Migration Defects of Leukocytes from Alpha4 or Beta2 Integrin-Deficient Mice during Aseptic Peritonitis.

Abstract

Leukocyte migration to the inflammatory sites involves a dynamic and coordinated sequence of adhesion/ migration events, in which the α4 and β2 integrins in leukocytes play critical roles. In a model of aseptic peritonitis, normal mice respond with an initial influx of neutrophils followed by later recruitment of mononuclear cells. Previous studies used antibodies to inhibit α4 integrins in this model and provided only early observations (up to 24 hrs). Events at later stages were not addressed. To clarify the role of α4 integrins in this process, we induced aseptic peritonitis in α4-ablated (α4−/ −) mice and compared the results to similarly treated wild type (WT), and β2 integrin-deficient (β2−/ −) animals. A 4, 16 and 96 hrs post thioglycollate injection, leukocytes, harvested from the peritoneal cavity, were counted and evaluated by FACS and by differentials in smears. 3-5 animals per time point per genotype were analyzed. Early neutrophil accumulation in the peritoneum was similar in WT and α4−/ − mice (Table 1). β2−/ − mice were slow to accumulate neutrophils, but at 16 hrs the peaks were not different from α4−/ − or WT animals. However at later times in both α4−/ − and β2−/ − mice neutrophils persisted longer, suggesting either increased survival or prolonged recruitment. At 16 hrs large numbers of monocyte/ macrophages accumulated in the peritoneum and they were similar in all groups of mice up to 96 hours. The cumulative number of leukocytes in the peritoneum was compared to the concurrent circulating pool of leukocytes in each mouse group. At the peak time (16 hrs) the fraction recovered in the WT represented 394±148%, while in the α4−/ − mice it was 98±33% and in the β2−/ − mice it was 40±19%. Thus, although sufficient numbers of leukocytes accumulated in the peritoneum of both integrin-deficient mice, migration in the WT mice was significantly higher than that observed in the other two genetic models, suggesting impairment in animals with either integrin deficiency. Furthermore, lymphocyte accumulation was dramatically decreased at all time points in the α4−/ − mice (Table 2). Preliminary data in a model of combined, α4- and β2-integrin deficiency, generated in our laboratory, showed that only 3±2% of circulating leukocytes migrated into the peritoneum. Taken together, our results indicate that (1) for migration into peritoneum, neutrophils and monocyte/ macrophages can use α4 and β2 integrins interchangeably, (2) for optimal migration both integrins are needed, and (3) lymphocytes have an absolute requirement of α4 integrins for migration. These data extend previous knowledge on kinetic changes of leukocytes accumulating in the aseptic peritonitis model. Whether different kinetic changes are seen in other inflammatory models using our integrin-deficient animals requires further studies.Accumulation of myeloid cells in peritoneumNeutrophilsMonocyte/macrophages4 hrs16 hrs96 hrs4 hrs16 hrs96 hrsWT5.5±0.59.8±7.40.2±0.043.1±0.617.7±3.911.0±5.2β2−/ −2.5±0.810.5±5.33.7±1.64.7±1.312.8±5.211.0±4.0α4−/ −5.2±2.19.8±7.40.8±0.44.6±1.111.0±5.212.6±5.1Lymphocyte accumulation in peritoneum4 hrs16 hrs96 hrsWT2.7±1.22.5±0.33.1±0.5β2−/ −6.7±1.74.7±1.23.3±1.0α4−/ −0.5±0.10.4±0.10.6±0.1Cell number (x106) in peritoneum at various time points; in bold are the numbers that significantly differ from the WT for each time point (p<0.05)