CD47 associates with alpha 5 integrin and regulates responses of human articular chondrocytes to mechanical stimulation in an in vitro model.

BACKGROUND: Recent studies provide evidence of roles for integrins in mechanical signalling in bone and cartilage. Integrin signalling is modulated by various mechanisms, including interaction with other transmembrane proteins. We aimed to identify whether one such protein, integrin-associated protein (CD47/IAP), is expressed by chondrocytes and whether it may regulate integrin-dependent mechanotransduction. METHODS: Chondrocytes, isolated from macroscopically normal and osteoarthritic articular cartilage of human knee joints, were studied in a resting state or following mechanical stimulation at 0.33 Hz. CD47/IAP expression and associations were confirmed by immunohistology, reverse transcription-polymerase chain reaction, Western blotting, and immunoprecipitation. Roles in mechanotransduction were studied by assessing effects of function-blocking antibodies on a range of electrophysiological, cellular, and molecular responses of primary chondrocytes and responses of CD47/IAP-null cell lines transfected with CD47/IAP. RESULTS: Human articular chondrocytes were shown to express CD47/IAP, predominantly the type 2 isoform. Immunoprecipitation showed association of CD47/IAP with alpha5 integrin and thrombospondin but not SIRPalpha (signal-regulatory protein-alpha). The function-blocking anti-CD47/IAP antibody Bric 126 inhibited changes in membrane potential, tyrosine phosphorylation, and elevation of relative levels of aggrecan mRNA induced by mechanical stimulation, whereas in the presence of B6H12, an antibody that has partial agonist activity, a membrane depolarisation rather than a membrane hyperpolarisation response was induced by mechanical stimulation. CD47-null cell lines did not show changes in cell membrane potential following mechanical stimulation. Changes in cell membrane potential following mechanical stimulation were seen when CD47-null cells were transfected with CD47/IAP expression vectors but were not seen following mechanical stimulation of cells transfected with vectors for the extracellular immunoglobulin variable (IgV) domain of CD47/IAP in the absence of the transmembrane and intracellular domains. CONCLUSION: CD47/IAP is necessary for chondrocyte mechanotransduction. Through interactions with alpha5beta1 integrin and thrombospondin, CD47/IAP may modulate chondrocyte responses to mechanical signals.

Lethal autoimmune hemolytic anemia in CD47-deficient nonobese diabetic (NOD) mice.

The glycoprotein CD47 (integrin-associated protein, IAP) is present on the surface of virtually all cells, including red blood cells (RBCs). CD47 acts like a marker of self by ligating the macrophage inhibitory receptor signal regulatory protein alpha (SIRPalpha). In this manner mild reactivity of wild-type RBCs with macrophage phagocytic receptors is tolerated, whereas otherwise identical CD47-deficient RBCs are rapidly eliminated. We show here that virtually all CD47-deficient nonobese diabetic (NOD) mice spontaneously develop severe lethal autoimmune hemolytic anemia (AIHA) at 180 to 280 days of age, whereas none of the control CD47(+) NOD mice develop lethal AIHA at least during the first year of life. This phenotype is at least partially due to a markedly increased rate of elimination of opsonized CD47(-/-) compared to CD47(+) RBCs. Similarly, CD47(-/-)C57BL/6 mice were much more sensitive than their wild-type counterparts to experimental passive AIHA induced by anti-RBC monoclonal antibodies. Thus, CD47-SIRPalpha signaling can have a profound influence on the severity of AIHA, making manipulation of this signaling pathway a theoretically appealing avenue in the treatment of the disease.