of treatment of the both chambers; *p < 0.05 compared to control by paired t-test. In C, data are the mean ± SEM from 4 experiments.

Fig. 9

Effects of combinations of antibodies against integrins on transmigration of neutrophils through TNF-treated HUVEC cultured for 4 or 20 days. Antibodies against β₁- and β₃-integrins were added to the upper and lower chambers, and antibody against β₂-integrin was added to the lower chambers only. Results are shown for 4-day () and 20-day () cultures, expressed relative to untreated control. Data are the mean ± SEM from 4 experiments. **p < 0.01 compared to control by paired t-test. ⁺p < 0.05 for comparison between 4-day and 20-day cultures by paired t-test.

Fig. 10

Effects of inhibiting integrin function on migration velocity of neutrophils after migration underneath HUVEC cultured for 4 or 20 days. HUVEC were cultured for 4 or 20 days, treated with 100 U/ml TNF for 4 h and transferred to a flow chamber before perfusion of neutrophils for 4 min, followed by washout. Migration velocity was measured between 11–16 min of washout. Antibody against β₁-integrin () or β₃-integrin () was added to neutrophils before perfusion, but was absent from washout medium. RGDS peptide () was added to neutrophils before perfusion and was also present in washout medium. Data are the mean ± SEM of the mean velocities relative to untreated controls from 3 experiments. *p < 0.05, **p < 0.01 compared to untreated cells by paired t-test.

Fig. 11

Protein content of matrix from HUVEC cultured for 4 or 20 days, and effects of integrin-inhibition on adhesion of neutrophils to matrix or purified proteins. A. shows absorbance values from ELISA for vitronectin (Vn), laminin (Lam), α4-chain of laminin (Lam α4), α5-chain of laminin (Lam α5) on matrices deposited by HUVEC after culture for 4 days () or 20 days (). B. shows effect of RGDS peptide on adhesion of neutrophils to 4-day or 20-day BM, collagen type IV (Coll), laminin (Lam), fibronectin (Fn) or vitronectin (Vn). C. shows effect of antibody against β₃-integrin on adhesion to 4-day or 20-day BM, fibronectin (Fn) or vitronectin (Vn). In B and C, adhesion is expressed relative to untreated control. Data are the mean ± SEM from 3 or more experiments. *p < 0.05, **p < 0.01 compared to 4-day in A, or untreated control in B and C, by paired t-test.