American Society of Hematology

Figure 3

$Figure 3. CCL2 inhibits firm integrin-mediated lymphocyte adhesion to HEV walls in mouse PLNs. (A) Intravital micrograph and sketch showing a typical subiliac LN at low (5×/0.12 NA, N Plan, water immersion) magnification. LN venous blood drains into an extralymphoid side branch of the superficial epigastric vein via the LOVs (orders I, II, and some order III venules) and the HEVs (orders III, IV and V venules). (B) Rolling and (C-H) sticking fractions of calcein-labeled naive control or CCL2-treated lymphocytes (C-E) or CCR2−/− control or CCL-2 treated lymphocytes (F-H) in the venular tree of subiliac LNs. Lymphocytes were pretreated with CCL2 (1 ng/mL, 30 minutes) or left untreated and injected into the femoral artery. Rolling fraction = 100 × number of rolling cells/number of fast cells. Sticking fraction = 100 × number of arrested cells (more than 10 seconds or 30 seconds or 1 minute) / number of rolling cells. Data shown are means plus or minus SEM of 3 to 5 venules per mouse (n = 3 animals analyzed). (I-L) Rolling (I) and sticking fractions (J-L) of calcein-labeled naive CCL2-treated WT or CCR2−/− lymphocytes in the venular tree of subiliac LNs. Wild-type or CCR2−/− lymphocytes were pretreated with CCL2 (1 ng/mL, 30 minutes) and injected into the femoral artery of mice. Rolling fraction = 100 × number of rolling cells / number of fast cells. Sticking fraction = 100 × number of arrested cells (more than 10 seconds or 30 seconds or 1 minute) / number of rolling cells. Data shown are means plus or minus SEM of 3 to 9 venules per mouse (n = 3 animals analyzed for wild-type lymphocytes and n = 4 animals analyzed for CCR2−/− lymphocytes). Percentages of inhibition are indicated. (M,N) Intravital micrographs (10×/0.3 NA, HCX APO, water immersion) of lymphocyte arrest in the PLN venular tree. The accumulation of CCL2-treated (M) or control (N) lymphocytes in PLN venules was analyzed 30 minutes after intravenous injection of the fluorescently labeled cells. All photomicrograph images (A,M,N) were obtained with a Leica INM 100 microscope (Leica Microsystems SA, Rueil-Malmaison, France) coupled to a silicon-intensified target camera (Hamamatsu Photonics, Massy, France); videos were recorded on DVCAM video tapes (DSR-11 Sony; IEC-ASV, Toulouse, France) and images were obtained with an image processing unit (Argus 20; Hamamatsu Photonics) and numerized with DPS velocity software (Digital Processing System, Hicksville, NY).$

CCL2 inhibits firm integrin-mediated lymphocyte adhesion to HEV walls in mouse PLNs. (A) Intravital micrograph and sketch showing a typical subiliac LN at low (5×/0.12 NA, N Plan, water immersion) magnification. LN venous blood drains into an extralymphoid side branch of the superficial epigastric vein via the LOVs (orders I, II, and some order III venules) and the HEVs (orders III, IV and V venules). (B) Rolling and (C-H) sticking fractions of calcein-labeled naive control or CCL2-treated lymphocytes (C-E) or CCR2^−/− control or CCL-2 treated lymphocytes (F-H) in the venular tree of subiliac LNs. Lymphocytes were pretreated with CCL2 (1 ng/mL, 30 minutes) or left untreated and injected into the femoral artery. Rolling fraction = 100 × number of rolling cells/number of fast cells. Sticking fraction = 100 × number of arrested cells (more than 10 seconds or 30 seconds or 1 minute) / number of rolling cells. Data shown are means plus or minus SEM of 3 to 5 venules per mouse (n = 3 animals analyzed). (I-L) Rolling (I) and sticking fractions (J-L) of calcein-labeled naive CCL2-treated WT or CCR2^−/− lymphocytes in the venular tree of subiliac LNs. Wild-type or CCR2^−/− lymphocytes were pretreated with CCL2 (1 ng/mL, 30 minutes) and injected into the femoral artery of mice. Rolling fraction = 100 × number of rolling cells / number of fast cells. Sticking fraction = 100 × number of arrested cells (more than 10 seconds or 30 seconds or 1 minute) / number of rolling cells. Data shown are means plus or minus SEM of 3 to 9 venules per mouse (n = 3 animals analyzed for wild-type lymphocytes and n = 4 animals analyzed for CCR2^−/− lymphocytes). Percentages of inhibition are indicated. (M,N) Intravital micrographs (10×/0.3 NA, HCX APO, water immersion) of lymphocyte arrest in the PLN venular tree. The accumulation of CCL2-treated (M) or control (N) lymphocytes in PLN venules was analyzed 30 minutes after intravenous injection of the fluorescently labeled cells. All photomicrograph images (A,M,N) were obtained with a Leica INM 100 microscope (Leica Microsystems SA, Rueil-Malmaison, France) coupled to a silicon-intensified target camera (Hamamatsu Photonics, Massy, France); videos were recorded on DVCAM video tapes (DSR-11 Sony; IEC-ASV, Toulouse, France) and images were obtained with an image processing unit (Argus 20; Hamamatsu Photonics) and numerized with DPS velocity software (Digital Processing System, Hicksville, NY).

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