Increased endothelial cell-leukocyte interaction in murine schistosomiasis: possible priming of endothelial cells by the disease

Abstract

Background and aims: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro.

Methodology and principal findings: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups.

Conclusion/significance: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.

Figure 1. Schistosomiasis increases leukocyte migration into peritoneal cavity in vivo.

A) Number of infiltrated leukocytes obtained from control (white bar) and S. mansoni- infected (gray bar) mice. Data expressed as the mean and S.E.M. *P<0.05, Students t test. B) Differential counting of peritoneal infiltrated leukocytes obtained from control (white bars) and S. mansoni-infected (gray bars) mice. P<0.05 for a vs b, one-way ANOVA followed by Bonferroni's Multiple Comparison test, n = 5–9.

Figure 2. Schistosomiasis increases leukocyte adhesion to mesenteric endothelial cells in vitro.

Data expressed as the mean and S.E.M. A. Number of mononuclear leukocytes adhering to endothelial cell monolayer obtained from control (white bar) and S. mansoni-infected mice (gray bar). *P<0.05, Students t test. B. Number of mononuclear leukocytes adhering to endothelial cell monolayer both from control mice in the absence (open white bar) or presence of TNF (0.1 ng/ml), TNF (0.1 ng/ml) plus SNAP (1 µM) or L-NNA (300 µM) treatment for 4 h. *P<0.05 , One-way ANOVA followed by Bonferroni's Multiple Comparison test. C. Number of mononuclear leukocytes adhering to endothelial cell monolayer from S. mansoni-infected mice in the absence (open gray bar) or presence of TNF (0.1 ng/ml) or TNF (0.1 ng/ml) plus SNAP (1 µM) treatment for 4 h. *P<0.05, One-way ANOVA followed by Bonferroni's Multiple Comparison test. D. Number of mononuclear leukocytes adhering to endothelial cell monolayer. White open bar  =  control endothelial cells (EC) incubated with mononuclear leukocytes (mono) from S. mansoni-infected mice. Gray bar with horizontal lines  =  endothelial cells (EC) from infected mice incubated with control mononuclear leukocytes (mono). *P<0.05, Student's t test. n = 16 replicates of a typical experiment. TNF  =  tumor necrosis fator; SNAP  =  S-Nitroso-N-Acety-DL-Penicillamine; L-NNA  =  N^G-nitro-L-Arginine.

Figure 3. Nitric oxide production in cultured endothelial cells from control and S. mansoni-infected mice.

Nitric oxide (NO) production by confluent mesenteric endothelial cells in response to 100 µM ATP and 2 µM A23187 was measured in living cells using the fluorescent probe DAF-FM (2.5 µM) and a microplate fluorometer. Control mice: white bars (open, horizontal and vertical lines). Infected mice: gray bars (open, horizontal and vertical lines). Data are expressed as the mean and S.E.M. of 6–7 experiments performed in triplicate obtained from four different cultures and from different animals (ATP condition) or four replicates of a typical experiment (A23187 condition). Basal NO production observed in the absence of ATP or A23187 was considered as 100%. *P<0.05 vs. control mice; **P<0.05 vs. A23187 treatment in control mice, One-way ANOVA followed by Bonferroni's Multiple Comparison test.

Figure 4. Expression of eNOS and caveolin-1 in endothelial cells from control and S. mansoni-infected mice.

C  =  control; I  =  infected. A refers to eNOS expression; B refers to caveolin-1 (Cav-1) expression in confluent cultured mesenteric endothelial cells. Insert: Representative experiment of each experimental group. Rouge of Ponceau dye was used as an internal control of protein loading and did not differ among samples (data not shown). Data expressed as the mean and S.E.M. *P = 0.014 (Students t test), n = 3 (caveolin-1) or 5 (eNOS).

Figure 5. Expression of eNOS and caveolin-1 in endothelial cells from control and S. mansoni-infected mice.

Merged images of the immunocytochemical staining of cultured endothelial cells using an antibody against eNOS (red) and nuclear fluorescence using DAPI (blue) (400X). Upper and lower left panels: controls. Upper and lower right panels: S. mansoni-infected mice. Each image from control and infected groups was randomly chosen and obtained from a different plate.