Histamine activates p38 MAP kinase and alters local lamellipodia dynamics, reducing endothelial barrier integrity and eliciting central movement of actin fibers

Abstract

The role of the actin cytoskeleton in endothelial barrier function has been debated for nearly four decades. Our previous investigation revealed spontaneous local lamellipodia in confluent endothelial monolayers that appear to increase overlap at intercellular junctions. We tested the hypothesis that the barrier-disrupting agent histamine would reduce local lamellipodia protrusions and investigated the potential involvement of p38 mitogen-activated protein (MAP) kinase activation and actin stress fiber formation. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein-actin were studied using time-lapse fluorescence microscopy. The protrusion and withdrawal characteristics of local lamellipodia were assessed before and after addition of histamine. Changes in barrier function were determined using electrical cell-substrate impedance sensing. Histamine initially decreased barrier function, lamellipodia protrusion frequency, and lamellipodia protrusion distance. A longer time for lamellipodia withdrawal and reduced withdrawal distance and velocity accompanied barrier recovery. After barrier recovery, a significant number of cortical fibers migrated centrally, eventually resembling actin stress fibers. The p38 MAP kinase inhibitor SB203580 attenuated the histamine-induced decreases in barrier function and lamellipodia protrusion frequency. SB203580 also inhibited the histamine-induced decreases in withdrawal distance and velocity, and the subsequent actin fiber migration. These data suggest that histamine can reduce local lamellipodia protrusion activity through activation of p38 MAP kinase. The findings also suggest that local lamellipodia have a role in maintaining endothelial barrier integrity. Furthermore, we provide evidence that actin stress fiber formation may be a reaction to, rather than a cause of, reduced endothelial barrier integrity.

Keywords: actin cables; endothelial permeability; inflammation; lamellipodia; p38 MAPK.

Fig. 1.

Histamine alters the dynamics of lamellipodia protrusion and withdrawal. A: time-lapse images show human umbilical vein endothelial cells (HUVEC) expressing green fluorescent protein (GFP)-actin during baseline and 1, 12, and 20 min after addition of 10 μM histamine. Cells either not expressing GFP-actin or expressing very low levels occupy the dark spaces around this cluster of cells. The small arrows in the baseline image point to local lamellipodia. Within 1 min after histamine was added, there was a shift in GFP-actin, with some cells developing very intense labeling around large vesicle-like structures (arrows). At 12 and 20 min, there was apparent movement of cortical actin fibers toward the center of the cell, eventually resembling actin stress fibers (arrows). The entire video is available in the supplemental material. B: time courses of histamine-induced changes in mean protrusion frequency (PF), distance (PD), persistence (PP), and velocity (PV) of endothelial local lamellipodia were obtained by kymograph analysis of n = 9 cells from three different experiments. C: withdrawal distance (WD), time (WT), and velocity (WV), and percentage of protrusions that had a WT greater than 5 min were also obtained from the same cells. Each time point represents an average of the previous 5-min period. *P < 0.05 vs. the 0-min time point, representing the period just before histamine addition.

Fig. 2.

Histamine promotes lateral migration of cortical actin fiber bundles to the center of the cell. A: in this kymograph generated from time-lapse images of an endothelial cell expressing GFP-actin, the location where individual actin fibers cross the kymograph line over time can be observed. The beginning of the time-lapse series is at the top. Arrowheads denote where one individual actin fiber crosses the kymograph line (horizontal dimension) over time (vertical dimension). The fibers often form at the periphery of the cell (denoted by X) and travel toward the center. After 10 μM histamine was added, more fibers moved toward the center of the cell. Additional fibers also appeared due to the formation and movement of bifurcations. *Denotes where a stress fiber bifurcation crossed the kymograph line. B: mean lateral velocity of actin stress fibers over 5-min periods before and after treatment with 10 μM histamine was calculated using kymographs of nine different cells treated with histamine in three different experiments. C: mean number of actin fibers obtained from the same kymographs of histamine-treated cells for each 5-min period. In B and C, *P < 0.05 vs. baseline (0-min time point).

Fig. 3.

Inhibition of the p38 mitogen-activated protein (MAP) kinase, but not ERK-1/2, attenuates histamine-induced endothelial barrier disruption. A: histamine causes phosphorylation of the activation sites of both p38 MAP kinase and ERK-1/2 in endothelial cells. Histamine was applied at 10 μM for 2 min. Inhibitors were all applied for 30 min before histamine in the following concentrations: SB203580, 6 μM; PD98059, 10 μM; U0126, 5 μM. VEGF was used as a positive control and was applied for 5 min. The blots shown are representative of three separate experiments. B: time course of 10 μM histamine-induced decreases in transendothelial resistance (TER) in the absence of inhibitors or in the presence of 5 μM U0126, 10 μM PD98059, or 6 μM SB203580. C: maximal change in TER elicited by histamine in the presence of MAPK inhibitors. *P < 0.05 vs. no inhibitors. In B and C, n = 16 HUVEC monolayers in each group.

Fig. 4.

Inhibition of p38 MAP kinase reduces the impact of histamine on lamellipodia protrusion and withdrawal. A: time courses of lamellipodia PF, PD, PP, and PV during baseline, addition of 6 μM SB203580, and addition of 10 μM histamine. B: time course changes in lamellipodia WD, WT, and WV, along with the percentage of protrusions with a WT greater than 5 min during baseline, SB203580 (SB) treatment both before and after histamine was added. C: maximum changes in the aforementioned protrusion and withdrawal parameters are directly compared between cells that were treated with histamine in the absence or presence of SB203580. *P < 0.05, in A compared with baseline (−20 min time point), and in C between the two groups. Measurements came from n = 9 cells from 3–4 different experiments in each group.

Fig. 5.

Inhibition of p38 MAP kinase attenuates histamine-induced actin stress fiber formation. A: time course of changes in lateral velocity and number of actin fiber cables during baseline, after addition of 6 μM SB203580, and after addition of 10 μM histamine. B: direct comparisons of maximum changes in lateral velocity and numbers of actin fibers are shown for histamine-treated cells with or without SB203580 pretreatment. *P < 0.05 between groups. Measurements came from n = 9 cells from 3–4 different experiments in each group.