Unique and redundant roles of alpha4 and beta2 integrins in kinetics of recruitment of lymphoid vs myeloid cell subsets to the inflamed peritoneum revealed by studies of genetically deficient mice

Abstract

Objective: Leukocyte recruitment to inflammatory sites is a prominent feature of acute and chronic inflammation. Instrumental in this process is the coordinated upregulation of leukocyte integrins (among which alpha4beta1 and beta2 integrins are major players) and their cognate receptors in inflamed tissues. To avoid the ambiguity of previous short-term antibody-based studies and to allow for long-term observation, we used genetically deficient mice to compare roles of alpha4 and beta2 integrins in leukocyte trafficking.

Materials and methods: Aseptic peritonitis was induced in alpha4 or beta2 integrin-deficient (conditional and conventional knockouts, respectively) and control mice, and recruitment of major leukocyte subsets to the inflamed peritoneum was followed for up to 4 days.

Results: Despite normal chemokine levels in the peritoneum and adequate numbers, optimal recruitment of myeloid cells was impaired in both alpha4- and beta2-deficient mice. Furthermore, clearance of recruited neutrophils and macrophages was delayed in these mice. Lymphocyte migration to the peritoneum in the absence of alpha4 integrins was drastically decreased, both at steady state and during inflammation, a finding consistent with impaired lymphocyte in vitro adhesion and signaling. By contrast, in the absence of beta2 integrins, defects in lymphocyte recruitment were only evident when peritonitis was established.

Conclusions: Our data with concurrent use of genetic models of integrin deficiency reveal nonredundant functions of alpha4 integrins in lymphocyte migration to the peritoneum and further refine specific roles of alpha4 and beta2 integrins concerning trafficking and clearance of other leukocyte subsets at homeostasis and during inflammation.

Figure 1. Both α4 and β2 integrins are required for efficient neutrophil migration to the peritoneum

Mice were injected with TG, peritoneal leukocytes were harvested 4 and 16 hours post injection and stained with various leukocyte markers (see Materials and Methods). Neutrophil (Gr-1+,F4/80-) numbers (A) and recruitment indices (B) were calculated. Note that although sufficient numbers of neutrophils are present in the peritoneal cavity of α4Δ/Δ and β2-/- mice, there is a significant decrease in recruitment index at each time point. Asterisk (*) indicates significant difference compared to controls, P<0.05. Mice used per group: WT, n=5; β2-/-, n=4; α4Δ/Δ, n=4.

Figure 2. Recruitment of inflammatory monocytes to the peritoneum is impaired in α4Δ/Δ and β2-/- mice

Aseptic peritonitis was induced in mice by TG injection and 4 and 16 hours later, numbers of inflammatory monocytes were counted. (A) Typical FACS profile of peritoneal exudate of a WT mouse 4 hours (left panel) and 16 hours (right panel) after TG injection. Neutrophils are Gr-1+F4/80- (R1), inflammatory monocytes are Gr-1+F4/80+ (R2), and non-inflammatory monocytes/macrophages are Gr-1-F4/80+ (R3); Numbers of inflammatory monocytes in WT (n=5), β2-/- (n=4), and α4Δ/Δ (n=4) mice were determined in the peritoneal cavity (B) and in peripheral blood (C). Recruitment indices (D) were calculated to assess the efficiency of migration from the circulation. Asterisk (*) indicates significant difference over control (P<0.05).

Figure 3. Macrophages use α4 and β2 integrins interchangeably to maintain sufficient numbers in peritoneum during inflammation

(A) Peritonitis was induced in WT (n=5), β2-/- (n=4), and α4Δ/Δ (n=4) mice and 4, 16, and 96 hours later numbers of macrophages (F4/80+) and (B) numbers of α4-positive and -negative macrophages in a4Δ/Δ peritoneal lavage were determined by FACS. Asterisk (*) indicates significant difference over control (P<0.05); Mφ denotes macrophages.

Figure 4. Lymphocyte migration to the inflamed peritoneum is absent in α4Δ/Δ and decreased in β2-/- mice

(A) Aseptic peritonitis was induced in WT (n=5), β2-/- (n=5) and α4Δ/Δ (n=5) mice. Lymphocyte numbers were determined by FACS, before and 48 hours after TG injection. (B) Efficiency of migration, as indicated by recruitment index, was calculated at 48 hours after TG injection. Asterisk (*) indicates significant difference from control mice at the corresponding hour of peritonitis (P<0.05).

Figure 5. Alpha4 and beta2 integrins are necessary and sufficient for leukocyte migration to the inflamed peritoneum

Total leukocyte numbers in peritoneum of WT (n=5), β2-/- (n=5), α4Δ/Δ (n=5), and α4Δ/Δ β2-/- double deficient (n=4) mice at various times after induction of aseptic peritonitis. Note that, most of the time leukocyte numbers in mice deficient in a single integrin are similar to that of controls, whereas no recruitment of double deficient leukocytes occurs. Asterisk (*) indicates significant difference from control (P<0.05)

Figure 6. Alpha-4 integrins are essential for lymphocyte adhesion and migration in vitro

(A) Adhesion of control (n=5) and α4Δ/Δ (n=3) splenocytes to endothelial cells. Splenocytes were labeled with CFSE and allowed to adhere to either non-stimulated or TNFα-stimulated bEND3 monolayer. Fold increase of adhesion to stimulated over non-stimulated endothelium is displayed. (B) Trans-well and (C) trans-endothelial migration of B-lymphocytes towards SDF-1α. Note that migration of α4Δ/Δ B cells (and T cells, not shown) through VCAM-1-expressing endothelium (TNFα) did not differ from that of non-stimulated endothelium, in contrast to control and β2-/- cells. Asterisk (*) indicates significant difference over controls; ‡ indicates significant difference over non-stimulated endothelium (P<0.05). Three mice per group were used.

Figure 7. Lymphocyte early responses to chemoattractant SDF-1α

(A) Actin polymerization in WT, α4Δ/Δ and β2-/- lymphocytes. Splenocytes were stained for lymphocyte markers (CD3 and B220), stimulated with SDF-1α for indicated amount of time and phalloidin staining was performed. Fold increase over non-stimulated cells was calculated. (B) SDF-1α-induced Ca²⁺ mobilization. Splenocytes were loaded with Indo-1 and stained for lymphocyte markers. Ca²⁺ flux was assessed following stimulation with SDF-1α or ionomycin. Results are displayed as percent of the peak stimulation levels over baseline. Asterisk (*) indicates significant difference over control (P<0.05). Three mice per group were used.