Raimund W. Kinne

Apoptotic cell death in activated monocytes following incorporation of clodronate-liposomes.

The present study was performed to elucidate whether sterically stabilized liposomes laden with clodronate, which lead to depletion of macrophages (Mɸs) and amelioration of experimental autoimmune arthritis in vivo, selectively affect cells of the mɸ lineage in vitro. The rates of incorporation of drug‐free, fluorescent liposomes and the rates of cell death following exposure to clodronate‐liposomes were assessed in human peripheral blood monocytes, as well as in polymorphonuclear leukocytes (PMNs), T cells, endothelial cells, and fibroblasts, both at rest and following activation. Gel electrophoresis of nuclear extracts and ultrastructural analyses were performed to identify the modality of cell death. Monocytes, particularly upon activation, were more efficient in incorporating sterically stabilized liposomes than all other cells except PMNs. Twenty percent of resting monocytes and up to 65% of activated monocytes died within 24 h of exposure to clodronate‐liposomes, whereas the other cell types, including PMNs, remained unaffected. Activated monocytes exposed to clodronate‐liposomes, but not resting or activated monocytes exposed to drug‐free liposomes, showed clear signs of apoptotic cell death. In most of the assays, sterically stabilized liposomes were more efficient than conventional phosphatidylcholine‐liposomes. Sterically stabilized clodronate‐liposomes preferentially affect cells of the mɸ lineage, particularly if activated. Selective elimination of activated Mɸs by apoptosis may explain both therapeutic efficacy and safety of clodronate‐liposomes in experimental models of autoimmunity. J. Leukoc. Biol. 60: 230–244; 1996.

Imaging rheumatoid arthritis joints with technetium-99m labelled specific anti-CD4- and non-specific monoclonal antibodies.

A direct comparison of the joint-imaging properties of inflammation-specific- and non-specific monoclonal antibodies (Mabs) was possible in a patient suffering from long-standing, severe rheumatoid arthritis (RA). This patient received an anti-CD4− and an anti-carcinoembryonic antigen (anti-CEA) Mab, both labelled with technetium-99m, 9 days apart from each other. The anti-CD4 Mab was superior to the isotype-matched anti-CEA Mab in imaging inflamed joints. In the knee joint, the target-to-background ratio of the synovial membrane (SM) activity in comparison to that of adjacent large vessels was 1.22 (SM/muscle 1.55) for the anti-CD4 Mab and 0.53 (SM/muscle 0.92) for the anti-CEA Mab, in both cases 4 h after injection of the immunoglobulin. Since the CD4 antigen is present on the surface of T-helper lymphocytes and macrophages infiltrating the inflamed synovial membrane, imaging with the anti-CD4 Mab may allow more specific detection of inflammatory infiltrates in RA.

ADAM15 Adds to Apoptosis Resistance of Synovial Fibroblasts by Modulating Focal Adhesion Kinase Signaling

OBJECTIVE To study the contribution of ADAM15, a disintegrin metalloproteinase that is up-regulated in the rheumatoid arthritis (RA) synovial membrane, to the characteristic resistance of RA synovial fibroblasts (RASFs) to apoptosis induction by genotoxic stress or stimulation with proapoptotic FasL, which is present at high concentrations in RA synovial fluid. METHODS Caspase 3/7 activity and the total apoptosis rate in RASFs upon exposure to the DNA-damaging agent camptothecin or FasL were determined using enzyme assays and annexin V staining. Phosphorylated signaling proteins were analyzed by immunoblotting. RNA interference was used to silence ADAM15 expression. NF-κB activity was determined by enzyme-linked immunosorbent assay. RESULTS RASFs displayed significantly higher caspase 3/7 activity upon camptothecin and FasL exposure when ADAM15 had been down-regulated by specific small interfering RNAs. Upon FasL stimulation, RASFs phosphorylated focal adhesion kinase (FAK) and c-Src (Src), and activated phosphatidylinositol 3-kinase as well as the transcription factor NF-κB. This ADAM15-dependent, FasL-induced activation of antiapoptotic kinases and NF-κB was demonstrated by a marked reduction of apoptosis upon knockdown of ADAM15 protein expression. Inhibitors specifically interfering with FAK and Src signaling, such as FAK inhibitor 14 and dasatinib, potently induce apoptosis in RASFs, with significant enhancement by the silencing of ADAM15. CONCLUSION ADAM15 contributes to apoptosis resistance in RASFs by activating the Src/FAK pathway upon FasL exposure, rendering the FAK/Src signaling pathway an interesting target for potential therapeutic intervention in RA.

Magnetic targeting of a therapeutic antibody using magnetotropic microspheres of the interpolyelectrolyte coacervation complex.

Abstract The therapeutic monoclonal anti-rat CD4 antibody OX35 was successfully localized in the ankle joint of arthritic rats in vivo using a permanent magnet and magnetotropic microspheres composed of a protamine-chondroitin sulphate complex. The microspheres formed at nearly stoichiometric quantities of the polyelectrolytes and quantitatively incorporated both magnetite and antibody. The release of immunologically active antibody from the microspheres was controlled by glutaraldehyde treatment.

Imaging of RA Inflamed Joints with 99mTc-Labeled Specific Murine Anti-CD4- and Nonspecific Human Immunoglobulin

The presence of CD4-molecules on T-helper cells and macrophages, both abundantly present in human rheumatoid arthritis (RA) inflammatory infiltrates, provides the rationale for the use of radiolabeled anti-CD4 monoclonal antibodies (mAbs) to image RA arthritic joints (Becker et al. 1990). The imaging properties of a 99mTechnetium (99mTc)-labeled murine anti-human CD4 mAb (MAX.16H5; 200-300 μg, 370-550 MBq) were compared to those of polyclonal human immunoglobulin (HIG; TechnescanR, MDH-67, Mallinckrodt Diagnostica; 1 mg, 370 MBq) after intravenous injection into 8 patients with severe, active RA; in one RA patient the anti-human CD4 mAb was compared to an isotype-matched murine control mAb (BW 431/26; anti-human carcino-embryonic-antigen; Behringwerke; 2 mg; 810 MBq), and to HIG on separate occasions. Whole body and joint scans in anterior and posterior views were obtained 1, 4 and 24 h after injection.