Monocyte ADAM17 promotes diapedesis during transendothelial migration: identification of steps and substrates targeted by metalloproteinases

Abstract

Despite expanded definition of the leukocyte adhesion cascade and mechanisms underlying individual steps, very little is known about regulatory mechanisms controlling sequential shifts between steps. We tested the hypothesis that metalloproteinases provide a mechanism to rapidly transition monocytes between different steps. Our study identifies diapedesis as a step targeted by metalloproteinase activity. Time-lapse video microscopy shows that the presence of a metalloproteinase inhibitor results in a doubling of the time required for human monocytes to complete diapedesis on unactivated or inflamed human endothelium, under both static and physiological-flow conditions. Thus, diapedesis is promoted by metalloproteinase activity. In contrast, neither adhesion of monocytes nor their locomotion over the endothelium is altered by metalloproteinase inhibition. We further demonstrate that metalloproteinase inhibition significantly elevates monocyte cell surface levels of integrins CD11b/CD18 (Mac-1), specifically during transendothelial migration. Interestingly, such alterations are not detected for other endothelial- and monocyte-adhesion molecules that are presumed metalloproteinase substrates. Two major transmembrane metalloproteinases, a disintegrin and metalloproteinase (ADAM)17 and ADAM10, are identified as enzymes that control constitutive cleavage of Mac-1. We further establish that knockdown of monocyte ADAM17, but not endothelial ADAM10 or ADAM17 or monocyte ADAM10, reproduces the diapedesis delay observed with metalloproteinase inhibition. Therefore, we conclude that monocyte ADAM17 facilitates the completion of transendothelial migration by accelerating the rate of diapedesis. We propose that the progression of diapedesis may be regulated by spatial and temporal cleavage of Mac-1, which is triggered upon interaction with endothelium.

Figure 1. Net migration of mononuclear cells across endothelial monolayers is reduced by the broad-spectrum metalloproteinase inhibitor GM6001

A and B. Peripheral blood mononuclear cells were co-incubated for 4 h with HUVECs grown to confluence on fibronectin-coated porous transwell inserts to assess migration in the presence of 50 μM GM6001 or 0.1% DMSO (vehicle control). Before co-incubation, the HUVEC monolayers and leukocytes were separately pre-incubated for 30 min with vehicle or GM6001. HUVECs were unactivated (A) or pre-activated with 0.1 ng/ml TNFα for 4 h (B). Representative data from at least 3 different experiments are shown, *p <0.02, **p<0.002 (unpaired t test). C. Monocyte migration across pre-activated HUVEC monolayers (10 ng/ml TNF-α) was determined by FACS analysis for mononuclear cell transendothelial migration as shown in B. Monocyte numbers in the presence of GM6001 are expressed as ratios relative to those of DMSO. Means ± SD of 6 experiments using different mononuclear cell donors are shown, ***p <0.001 (paired t test).

Figure 2. Metalloproteinase blockade by GM6001 delays monocyte transendothelial migration under static and flow conditions

A. Human monocytes were added to unactivated HUVEC monolayers in the presence of DMSO or GM6001 under static conditions and observed using time-lapse video microscopy every 37.8 s. Individual monocytes migrating on the apical surface of the monolayers were tracked until they complete migration across the monolayers. Monocytes that complete their transendothelial migration up to each time point are expressed as % of total monocytes in the field. In one experiment, an average of 50 monocytes were evaluated for DMSO and 46 for GM6001. B. Monocyte transendothelial migration under flow conditions in the presence of DMSO (Video 1) or GM6001 (Video 2) was analyzed as described in A. Monocytes were drawn across pre-activated HUVEC monolayers (10 ng/ml TNF-α for 4–6 h) at 0.5 dyn/cm² in flow medium containing DMSO or GM6001. Pretreatment with DMSO or GM6001 was 30 min for HUVECs and 5 min for monocytes. Time-lapse video microscopy was performed every 10 s for 30 min. Means ± SD of 3 different experiments are shown, *p < 0.05, **p <0.01, and ***p <0.005 (paired t test).

Figure 3. Monocyte diapedesis is prolonged by metalloproteinase inhibition under both static and flow conditions

A. Sequential time-lapse images from the beginning to the end of diapedesis in the presence of DMSO (top, Video 3) and GM6001 (bottom, Video 4) are shown every 75.6 s. The beginning of diapedesis (Start) was defined as the image frame where the cell body or monocyte membrane protrusion reached the site of diapedesis (asterisk). Completion of diapedesis (End) was denoted as the image frame where the remnant of the monocyte cell body disappeared from the apical surface of HUVEC monolayer. Arrows indicate the direction of monocyte migration. B. Histograms of the diapedesis duration in DMSO and GM6001 are shown for unactivated HUVEC monolayers. Diapedesis duration is defined as the interval between the start and the end frames of diapedesis, as defined in A. A representative data set from 3 different experiments is shown. Medians for this data sets are 8.8 min, n=47 monocytes for DMSO; 21.7 min, n=26 monocytes for GM6001, p <0.0001 (Mann-Whitney test). C. Means ± SD of diapedesis duration from different experiments on unactivated (n=3) and TNF-activated HUVEC (0.1 ng/ml for 4 h, n=4) are shown for monocytes prepared from different donors. * p < 0.05; ** p < 0.02 (paired t test). D. Duration of diapedesis under flow conditions in the presence of DMSO or GM6001 was evaluated as described above. Means ± SD of medians obtained from 3 different experiments are shown, ***p <0.005 (paired t test). In a single experiment, an average of 45 monocytes were evaluated for DMSO and 37 for GM6001.

Figure 4. CD18 integrins are shed from human monocytes in a metalloproteinase-dependent manner both constitutively and inducibly upon interaction with endothelial cells

A and B. Cell-surface expression of CD11a (A), and CD11b (B) on monocytes was determined by FACS analysis after co-incubation with TNF-activated HUVEC monolayers (EC+PBMC) or separate incubation (PBMC alone) for 2 h in the presence of DMSO or GM6001. For analysis of cell surface expression on monocytes, first green-fluorescence endothelial cells were gated out and then CD14+ positive cells were analyzed further. At least 5,000 events were collected. The expression levels are presented as ratios relative to separate incubation with DMSO. Each column represents mean ± SD from 3 different experiments for CD11a, and 5 for CD11b, *p <0.05, **p <0.01 (paired t test). C. Platelet-free monocytes were incubated in Opti-MEM at 10⁶/ml in the presence of DMSO (−) or GM6001 (+) for 16 h at 37°C. Cells were lysed on ice with NP-40 buffer supplemented with proteinase inhibitors and conditioned media (CMs) were concentrated using YM-30 membrane (Millipore) following centrifugation. CMs (40x concentrated, 40 μl) and cell lysates (4 μg) were resolved by 7.5% SDS-PAGE, and evaluated by Western blotting with antibody to the ectodomain of human CD18. Arrows indicate full-length CD18 in the lysates and shed forms (soluble) of CD18 in CM. D. CD18 integrin complexes shed in 16-h CM were quantitated by sandwich ELISA using antibodies for CD11a and CD11b for capture and CD18 for detection. See Table SI for antibodies used.

Figure 5. Depletion of ADAM17 and ADAM10 from primary monocytes decreases shedding of CD11b/CD18

Human primary monocytes were transfected with siRNAs for ADAM17, ADAM10 and a non-specific control sequence. A. Knockdown of ADAM17 and ADAM10 was evaluated by immunoblotting of cell lysates 16 h post-transfection. Cells were lysed with NP-40 lysis buffer and 10 μg/lane were separated by 7.5% SDS-PAGE under reducing conditions and probed with rabbit polyclonal antibodies against the cytoplasmic domains of ADAM10 and ADAM17 or Pan-actin antibody (see Table SI for antibodies). Arrows indicate specific bands for each protein. B–C. Soluble CD11a/CD18 (B) and CD11b/CD18 (C) integrins was evaluated by sandwich ELISA of 16-h CMs prepared from siRNA-transfected monocytes as described in Figure 4D. Representative data are shown from 2 independent experiments. Means±SD are calculated from measurements of triplicate wells, *p< 0.005, and **p<0.0001 (unpaired t test). For comparison between control and ADAM10 in B, p< 0.05 (unpaired t test).

Figure 6. Depletion of ADAM17, but not ADAM10, from monocytes prolongs the diapedesis step

A. Untransfected monocytes and monocytes transfected with control siRNA were generated as described in the Materials and Methods, and subjected to time-lapse video analysis on TNF-activated endothelium in the presence of DMSO or GM6001. Representative data from 2 independent experiments are expressed as means ± SEM, **p <0.0005 (unpaired t test with Welch correction; n=19, 37, and 27 monocytes for untransfected, DMSO, and GM6001, respectively). B. Duration of monocyte diapedesis was determined for monocytes transfected with control, ADAM10 or ADAM17 siRNA. Means ± SD from 3 different experiments are shown, *p <0.01 (paired t test). All monocyte preparations show compatible viability (>95% as determined by trypan blue exclusion).