We present a simple method to identify the recruitment of leukocyte subsets and determine concurrent surface-receptor clustering in live mice. We show that CD45+ F4/80− Gr-1+ neutrophils are robustly recruited in surgery-activated cremasteric venules, whereas adherent CD45+ B220+ B lymphocytes were dominant in bone marrow venules. Most adherent Gr-1+ leukocytes are not firmly stationary but actively migrate on TNF-α–activated cremasteric venular endothelium and exhibit marked polarization of surface PSGL-1, but not LFA-1, to the trailing edge.
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This work was supported by the US National Institutes of Health R01 HL69438 (P.S.F.), T32 HL07824 (J.C.), a Glorney-Raisbeck fellowship from the New York Academy of Medicine (E.Y.C.) and a fellowship from the Charles Revson Foundation (A.H.). P.S.F. is an Established Investigator of the American Heart Association.
Low doses of fluorescence-conjugated monoclonal antibodies to CD45 (red), Gr-1 (green), and F4/80 (blue) were injected into a wild-type animal to identify adherent leukocyte subsets. Several images were captured from cremasteric venules in the brightfield and three fluorescence channels (Cy3, FITC and Cy5) over 1 min to distinguish firmly adherent from rolling leukocytes; this representative video in 10× time-lapse shows 1 lymphocyte (red), 2 PMNs (green-yellow) and 1 monocyte (blue). Actual time is displayed in the left upper corner.
Fluorescence-conjugated monoclonal antibodies against PSGL-1 (red), LFA-1 (blue), and Gr-1 (green) were injected into wild-type mice to determine receptor distribution on adherent leukocytes in cremasteric venules. Images were captured in brightfield and three fluorescence channels (Cy3, FITC and Cy5) over 3 min; a representative movie shown is in 10x time-lapse.
Migrating adherent leukocytes in cremasteric venules of untreated (first segment) and antibody-treated (second segment) mice. TNF-α-stimulated wild-type mice were left unjected or injected with the maximal dose of antibody used in this study (0.22 mg / kg or ∼5.5 μg per mouse: 0.12 mg / kg of APC-anti-Gr-1, 0.08 mg / kg of FITC-anti-LFA-1 and 0.02 mg / kg of anti-PSGL-1) to evaluate whether the antibody injection alters leukocyte behavior. A representative series of images, recorded in 10× time-lapse over 3 min, revealed similar motility of adherent leukocytes on inflamed endothelium between both groups, indicating that leukocyte migration on inflamed endothelium is not triggered by antibody injection.
PSGL-1 clusters at the trailing edge of migrating leukocytes. Fluorescence-conjugated monoclonal antibodies against PSGL-1 (red), LFA-1 (not shown), and Gr-1 (green) were injected into wild-type mice to determine receptor distribution on individual adherent leukocytes in inflamed cremasteric venules. Images were captured in the brightfield and three fluorescence channels (Cy 3, FITC, and Cy5) over 3 min to assess leukocyte behavior over time; a representative movie shows a migrating Gr-1+ leukocyte (arrow) with clustered PSGL-1 always at the trailing edge during a 180° change in direction. Actual recording time of a 10× time-lapse movie is displayed on left upper corner.