Analysis of early calcium signals related to the differntiation of macrophages from monocytes

Monocytes/macrophages(MOs/Mφs)are the multi-functional cell system that responds to inflammatory stimuli via receptors and accomplishes migration，adhesion，phagocytosis，immune cell activation，and cytotoxicity。The stimulated MOs differentiate into Mφs as they leave the circulation to various tissues and organs。In a wide variety of cells，Ca I D 12+I D 1 is the intracellular signal that couples receptor stimulation to cell activation.The present study aimed to examine the early response of MOs/Mφs to stimuli via purinoceptors and CD14 receptors and to lipopolysaccharide(LPS；endotoxin of gram-negative bacteria)and its change during differentiation into Mφs，in terms of an increase in intracellular Ca I D 12+i D 1 concentration([Ca I D 12+I D 1]I D 2 i D 2)。Human peripheral blood MOs were isolated by adhesion to glass dishes and cultured in vitro.[Ca I D 12+I D 1]I D 2 i I D 2 was measured in single cells using a Ca I D 12+I D 1 imaging method with the Ca I D 12+I D 1 indicator dye fura-2.The foll…More owing results were obtained during the term of the project.1)MOs/Mφs exhibited a transient[Ca I D 12+I D 1]I D 2 i i D 2 rise in response to extracellular ATP via P I D 22 U I D 2 and P I D 22 Y I D 2 purinoceptors.The similar Ca I D12+I D1 response was induced by application of supernatant from tumor cells lysed by hypoosmotic treatment。A[Ca I D 12+I D 1]I D 2 i i d 2 rise occurred in MOs/Mφs in the vicinity of a single tumor cell that was attacked and permeabilized by a natural killer cell in a dish.These results support the view that MOs/Mφs respond to signal messengers discharged from damaged or dying cells to be ingested，and ATP is at least one of the messengers and causes a[Ca I D12+I D1]I D 2 i I D 2 rise via P I D 22 U I D 2 and P I D 22 Y I D 2 purinoceptors.2)CD 14 is known as the recognition site for LPS leading to production of cytokines.Stimulation of CD14 receptors by anti-CD14 monoclonal antibody caused[Ca I D12+I D 1]I D 2 i I D 2 rises in MOs/Mφs in the form of damping oscillations.The signal transduction to the[Ca I D 12+I D 1]I D 2 i i d 2 rise through CD 14 is interesting for future studies，Since CD14molecules are anchored in the lipid bilayer of the plasma membarne without the intracellular domain.3)LPS on its own at concentrations between 1 ng/ml and 100µg/ml did not cause any substantial[Ca i D12+i D1]I D 2 i d 2 rise in MOs/Mφs.Involvement of LPS-binding protein has been proposed in interaction between LPS and CA 14.4)Macrophage-colony stimating factor(M-CSF)proding protein has been proposed in interaction between LPS and CA14.4)Macrophage-colony stimulating factor(M-CSF)produced protein has been proposed in interaction.The percentage of responding cells to anti-CD14 antibody as well as the magnitude of Ca I D12+I D1 responses was augmented during culture.Interestingly，about45%of Mφs showed[Ca I D12+I D 1]I D 2 i i d 2 rises in responses to LPS.It is likely that expression of CD14molecules and/or the binding capacity of CD14is enhanced during differentiation of Mφs.Less

Monocytes/macrophages (MOs/Mφs) are the multi-functional cell system that responds to inflammatory stimuli via receptors and accomplishes migration, adhesion, phagocytosis, immune cell activation, and cytotoxicity. The stimulated MOs differentiate into Mφs as they leave the circulation to various tissues and organs. In a wide variety of cells, CaィイD12+ィエD1 is the intracellular signal that couples receptor stimulation to cell activation. The present study aimed to examine the early response of MOs/Mφs to stimuli via purinoceptors and CD14 receptors and to lipopolysaccharide (LPS ; endotoxin of gram-negative bacteria) and its change during differentiation into Mφs, in terms of an increase in intracellular CaィイD12+ィエD1 concentration ([CaィイD12+ィエD1]ィイD2iィエD2). Human peripheral blood MOs were isolated by adhesion to glass dishes and cultured in vitro. [CaィイD12+ィエD1]ィイD2iィエD2 was measured in single cells using a CaィイD12+ィエD1 imaging method with the CaィイD12+ィエD1 indicator dye fura-2. The foll … More owing results were obtained during the term of the project.1) MOs/Mφs exhibited a transient [CaィイD12+ィエD1]ィイD2iィエD2 rise in response to extracellular ATP via PィイD22UィエD2 and PィイD22YィエD2 purinoceptors. The similar CaィイD12+ィエD1 response was induced by application of supernatant from tumor cells lysed by hypoosmotic treatment. A [CaィイD12+ィエD1]ィイD2iィエD2 rise occurred in MOs/Mφs in the vicinity of a single tumor cell that was attacked and permeabilized by a natural killer cell in a dish. These results support the view that MOs/Mφs respond to signal messengers discharged from damaged or dying cells to be ingested, and ATP is at least one of the messengers and causes a [CaィイD12+ィエD1]ィイD2iィエD2 rise via PィイD22UィエD2 and PィイD22YィエD2 purinoceptors.2) CD14 is known as the recognition site for LPS leading to production of cytokines. Stimulation of CD14 receptors by anti-CD14 monoclonal antibody caused [CaィイD12+ィエD1]ィイD2iィエD2 rises in MOs/Mφs in the form of damping oscillations. The signal transduction to the [CaィイD12+ィエD1]ィイD2iィエD2 rise through CD14 is interesting for future studies, since CD14 molecules are anchored in the lipid bilayer of the plasma membarne without the intracellular domain.3) LPS on its own at concentrations between 1 ng/ml and 100 μg/ml did not cause any substantial [CaィイD12+ィエD1]ィイD2iィエD2 rise in MOs/Mφs. Involvement of LPS-binding protein has been proposed in interaction between LPS and CA14.4) Macrophage-colony stimulating factor (M-CSF) produced CaィイD12+ィエD1 responses in about 45% of MOs/Mφs. MOs/Mφs differentiated to large Mφs, when cultured in the presence of M-CSF. The percentage of responding cells to anti-CD14 antibody as well as the magnitude of CaィイD12+ィエD1 responses was augmented during culture. Interestingly, about 45% of Mφs showed [CaィイD12+ィエD1]ィイD2iィエD2 rises in responses to LPS. It is likely that expression of CD14 molecules and/or the binding capacity of CD14 is enhanced during differentiation of Mφs. Less