Pamela J. Mansfield

Interleukin 18 induces angiogenesis in vitro and in vivo via Src and Jnk kinases

Background Interleukin 18 (IL-18) is a novel mediator of angiogenesis in rheumatoid arthritis (RA). Objective To examine the role of IL-18 in RA angiogenesis and the signalling mechanisms involved. Methods Human dermal microvascular endothelial cell (HMVEC) chemotaxis, capillary morphogenesis assays and Matrigel plug angiogenesis assays were performed in vivo using IL-18 with or without signalling inhibitors. A novel model of angiogenesis was devised using dye-tagged HMVECs to study their homing into RA and normal (NL) synovial tissues (STs) engrafted in severe combined immunodeficient (SCID) mice. Results IL-18-mediated angiogenesis depended on Src and Jnk, as the inhibitors of Src and Jnk blocked IL-18-induced HMVEC chemotaxis, tube formation and angiogenesis in Matrigel plugs. However, inhibitors of Janus kinase 2, p38, MEK, phosphatidylinositol-3-kinase and neutralising antibodies to vascular endothelial growth factor or stromal derived factor-1α did not alter IL-18-induced HMVEC migration. These results were confirmed with Jnk or Src sense or antisense oligodeoxynucleotides. Moreover, IL-18 induced phosphorylation of Src and Jnk in HMVECs. As proof of principle, IL-18 null mice had a significantly decreased angiogenesis compared with wild-type mice in Matrigel plug angiogenesis assays in vivo. IL-18 markedly enhanced mature HMVEC homing to human RA ST compared with NL ST in SCID mice, confirming the role of IL-18-induced angiogenesis in RA ST in vivo. Conclusion Targeting IL-18 or its signalling intermediates may prove to be a potentially novel therapeutic strategy for angiogenesis-dependent diseases, such as RA.

NEUTROPHIL THROMBOSPONDIN RECEPTORS ARE LINKED TO GTP-BINDING PROTEINS

The extracellular matrix (ECM) protein thrombospondin (TSP) binds to specific receptors on polymorphonuclear leukocytes (PMNs) and stimulates motility. TSP can also enhance the response of PMNs to the formylated peptide, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP). Our initial evidence suggesting that PMN TSP receptors were linked to GTP‐binding proteins (G‐proteins) came from studies using pertussis toxin (PT) and cholera toxin (CT) to inhibit TSP‐mediated motility. Both PT and CT inhibited TSP‐mediated chemotaxis and substrate‐associated random migration. Inhibition was not indirectly caused by a rise in cAMP since neither dibutyryl cAMP (300 μM) nor 8‐bromo‐cAMP (300 μM) significantly affected TSP‐mediated motility. In fact, TSP itself caused a significant rise in intracellular cAMP levels (from 7.2 ± 0.3 to 14.2 ± 0.1 pmol/106 cells). Although we could not test the PT sensitivity of TSP priming for FMLP‐mediated chemotaxis (as PT inhibits FMLP‐mediated chemotaxis itself), we evaluated the effect of CT on this response. CT completely abolished TSP‐dependent priming of FMLP‐mediated chemotaxis. Direct evidence for an interaction between TSP receptors and G‐proteins was obtained by examining the effect of TSP on α‐subunit ADP‐ribosylation, GTPase activity, and GTPγS binding. We observed a decrease in the ability of FMLP to stimulate GTPase activity on membranes isolated from PMNs incubated with TSP. Furthermore, the PT‐dependent ribosylation of Giα2,3 stimulated by FMLP was eliminated by TSP treatment. These data indicated that the two receptors share a pool of G‐proteins. However, TSP did not block the CT‐dependent ribosylation stimulated by FMLP, suggesting that TSP receptors may also interact with a different pool of Giα2,3. TSP itself significantly (P < 0.005) increased GTP hydrolysis in PMN membranes (to 110.6 ± 2.7% of control values). In addition, GTPγS binding to membranes increased significantly (P < 0.005) following exposure to 10 nM TSP (to 108 ± 1.4% of control values). Conversely, GTP treatment reduced the affinity of TSP for its receptor without altering total binding. These data demonstrate that TSP receptors are linked to G‐proteins, a subpopulation of which also associates with FMLP receptors.

H-2g, a glucose analog of blood group H antigen, mediates mononuclear cell recruitment via Src and phosphatidylinositol 3-kinase pathways

OBJECTIVE Monocyte recruitment by proinflammatory cytokines is a hallmark of rheumatoid arthritis (RA). Lewis(y-6) and H (Le(y)/H) are blood group antigens up-regulated on RA synovial endothelium. We have previously shown that both soluble Le(y)/H and a glucose analog of H, H-2g, are angiogenic and mediateleukocyte-endothelial adhesion via induction of intercellular adhesion molecule 1. We hypothesized that soluble Le(y)/H plays an important role in monocyte recruitment in RA. METHODS We examined the role of H-2g in monocyte chemotaxis in vitro. We used an RA synovial tissue (ST)-SCID mouse chimera model to evaluate the role of H-2g in monocyte recruitment in vivo. We used Western blots to examine signaling molecules activated by H-2g in monocytes. RESULTS H-2g induced human monocyte migration in vitro, which was mediated by Src and phosphatidylinositol 3-kinase (PI 3-kinase), since inhibitors and antisense oligodeoxynucleotides (ODNs) of Src and PI 3-kinase significantly decreased H-2g-induced monocyte migration (P < 0.05). H-2g significantly increased mononuclear cell (MNC) homing in vivo into an RA ST-SCID mouse chimera (P < 0.05). Transfection of MNCs with Src antisense ODNs blocked H-2g-induced MNC recruitment into the RA ST-SCID mouse chimera. Additionally, H-2g induced marked phosphorylation of protein kinase CalphaI/betaII (PKCalphaI/betaII), Src, IkappaBalpha, and Akt in monocytes. Src, Akt, and NF-kappaB were shown to be downstream targets of PKCalphaI/betaII, since an inhibitor of PKCalphaI/betaII reduced H-2g-mediated phosphorylation of Src, Akt, and NF-kappaB in monocytes. CONCLUSION These data suggest that H-2g may be a novel mediator of monocyte recruitment in chronic inflammatory diseases like RA.

Reproduction by Lake Michigan Fishes in a Tributary Stream

Abstract Three common Lake Michigan species (spottail shiner Notropis hudsonius, alewife Alosa pseudoharengus, and white sucker Catostomus commersoni) migrated into Little Pigeon Creek, a Lake Michigan tributary with a marsh upstream, to spawn during 1980. Spottail shiners, not previously documented as spawning in Lake Michigan tributaries, spawned when stream water temperatures reached approximately 18 C (7 C warmer than the 1-m depth contour in Lake Michigan), about 1 month earlier in this tributary than in Lake Michigan. Densities of spottail shiner larvae were higher in the stream than in the Lake Michigan beach zone, most likely due to high productivity of the stream and concentration of spawning adults in one area. Little Pigeon Creek produced an estimated 100,000 spottail shiner larvae per day at times of peak hatch, over a 12-day period. White sucker larvae were pelagic in surface waters of the marsh for 1–2 months, then drifted downstream into bottom waters of the stream. Alewife larvae occurred ...

Response of bacteria and phytoplankton to contaminated sediments from Trenton Channel, Detroit River

Several types of bioassays were used in 1986 and 1987 to investigate the effect of contaminated sediments on natural populations of bacteria and phytoplankton from the Trenton Channel, Detroit River. The approach included the measurement of uptake of 3H-glucose or 3H-adenine by bacteria and 14C-bicarbonate by phytoplankton in the presence of different amounts of Trenton Channel and Lake Michigan (control) sediments. Trenton Channel sediments are contaminated by high levels of toxic organic compounds and metals, especially zinc, lead, and copper. Because levels of biomass of bacteria and phytoplankton varied widely among the different bioassays, it was necessary to adjust uptake rates for biomass. Biomass adjustments were made using acridine orange counts for bacteria and chlorophyll measurements for phytoplankton. The results show a statistically significant suppression of uptake of substrates for both bacteria and phytoplankton with increasing amounts of sediment. Uptake was suppressed as much as 90 percent for bacteria and 93 percent for phytoplankton at 1200 mg l-1 of Trenton Channel sediments compared to bioassays without sediment. Uncontaminated Lake Michigan sediment suppressed uptake much less than Detroit River sediment; the difference in suppression of uptake between the two types of sediment was statistically significant for both bacteria and phytoplankton.

THROMBOSPONDIN (TSP)-DEPENDENT MIGRATION OF GLIOBLASTOMA CELLS: RELATIONSHIP TO INVASIVE POTENTIAL AND INVOLVEMENT OF TSP STRUCTURAL DOMAINS.▴ 913

THROMBOSPONDIN (TSP)-DEPENDENT MIGRATION OF GLIOBLASTOMA CELLS: RELATIONSHIP TO INVASIVE POTENTIAL AND INVOLVEMENT OF TSP STRUCTURAL DOMAINS. ▴ 913