Figure 4
Peripheral blood monocytes injection. (A) PKH26 red fluorescent dye-tagged human peripheral blood (PB) monocytes (5 × 106) were injected i.v. into SCID mice engrafted for 4 to 6 weeks with human rheumatoid arthritis synovial tissue (RA ST). Before administering cells, ST grafts were injected with rhuIL-18 (1,000 ng/graft) or sham injected (PBS stimulus). At 48 hours, grafts and inguinal lymph nodes (LNs) were harvested, and tissue sections were examined with immunofluorescence microscopy at 550 nm (100 ×). The top panel shows PKH26 dye-tagged monocytes migrating into PBS or rhuIL-18 injected RA ST. (B) The lower portion of the same panel shows an image of the local LNs containing recruited monocytes from the same mice. The number of dye-tagged cells migrating to engrafted RA ST or LN tissue in response to rhuIL-18 is graphed in the next panel. As shown, SCID mice receiving intragraft injections of rhuIL-18 showed significant recruitment of human monocytes to both engrafted RA ST and murine LNs. Monocyte migration was quantified by dividing the number of cells per hpf/tissue section at 100 × (n = number of tissue sections counted ± SEM). (C) LNs from rhuIL-18 simulated SCID chimeric mice were harvested and evaluated for human monocyte recruitment. LNs were stained for CD11b/Mac-1 with fluorescence histology. The primary antibody was a mouse anti-human mAb, followed by blocking with goat serum and the addition of a goat anti-mouse FITC-tagged secondary antibody. (a) Human monocytes expressing CD11b/Mac-1 migrate to murine LNs (fluorescent green cells, see arrow). (b) Fluorescent dye-tagged human cells in murine LNs. (c) Merger of (a) and (b), showing that the migrating cells are expressing human CD11b/Mac-1 (fluorescent yellow staining; see arrow). (d) DAPI staining showing cell nuclei (fluorescent blue cells, see arrow). (e) Negative-control staining for CD11b/Mac-1 (nonspecific IgG was used as the primary mAb). (f) Murine LN showing recruited cells (red fluorescent staining, see arrow). (g) Merger of (e) and (f) showing a lack of nonspecific cellular staining. (h) DAPI staining showing cell nuclei (original magnification, 400 ×).