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Abstract
Objective
DICAM, a dual Ig domain containing adhesion molecule, is involved in cell-cell adhesion through direct interaction with αvβ3 integrin. In our previous study showing the inhibitory role of DICAM in osteoclast differentiation, we found that DICAM also has a suppressive role in macrophage, the precursor cell of osteoclast. The role of DICAM in macrophage activation at the inflammatory milieu, however, remains obscure.
Methods
Expression pattern of DICAM by inflammatory cytokines and lipopolysaccharide (LPS) was studied with RAW264.7, a murine macrophage cell line. To study the role of DICAM on macrophage activation, we stably transduced DICAM, or empty vector, into RAW264.7, and then compared the LPS-mediated activation such as spreading and TNF-α production.
Results
DICAM was abundantly expressed in the synovial tissue of collagen-induced arthritis. When we assessed the expression of DICAM in RAW264.7 cells by mediators of inflammation, inflammatory cytokines, such as TNF-α, IL-1β, and IFN-γ, and M-CSF increased the expression of DICAM; however, LPS decreased. Functionally, DICAM that stably transduced-RAW264.7 cells showed attenuation of LPS-mediated macrophage activation including spreading and TNF-α production. DICAM decreased the phosphorylation of JNK MAP kinase by M-CSF and LPS stimulation, which was corroborated by a decrease in the expression of ITAM-associated receptors including Trem2, Pira1, and Oscar. Finally, a recombinant ectodomain of DICAM suppressed LPS-induced activation of RAW264.7 cells.
Figures and Tables
Figure 1
In vivo expression of DICAM in synovial tissue. Immunohistochemical analyses of DICAM in the ankle joint of control and CIA mice. The ankle joints of the CIA were collected for preparation of paraffin blocks. They were treated with anti-DICAM and anti-CD14, the marker for monocyte/macrophage. Bound Abs was visualized with HRP. Original magnification, ×100 (s: synovial tissue, b: bone).
Figure 2
Regulation of DICAM expression in monocyte by inflammatory cytokines. (A, B) qRT-PCR analyses of DICAM in RAW264.7 cells treated with pro-inflammatory cytokines. The expression of m-RNA of DICAM in RAW264.7 cells was analyzed after 1 day treatment of LPS (1 µg/mL), TNF-α (25 ng/mL), IL-1β (5 ng/mL), IFN-γ (50 ng/mL), and M-CSF (10 ng/mL). (C) Western blot analyses of DICAM expression by LPS. RAW264.7 cells were treated with various dose of LPS for 8 hr, and then harvested for immunoblotting. (D) LPS suppresses the expression of DICAM by M-CSF. Bone marrow-derived macrophages (BMMs) from C57BL6/J were treated with 50 ng/mL of M-CSF with or without 100 ng/mL of LPS for 3 days, and the expression of DICAM was analyzed with Western blot analysis. *TB: total BMMs.
Figure 3
DICAM attenuates activation of RAW264.7 cells and production of TNF-α. (A) RAW264.7 cells (2.5×103) stably transfected with control vector, or DICAM was treated with 102 ng/mL and 104 ng/mL of LPS in a 96-well culture plate for 2 days. Then, cell morphology was evaluated by inverted microscopy. (B) Activated spreading cells were guantified under high power field (×200). (C) Levels of TNF-α in the supernatant were measured by ELISA. n=5. *p<0.05, †p<0.01, Mann-Whitney U test.
Figure 4
DICAM regulates LPS-induced activation of monocyte by modulating the phosphorylation of JNK and expression of c-Fos. (A) BMMs infected with retrovirus expressing DICAM (pMx-DICAM) and control vector (pMx-vector) were stimulated with M-CSF (20 ng/mL) for 3 days, and then starved overnight and stimulated with 100 ng/mL of M-CSF for the indicated time period (0, 5, 30 min). Activation of ERK and JNK MAP kinase and Akt were assessed for phosphorylation by Western blot analysis. (B) Starved BMMs were treated with 1µg/mL of LPS for the indicated time period (0, 5, 30 min), and then harvested for immunoblotting. Activations of ERK and JNK MAP kinase, and IκB, and the expression of c-Fos were assessed by Western blot analysis.
Figure 5
DICAM attenuates the expression of ITAM-associated receptor, such as Trem2, Pira1, and Oscar. (A∼F) BMMs transduced with pMx-empty vector or pMx-DICAM were cultured for 3 days in the presence of M-CSF (20 ng/mL). Then, total m-RNA were subjected to real-time PCR for evaluating the expression of DICAM and ITAM-associated receptor, such as Trem2, Pira1, Oscar, Dap12, and FcRγ. *p<0.05, †p<0.01, Mann-Whitney U test.
Figure 6
Extracellular DICAM inhibits macrophage differentiation. (A) RAW264.7 (2.5×103) cells were cultured for 3 days in the presence of 104 ng/mL of recombinant Ecto-DICAM purified from E. coli and 100 ng/mL of LPS in a 96-well culture plate. The spreading cells were counted for quantification. Fc: Fc (constant) region of immunoglobulin as a negative control, D-Fc: recombinant Ecto-DICAM which has Fc portion for purification. (B) Quantification of spreading cells was presented as a graph. *p<0.05, Mann-Whitney U test.
Figure 7
A schematic model of the role of DICAM during macrophage differentiation. Our previous results revealed that DICAM directly interacts with integrin αVβ3 and it may suppress the phosphorylation of JNK MAP Kinase by LPS, leading to a down-regulation of c-Fos. It is accompanied by a down-regulation of ITAM-associated receptor, such as Trem2, Pira1, and Oscar, which has a major role in calcium signaling in monocyte activation.
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