Burn Injury-Associated MHCII+ Immune Cell Accumulation Around Lymphatic Vessels of the Mesentery and Increased Lymphatic Endothelial Permeability Are Blocked by Doxycycline Treatment

Abstract

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII⁺ immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Keywords: burn; lymphatic; macrophage; permeability.

FIG. 1.

Images of mesenteric lymphatic vessels from sham (a), and burn (b) rats stained for MHCII (green) (40 × ). Quantification of cells interacting with the vessel (white line) (c), in the periphery (d), and the ratio of the two (likelihood of a cell being on the vessel) (e). N = 6 animals with three images per animal all data analyzed by ANOVA with Dunnett's post-test. *denotes significant change from sham (p < 0.05).

FIG. 2.

Morphometric analysis of cells on lymphatic vessels from sham (a) and burn (b) (40 × with 2 × zoom) (red line shows cell border). Measurements were made of area (c) and shape (d–f). N = 6 animals with 3 images per animal and 10 cells per vessel analyzed. All data analyzed by ANOVA with Dunnett's post-test. *denotes significant change from sham (p < 0.05).

FIG. 3.

Data from lymphatic monolayer permeability studies. Inserts treated with sham, sham treated with doxycycline, burn or burn with doxycycline serum, and measures of FITC-BSA leakage were made N = 9. All data were analyzed by ANOVA with Dunnett's post-test. *denotes significant change from sham (p < 0.05).

FIG. 4.

Data from VE-cadherin staining of rat mesenteric lymphatic endothelial cells. The number of breaks in the cell to cell junctions was quantified per 40 × field of view. N = 3 with each N made up of the average of three images per sample. All data were analyzed by ANOVA with Dunnett's post-test. *denotes significant change from sham (p < 0.05).

FIG. 5.

Data from sham, burn, and burn treated with doxycycline-treated rats for MHCII accumulation on and off the vessel (a, b), the ratio of the two (c), and the size change of the cell associated with the vessel (d). N = 6 with 3 images per animal and 10 cells per vessel. All data were analyzed by ANOVA with Dunnett's post-test. *denotes significant change from sham (p < 0.05).

FIG. 6.

Overview of the theorized changes that occur around lymphatic vessels during burn injury. As a quiescent state changes to an inflamed state due to burn injury, lymphatic permeability increases allowing lymph contents escape the vessel to form a chemoattractive gradient that guides cells that escape from nearby blood vessels to the lymphatic vessel.