Human immunodeficiency virus envelope‐dependent cell‐cell fusion: A quantitative fluorescence cytometric assay

Abstract

Objective To examine the effects of a fibronectin (FN) fragment containing the COOH‐terminal heparin‐binding domain (HBFN‐f) on chondrocyte‐mediated type II collagen (CII) cleavage by collagenase and proteoglycan (PG) degradation in articular cartilage in explant culture. Methods Intact FN or HBFN‐f was added to explant cultures of mature bovine articular cartilage. We investigated collagenase‐mediated cleavage of CII caused by HBFN‐f in explant cultures using a new immunoassay for detection and measurement of the primary collagenase cleavage site of CII. CII denaturation in cartilage was also measured using a specific enzyme‐linked immunosorbent assay. Degradation of PG (principally aggrecan) was analyzed by a dye‐binding assay. APMA and/or a matrix metalloproteinase 13 (MMP‐13) preferential inhibitor or interleukin‐1 receptor antagonist (IL‐1Ra) were added to some cultures to examine the presence of latent procollagenase or the involvement of MMP‐13 or IL‐1, respectively, in cartilage breakdown induced by HBFN‐f. Secretion of MMP‐3 and MMP‐13 into media was detected by immunoblotting. Results In contrast to intact FN, HBFN‐f was shown to stimulate CII cleavage by collagenase in a dose‐dependent manner following PG degradation, similar to cartilage breakdown induced by IL‐1. Treatment with HBFN‐f also resulted in elevated denaturation of CII. Immunoblotting demonstrated that HBFN‐f enhanced pro–matrix metalloproteinase 13 (proMMP‐13) production as well as that of proMMP‐3. APMA, which activates latent proMMPs, enhanced the HBFN‐f–mediated cleavage of CII by collagenase. An MMP‐13 preferential inhibitor or IL‐1Ra suppressed HBFN‐f–induced collagen cleavage to control levels. Conclusion Our data demonstrate that HBFN‐f can induce early PG degradation and subsequent CII cleavage. The latter is probably mediated by early proMMP‐13 induction involving an IL‐1–dependent pathway. Activation of latent collagenase is delayed. This new information, together with existing data on other FN fragments, reveals that increased levels of these fragments, found in diseased joints such as in osteoarthritis and rheumatoid arthritis, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the present study.

Intimate Cell Conjugate Formation and Exchange of Membrane Lipids Precede Apoptosis Induction in Target Cells during Antibody-Dependent, Granulocyte-Mediated Cytotoxicity

“…Recently, cellcell fusion between cancer cells and myeloid cells has been described (42), and cell-cell fusion was detected as double fluorescent events in flow cytometric studies (38). Interestingly, during intimate contact formation we observed cytoplasmic communication between effector and target cells (Fig.…”

“…These data were further supported by flow cytometry showing clusters between PMN and tumor cells. We adapted a commonly used assay for the quantification of aggregates (38). The plasma membranes of PMN effector and Raji H cells were labeled with the fluorescent carbocyanines DiO or DiI, respectively.…”

Respiratory Syncytial Virus-Neutralizing Monoclonal Antibodies Motavizumab and Palivizumab Inhibit Fusion

“…A cell-to-cell fusion assay was developed much like other assays that have been reported for RSV (8,16,19), as well as for HIV (24). TREx-F green and red cells were plated together at 5 ϫ 10 5 each/well in a six-well plate.…”

“…In order to better assess the process of fusion by F protein, a quantitative assay to measure F protein mediated cell-to-cell fusion was developed in a manner similar to that described in previous reports for RSV and other viruses (8,16,19,24). The TREx-F cell line was created, which maintains tetracycline-inducible expression of RSV F protein.…”

Rapid Fusion and Syncytium Formation of Heterologous Cells upon Expression of the FGFRL1 Receptor

“…Notably, we did not observe background fusion of HEK cells with wild-type CHO cells, suggesting that CHO cells do not generally fuse with other cells. This is supported by the fact that CHO cells are routinely used as components in cell-cell fusion assays to assess the fusogenic activity of viral glycoproteins (39,40). If CHO cells had a pronounced propensity to fuse with each other or with other cells, they would not be suitable for quantitative fusion assays.…”