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Phagocytosis mediated by Yersinia invasin induces collagenase-1 expression in rabbit synovial fibroblasts through a proinflammatory cascade

Erica Werner^1,*, Farrah Kheradmand^1,, Ralph R. Isberg² and Zena Werb^1,

¹ Department of Anatomy, University of California, San Francisco, CA 94143-0452, USA
² Department of Microbiology and Molecular Biology, Tufts University School of Medicine, Boston, MA 02111, USA
^* Present address: Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA
Present address: Department of Medicine, Baylor College of Medicine, Houston, TX 77025, USA

View larger version (14K): [in a new window]   Fig. 1. Context-dependent induction of CL-1 expression by invasin. RSFs were plated on FN- or invasin-coated dishes. Beads coated with INV497 or fibronectin were added to cells spreading on FN or INV497. CL-1 expression was measured in the supernatant by slot blot 24 hours later. The error bars represent ± s.e.m. of two or more independent experiments. INV497-coated beads produced a large increase in CL-1 secretion, whereas FN-coated beads did not in cells plated on FN. The difference in CL-1 produced in response to FN-coated beads in cells plated on FN, compared to cells plated on INV497 was small, but significant (P=0.046).

View larger version (30K): [in a new window]   Fig. 2. Effect of affinity of invasin binding on bead phagocytosis and CL-1 induction. RSF were incubated for 2 or 24 hours with beads coated with MBP (nonspecific binding control), INV497D911E (low-affinity binding) or INV497 (high-affinity binding). Bead phagocytosis was analyzed after 3 hours and CL-1 expression was measured in the supernatant by slot blot after 24 hours. (A) Quantitative representation of bead binding and uptake after 3 hours and CL-1 expression after 24 hours for each type of coating. (B) Micrographs of the phagocytosis assay. The green beads are outside and the red beads are inside the cells. Error bars represent ± s.e.m. of 3 independent experiments. Bar, 50 µm.

View larger version (18K): [in a new window]   Fig. 3. Invasin induction of CL-1 expression is integrin dependent. (A) INV497 coated beads binding was inhibited by the addition of soluble GRGDSP peptide, but not by GRGESP peptide. As a control, GRGDSP peptides were used to compete for the binding of anti-5 integrin antibody. (B) Beads coated with other integrin ligands of high-affinity binding induce CL-1 expression. Beads coated with integrin antibodies: anti-5 integrin mAb (BIIG2), anti-4 integrin mAb (P4G6), function-blocking anti-ß1 integrin mAb (AIIB2) were compared with FN or 120FN in their potency to induce CL-1 expression. CL-1 expression was measured in the supernatant by slot blot after 24 hours of bead addition. Error bars represent ± s.e.m. of three independent experiments. (C) High-affinity integrin ligands recruit AP-2 to the bead-associated membrane fraction. Membranes associated with beads coated with BSA, INV497D911A, INV497D911E, INV497, FN and 120FN were analyzed for the presence of AP-2 after 1 and 6 hours of binding.

View larger version (13K): [in a new window]   Fig. 4. RhoA is required for phagocytosis and bead-induced CL-1 expression. (A) Anti-5 mAb-coated beads were added to cells transfected with dominant-negative mutants of Rac 1, RhoA or Cdc42 and a reporter construct of CL-1 promoter driving luciferase expression. Luciferase activity was measured 24 hours after bead addition. (B) CL-1 promoter driven luciferase activity was measured 24 hours after addition of invasin-coated beads to cells transfected with RhoAN19 or RhoAV14. Error bars represent ± s.d. of triplicates in one of two experiments. (C) Micrographs of the phagocytosis assay after 2 hours of bead addition to dominant-negative RhoA-transfected cells.

View larger version (64K): [in a new window]   Fig. 5. Bead phagocytosis induces NFB activation. (A) NFB (green) nuclear translocation was visualized by immunofluorescence 1 or 2 hours after the addition of anti-5 mAb-coated beads (red autofluorescence). Arrows mark cells positive for NFB translocation and with beads inside. Bar, 50 µm. (B) Activated NFB was detected by EMSA of nuclear extracts prepared from RSF incubated for 2 hours without beads (lane 5) or beads with anti-5 mAb-(lane 6) or beads coated with INV497 (lane 7). Lane 1, no extract; lane 2, HeLa extract; lane 3, HeLa extract plus 1.75 pmol of unlabelled specific probe; lane 4, HeLa extract plus 1.75 pmol nonspecific competitor.

View larger version (18K): [in a new window]   Fig. 6. Bead phagocytosis induces TNF- and IL-1 expression, however only IL-1 production is required for bead-induced CL-1 expression. (A) Time course of TNF-, IL-1 and CL-1 production after addition of anti-5 mAb-coated beads. The cytokines and CL-1 were measured in the culture supernatant by slot blot. Error bars represent ± s.d. from one of three independent experiments. (B) CL-1 expression was measured in the supernatant by slot blot 24 hours after the addition of beads coated with anti-5, IL-1 (2 ng/ml) or TNF- (1 µg/ml) in the absence or presence of IL-1 receptor antagonist (400 ng/ml), anti TNF- (8 µg/ml) or anti IL-1 (10 µg/ml). Error bars represent ± s.d. from one of three experiments. *, the effect of anti IL-1 on TNF-induced CL-1 expression was not assayed.

View larger version (122K): [in a new window]   Fig. 7. IL-1 activity in cis and in trans is required for bead induction of CL-1 expression. Micrographs of immunofluorescence staining for CL-1 expression (green) after 24 hours of anti-5 mAb-coated beads (red) addition in the absence (A, B and D) or presence of 10 µg/ml anti IL-1 (C). Arrows point to cells expressing CL-1, but with no beads. Bar, 50 µm.