Novel aspects of leukocyte trafficking during endotoxaemia

Ellen Hughes, Julia Buckingham, Felicity Gavins. Imperial College London, London, UK.

The inflammatory response to the Toll-like receptor (TLR) 4 ligand lipopolysaccharide (LPS) involves a sequence of intravascular leukocyte capture, rolling and adhesion, followed by their emigration into surrounding tissue which is often accompanied by plasma protein extravasation (PPE)1. Such effects are typically observed hours post LPS injection. However, we have chosen to investigate the earlier stages of leukocyte trafficking in response to LPS challenge, under the hypothesis that this is mediated by resident immune cells of the peritoneum such as the mast cell.

Male C57BL/6 mice aged 5-8 weeks were used and in all cases, the inflammatory response in terms of leukocyte-endothelial cell interactions within mesenteric venules was quantified in vivo under anaesthesia with i.p. ketamine (150mgkg-1) and xylazine (7.5mgkg-1), using the specialised technique of intravital microscopy. PPE was quantified by injection of FITC-albumin (0.25mgg-1, i.v.) as a ratio of fluorescence inside and outside the vessel. Excised mesenteries and livers were formalin-fixed, paraffin-embedded and sectioned at 3μm thickness for staining by haematoxylin and eosin (H&E)2. Data were statistically analysed using Student’s t-test for 2 groups and ANOVA with Bonferroni’s post-hoc test for >2 groups and are expressed as mean ± SEM.

Leukocyte emigration increased rapidly in response to LPS (10μg/mouse, i.p.) and had reached a plateau 2h post injection at 7.0 ± 1.7 cells (vs. 1.1 ± 0.2 cells with saline vehicle; P = 0.02). PPE followed the same pattern, increasing rapidly post-LPS and reaching a plateau within 2h at 36.09 ± 4.06% (vs. 20.50 ± 1.51% with vehicle; P = 0.005). The TLR2 ligand, zymosan (500g suspension/mouse, i.p.) also provoked rapid leukocyte emigration (5.5 ± 0.4 cells) and PPE (43.43 ± 2.50%) in line with LPS (5.8 ± 0.6 cells and 44.06 ± 2.82%, respectively), but the non-specific neutrophil activator, ammonium thioglycolate (250μl of a 3% solution, i.p.) did not, implicating TLR-specific mechanisms in LPS-induced leukocyte trafficking.

H&E analysis of mesenteric sections revealed the leukocyte population to consist primarily of neutrophils after LPS challenge. Leukocyte trafficking was also observed systemically in response to LPS: H&E sections of liver showed that intrasinusoidal leukocytic activity and small foci of infiltrating inflammatory cells had grown into microabscesses (consisting of neutrophils and B-lymphocytes) dispersed throughout the parenchyma within 2h of LPS injection.

Mesenteric leukocyte trafficking was associated with activation of peritoneal mast cells. Superfusion of buffer containing 0.001% ruthenium red, a dye selectively taken up by activated mast cells3, showed that mice in the early stages of an LPS-induced inflammatory response showed a greater (2.2x) uptake of ruthenium red by mast cells than saline-treated controls, indicating that LPS enhanced mast cell activation. The leukocyte infiltration was inhibited by pre-treatment 15 min before LPS administration with the general mast cell stabiliser cromolyn sodium (10mgkg-1, i.p.), which limited leukocyte emigration to 2.4 ± 0.1 cells, compared to saline vehicle pre-treatment at 5.8 ± 0.6 cells (P = 0.002).

In conclusion, our novel data suggest that mast cells play a role in LPS-induced peritoneal inflammation in terms of leukocyte emigration. Our finding that mast cell stabilisation blocks emigration but not PPE indicates that PPE occurs independently of the mechanisms by which the leukocyte chemotactic stimulus is generated. Further work is underway to determine the sequential mechanisms of cell types and mediators involved in these hyperacute stages of LPS-induced inflammation.

1. Ley, K., Laudanna, C., Cybulsky, M. & Nourshargh, S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7, 678-89 (2007).

2. Bancroft, J. Theory and practice of histological techniques. (Churchill Livingstone: London;New York, 2002).

3. Johnston, B., Gaboury, J., Suernatsu, M. & Kubes, P. Nitric oxide inhibits microvascular protein leakage induced by leukocyte adhesion-independent and adhesion-dependent inflammatory mediators. Microcirculation 6, 153-62 (1999).