Galectin-3 And Leukocyte Recruitment In The Inflamed Microcirculation

Beatrice Gittens, Dianne Cooper, Mauro Perretti. Centre for Biochemical Pharmacology, William Harvey Research Institute, London, EC1M 6BQ, UK.

Galectins are a highly conserved β-galactoside-binding family of animal lectins differentially expressed in many immune cells. Galectin-3 (Gal-3) has been implicated in a diverse group of human pathologies such as rheumatoid arthritis, S. Pneumoniae infection and stroke. A role for Gal-3 as a positive regulator of leukocyte recruitment has also been identified; Gal-3 was shown to significantly increase the number of neutrophils, monocytes and eosinophils recruited to murine air-pouches in vivo1. Furthermore, in a murine model of S. pneumoniae-infection, Sato et al. found that Gal-3 expression was increased in alveolar macrophages of infected animals. This correlated with an increased accumulation of Gal-3 in the alveolar space and the onset of neutrophil recruitment to the area2.

The current study has two objectives: to study the leukocyte-endothelium interactive events in post-capillary venules of Gal-3 knockout mice and secondly, to determine the pharmacological effects of Gal-3 itself. Intravital microscopy (IVM) of the cremaster muscle was used to visualize leukocyte recruitment in real time. Male mice from wild-type C57BL/6 or Gal-3 null strains weighing 24-28g were anesthetised using a mixture of xylazine (7.5 mg/kg) and ketamine (150 mg/kg). IVM was performed 2 or 4 hours after intrascrotal injection of PBS, IL-1β (30ng), TNF-α (300ng) and/or Gal-3 (500ng) in a final volume of 400μl. The tissue was perfused continuously with bicarbonate buffered saline at 37°C and segments of 100μm in 3-5 vessels per mouse and of 3-5 mice per group were analysed after an initial 30min stabilisation period. Rolling flux was recorded as the number of leukocytes rolling past a specific point, averaged over 5 min; rolling velocity was determined from the time taken for the leukocytes to travel 100μm; adherent cells were stationary on the vessel wall for 30 sec and transmigrated cells were those in the tissue 50μm by 100μm on either side of the vessel. Statistical significance (P<0.05) was assessed using unpaired t-tests and one-way ANOVA with Tukey’s post-test for multiple comparisons; results are expressed as mean ± SEM.

Treatment with IL-1β in Gal-3 knockout mice resulted in a significant increase in rolling velocity (27.07±2.67, 15.64±1.48) and a significant decrease in the number of emigrated cells (4.33±0.87, 9.82±1.67) when compared to their wild-type counterparts. In comparison leukocyte recruitment in Gal-3 knockout mice treated with TNF-α was comparable to levels observed in wild-type mice with the exception of rolling velocity (49.13±15.16, 3.96±0.33), which was significantly increased (KO, WT). Intra-scrotal administration of recombinant Gal-3 resulted in a significant reduction in leukocyte flux (29.05±5.81, 50.97±5.16) and rolling velocity (8.31±0.97, 25.71±1.91) and a significant increase in the number of adherent (6.57±0.50, 3.87±0.26) and emigrated (7.81±0.87, 4.29±0.54) leukocytes when compared to sham-treated animals (rGal-3, sham).

These data suggest that Gal-3 acts as a positive regulator of leukocyte recruitment in the inflamed microcirculation and that it may have differing roles in response to different inflammatory stimuli, such as IL-1β and TNF-α. In conclusion, this study joins many others in demonstrating the potential to direct further research and future therapies for inflammatory disorders towards the lectin Gal-3.

1) Sano H., Hsu D. K., Yu L., Apgar J. R., Kuwabara I., Yamanaka T., Hirashima M. and Liu F. T., 2000. Human galectin-3 is a novel chemoattractant for monocytes and macrophages. J Immunol 165, 2156-64.

2) Sato S., Ouellet N., Pelletier I., Simard M., Rancourt A. and Bergeron M. G., 2002. Role of galectin-3 as an adhesion molecule for neutrophil extravasation during streptococcal pneumonia. J Immunol 168, 1813-22.