Tyrosine kinase activation in LPS stimulated rat Kupffer cells.

Kupffer cells, a majority of the body's fixed macrophages, are a major site of bacterial lipopolysaccharide (LPS) metabolism and are mediators in the body's response to sepsis. Uptake of LPS is different in Kupffer cells than other macrophages. Signal transduction in other macrophages in response to LPS involves phosphorylation of proteins in the 50-60 kDa range. We hypothesized that Kupffer cells may have unique signal transduction pathways in response to LPS. Rat Kupffer cells were exposed to LPS (1 microgram/mL) for varying times ranging from 15 to 90 min. Cell lysates were Western blotted using an anti-phosphotyrosine antibody. The blots showed an increase in the amount of tyrosine phosphorylation on two proteins of 119 kDa and 83 kDa. The effects of varying LPS concentration (1 ng/mL-1 microgram/mL) showed an increasing amount of phosphorylation with increasing LPS concentration. To associate the importance of tyrosine phosphorylation in the response of Kupffer cells to LPS, the tyrosine kinase inhibitors, tyrphostin, lavendustin, and genisten were used to study the effects of inhibiting phosphorylation on TNF-alpha production. Kupffer cells were preincubated in the presence of the inhibitor and exposed to LPS (1 microgram/mL). TNF-alpha was measured in the conditioned media by ELISA. A 70% or greater decrease in TNF-alpha production was observed. When phagocytosis of latex beads by rat Kupffer cells was measured in vivo using intravital video microscopy, LPS treatment significantly increased uptake. This increase in phagocytosis was inhibited by tyrphostin. These results show what may be unique phosphorylation events in Kupffer cells that are related to LPS induced production of TNF-alpha.

Accuracy of corneal elevation with four corneal topography systems.

PURPOSE: To compare the elevation accuracy of placido-based videokeratoscopes with a rasterphotogrammetry-based topography system using models of an aspheric surface and a simulated myopic ablation. METHODS: Test surfaces representative of normal corneas and corneas after excimer ablation were fabricated from a material measurable using both reflective (placido) and projective (rasterstereographic) systems. The "normal" surface was a rotationally symmetric asphere, while the simulated photorefractive keratectomy ablation had a central 5.5 mm diameter clear zone with a 1 mm blend radius to eliminate surface discontinuity. The surfaces were measured with their axes of symmetry aligned with the instrument axis, and again with their axes tilted downward by 10 degrees to produce a nonsymmetric shape. RESULTS: All of the topography systems performed reasonably well in measuring the aspheric surface, with root mean square elevation error ranging from 1.2 to 14.3 microm. The rasterstereographic system had a root mean square error of 4.7 microm on the simulated ablation, while the placido systems had root mean square errors of 10.6 to 29.5 microm. Tilting the surfaces did not significantly alter the results. CONCLUSIONS: The tested placido videokeratoscopes do not provide reliable elevation data for shapes that have nonlinear changes in curvature. This may have serious implications if these systems are used for postoperative analysis or contact lens fitting on corneas that have been modified by refractive procedures.

High molecular weight kininogen binds to Mac-1 on neutrophils by its heavy chain (domain 3) and its light chain (domain 5).

High molecular weight kininogen (HK) binds specifically, saturably, and reversibly to neutrophils and also reciprocally inhibits the binding of fibrinogen to neutrophils. Since fibrinogen binds to the leukocyte integrin CD11b/18 (Mac-1, alpha M beta 2), we investigated whether HK bound to Mac-1 and whether the binding site was similar to that for factor X. We also examined whether one or both chains of cleaved HK (HKa) were involved. Two monoclonal antibodies, 2B5 (0.29 microM) to HK heavy chain domains 2 (D2) and 3 (D3), and C11C1 (0.26 microM) to HK light chain domain 5 (D5), inhibited by 99 and 93% the binding, respectively, of 125I-HK (8.3 nM) to neutrophils. To minimize steric hindrance, we further demonstrated that the Fab' fragments of 2B5 and C11C1 were able to inhibit the binding of this ligand to virtually the same extent as the intact antibody, indicating that, as in binding of HK to platelets and endothelial cells, both chains are involved. To directly demonstrate the involvement of each chain, we showed that the reduced alkylated light chain derived from HK and low molecular weight kininogen, which contains the same heavy chain as HK, each markedly inhibited the binding of HK to neutrophils. We localized the domain responsible for the binding in each chain by showing that recombinant D3 and D5 decreased the binding of HK to neutrophils. To define the receptor for HK, we employed three monoclonal antibodies to Mac-1: OKM1 and OKM10 to epitopes on the alpha M subunit and IB4 to an epitope on the beta 2 chain. OKM1, which can inhibit fibrinogen binding to neutrophils, inhibited HK binding by 79%, whereas the other antibodies inhibited HK binding less than 25%. Coagulation factor X also binds to Mac-1 on monocytes at a similar site to C3bi. Synthetic peptides which define noncontiguous surface loops in factor X that interact with Mac-1, failed to inhibit 125I-HK binding to neutrophils. We conclude that HK binds, via domains on its heavy chain, D3, and light chain, D5, to Mac-1 on the neutrophil surface, and HK occupies a site overlapping with fibrinogen and different from factor X.

Platelet thrombospondin interactions with human high and low molecular weight kininogens.

Multifunctional proteins, e.g. high molecular weight kininogen (HK, 120 kDa) and the homotrimer, thrombospondin (TSP, 540 kDa), which have more than one domain on a single polypeptide chain, are particularly well-suited to be structural elements of extracellular matrices because of their ability to bind to several macromolecules. We now demonstrate that 125I-high molecular weight kininogen (HKa) cleaved by purified kallikrein forms a complex with purified intact platelet TSP (540 kDa). HK also complexed with a proteolytic fragment (450 kDa) of TSP, lacking its three identical heparin-binding domains (HBD, 30 kDa), but failed to bind to a more extensively proteolysed molecule (210 kDa) lacking the C-terminal globular domain indicating that the binding on TSP-450 kDa is confined to the C-terminus. The binding of HK to intact TSP and to its 450 kDa fragment was of high affinity (Kd = 17-52 nM), specific, concentration dependent and saturable. Furthermore, we found both forms of the light chain (LC) of HK (56 and 46 kDa) resulting from cleavage by plasma kallikrein bound to both intact TSP and HBD independent of the presence of calcium ions. However, neither the epitope recognized by monoclonal antibody (MAb) C11C1 on domain 5 nor the prekallikrein binding site on domain 6 are involved, suggesting that the intervening proline-rich region may be the site of interaction. The heavy chain (HC) of HK required ionized calcium to bind to intact TSP or its 450 kDa homotrimer fragment.(ABSTRACT TRUNCATED AT 250 WORDS)