Frontline Science: Elevated nuclear lamin A is permissive for granulocyte transendothelial migration but not for motility through collagen I barriers

Abstract

Transendothelial migration (TEM) of lymphocytes and neutrophils is associated with the ability of their deformable nuclei to displace endothelial cytoskeletal barriers. Lamin A is a key intermediate filament component of the nuclear lamina that is downregulated during granulopoiesis. When elevated, lamin A restricts nuclear squeezing through rigid confinements. To determine if the low lamin A expression by leukocyte nuclei is critical for their exceptional squeezing ability through endothelial barriers, we overexpressed this protein in granulocyte-like differentiated HL-60 cells. A 10-fold higher lamin A expression did not interfere with chemokinetic motility of these granulocytes on immobilized CXCL1. Furthermore, these lamin A high leukocytes exhibited normal chemotaxis toward CXCL1 determined in large pore transwell barriers, but poorly squeezed through 3 μm pores toward identical CXCL1 gradients. Strikingly, however, these leukocytes successfully completed paracellular TEM across inflamed endothelial monolayers under shear flow, albeit with a small delay in nuclear squeezing into their sub-endothelial pseudopodia. In contrast, CXCR2 mediated granulocyte motility through collagen I barriers was dramatically delayed by lamin A overexpression due to a failure of lamin A high nuclei to translocate into the pseudopodia of the granulocytes. Collectively, our data predict that leukocytes maintain a low lamin A content in their nuclear lamina in order to optimize squeezing through extracellular collagen barriers but can tolerate high lamin A content when crossing the highly adaptable barriers presented by the endothelial cytoskeleton.

Keywords: chemokines; chemotaxis; granulocytes; inflammation; motility.

Figure 1.. Lamin A overexpression in CXCR2 expressing granulocyte-like dHL-60 cells does not affect the surface expression of receptors involved in trans-endothelial migration and does not alter CXCL1 driven chemokinesis.

(A) Adhesive and migratory phenotypes of granulocyte-like DMSO differentiated HL-60 (dHL-60) variants deficient in CXCR2 or stably expressing CXCR2 crossing inflamed endothelial monolayers under shear flow. The two dHL-60 cells were perfused over monolayers of confluent HDMVECs which had been stimulated with IL-β for 3 hrs to induce E-selectin, integrin ligands and multiple CXCR2 and CCR2 chemokines. (B) Intracellular FACS staining of lamins A/C in permeabilized CXCR2 dHL-60 cells stably expressing the pRetroX-PrelaminA-IRES-ZsGreen1 construct (green) or control CXCR2 dHL-60 cells (black). Dashed line depicts cell staining with an isotype matched control mAb. (C-H) FACS analyzed surface staining of CXCR2, the E-selectin carbohydrate ligand carrying the HECA-452 epitope, and the integrin subunits CD18, CD11a, CD11b and CD29 on sham (black) vs. ZsGreen1-lamin A/C overexpressing (green) dHL-60 cells. Cells were labeled as described in the Materials and Methods section with either PE conjugated anti-human mAbs or with unlabeled primary antibodies followed by a PE or APC conjugated secondary Ab. (I) Migration tracks of control and lamin A overexpressing dHL-60 cells settled on immobilized CXCL1 analyzed with Imaris 9.0.0 software. The migration tracks are plotted with a common origin (central black dot) and the color code depicts the start and end time points of each track. The average total distance mean ± SD travelled by individual granulocyte-like cells within the indicated experimental groups interacting with immobilized CXCL1 is depicted in parenthesis.

Figure 2.. Lamin A overexpression in granulocyte-like HL-60 cells restricts chemotaxis via small rigid pores.

(A) Chemotaxis of control and lamin A overexpressing (LaminA-OE) granulocyte-like CXCR2 expressing dHL-60 cells towards a CXCL1 gradient across transwell membranes with either 3 or 5 micron pore sizes. Cells were collected 30 mins after introduction to the upper wells. The assays were performed in triplicate. Results are representative of two independent experiments. (B) Representative images of Hoechst labeled CXCR2 control (black) and ZsGreen1-lamin A overexpressing (green, LaminA OE cells) dHL-60 cells settled on a PLL coated surface. (C,D) Scatter plots of nuclear circularity index determined for Hoechst labeled control and lamin A overexpressing dHL-60 cells attached to PLL coated surface (C) or immobilized CXCL1 (D). For more details, refer to the Materials and Methods section. The experiments in C and D are each representative of three. Error bars represent mean ± SD. *p < 0.02.

Figure 3.. Lamin A overexpression in granulocyte-like HL-60 cells is permissive for TEM across an inflamed IL-1β stimulated HDMVEC monolayer.

(A) Migratory phenotypes of control and lamin A overexpressing (Lamin A-OE) granulocyte-like differentiated CXCR2 expressing HL-60 cells interacting with HDMVECs stimulated for 3 hrs with IL-β under shear flow. Values represent the mean ± SD of three fields in each experimental group. The experiment shown is representative of three. (B) The percentage of granulocyte-like CXCR2 dHL-60 cells that completed transmigration across the inflamed HDMVEC monolayer at the indicated time points following the accumulation phase. Values represent the mean ± SEM of three fields in each experiment. The experiment shown is representative of three. * p< 0.02 for t= 3 mins. (C) Images of a representative Hoechst-labeled sham (control) and lamin A overexpressing (LaminA-OE) granulocyte-like dHL-60 cell during paracellular TEM taken 3 mins after the end of the accumulation phase. The green outline depicts the basolateral leading edge of the transmigrating dHL-60 cell. (D) The percentage of dHL-60 cells that projected a protrusive sub-endothelial leading edge underneath the monolayer at the indicated time points prior to nucleus crossing. (E) Images of a representative Hoechst-labeled sham (control) and lamin A overexpressing (LaminA-OE) dHL-60 cell taken 15 sec after the end of the accumulation phase. The green outline depicts the basolateral leading edge generated during the early stage of TEM. (F) TEM kinetics of individual control vs. lamin A overexpressing (LaminA-OE) granulocyte-like CXCR2 dHL-60 cells that crossed the inflamed HDMVEC monolayer measured from the first detectable protrusion of a sub-endothelial leading edge to the final detachment of the dHL-60 uropod from the apical endothelial aspect. Values were determined in multiple fields taken from three independent experiments. Error bars represent mean ± SD. **p < 0.002.

Figure 4.. Lamin A overexpressing granulocyte-like dHL-60 cells exhibit slower nuclear squeezing and generate larger endothelial gaps during paracellular TEM.

(A) Images taken from Supplemental Video 1 recording a representative Hoechst-labeled sham (control) and lamin A overexpressing (LaminA-OE) granulocyte-like CXCR2 dHL-60 cell squeezing through paracellular EC junctions. The green outline depicts the basolateral leading edge of the transmigrating dHL-60 cell and the red outline depicts the nuclear lobes inserted underneath the endothelial monolayer. The red circumference of the nucleus is highlighted in white dots at the first time point at which the entire nucleus of each of the transmigrating dHL-60 cells has completed its passage underneath the endothelial monolayer. The yellow asterisks denote the leukocyte uropods at the time of TEM completion. Time intervals are depicted in each image. Scale bar= 5 μm. (B) Nuclear passage duration in individual control and lamin A overexpressing (LaminA-OE) CXCR2 dHL-60 cells transmigrating across inflamed HDMVECs monolayers. Values represent cells from multiple fields taken from three independent experiments. Error bars represent mean ± SD. *p < 0.03. (C) The diameter of endothelial gaps generated by crossing granulocyte-like CXCR2 dHL-60 (control vs. LaminA-OE) cells determined as described in Materials and Methods. Values for cells from multiple fields were collected in three independent experiments. Error bars represent the mean ± SD. *p < 0.03.

Figure 5.. A setup for leukocyte motility on a chemokine-coated surface in the presence of distinct 3D collagenous barriers.

(A) A scheme depicting the experimental model used to assess leukocyte crossing of distinct collagenous barriers. Leukocytes suspended in cold matrigel or collagen I solutions were sedimented for 3 mins and incubated under the distinct collagen solutions for 30 min at 37°C to allow collagen polymerization. Leukocyte motility was recorded for 30 additional minutes. (B) Representative confocal reflectance images of matrigel (50% solution) and collagen 1 matrices (3.2 mg/ml) which underwent polymerization for 30 mins at 37°C. Scale bars= 10μm.

Figure 6.. Lamin A overexpression in granulocyte-like cells does not affect their chemokine driven motility through a permissive matrigel barrier.

Images from Supplemental Videos 2 and 3 depicting representative control (A) vs. lamin A overexpressing (LaminA-OE, B) Hoechst-labeled granulocyte-like dHL-60 cells migrating on immobilized CXCL1 either in aqueous medium (left) or when embedded inside a polymerized matrigel matrix (right). Time codes are depicted and scale bars= 5 μm. (C) Nuclear locations in control (Cont.) and lamin A (LaminA-OE) overexpressing Hoechst labeled granulocyte like dHL-60 cells migrating over immobilized CXCL1 either in medium or embedded in polymerized matrigel (50% solution). Results were determined for 40–50 cells from 3 independent experiments. (D) The velocities of individual control (Cont.) and lamin A overexpressing (LaminA-OE) granulocyte-like dHL-60 cells migrating over immobilized CXCL1 alone or when embedded in the polymerized matrigel. Values represent cells from multiple fields taken from three independent experiments. Error bars represent mean ± SD. *p < 0.05; **p < 0.007.

Figure 7.. Lamin A overexpression in granulocyte-like cells restricts nucleus squeezing and chemokine driven motility through a dense collagen I barrier.

(A) Images from Supplemental Video 4 depicting representative control vs. lamin A overexpressing (LaminA-OE) Hoechstlabeled granulocyte-like dHL-60 cells migrating on immobilized CXCL1 while being embedded inside a polymerized collagen I matrix (B). Time codes are depicted for each image. Scale bar= 5 μm. Yellow arrows depict the direction of granulocyte motility over the chemokine coated 2D surface. The red asterisk indicates a retracting leading edge. (B) Nuclear locations in control and lamin A overexpressing (LaminA-OE) Hoechst labeled granulocyte like dHL-60 cells migrating over immobilized CXCL1 through the collagen I barrier. Results were determined for 40–50 cells from 3 independent experiments. (C) Scatter plot of velocities of individual control and lamin A/C overexpressing (LaminA-OE) granulocyte-like dHL-60 cells migrating through polymerized collagen I matrices. Values were collected from three independent experiments. Error bars represent mean ± SD. **p < 0.004. (D) Kinetics of collagen I barrier crossing of individual control and lamin A overexpressing (LaminA-OE) granulocyte-like dHL-60 cells. The numbers of cells within each experimental group that successfully crossed a 20 μm long barrier of collagen I as a function of time. Values represent the mean ± SEM of three fields. ** p< 0.01 for t= 9 mins. The experiment shown is a representative of three.