Immune Netw.  2015 Oct;15(5):232-240. 10.4110/in.2015.15.5.232.

A New Triggering Receptor Expressed on Myeloid Cells (TREM) Family Member, TLT-6, is Involved in Activation and Proliferation of Macrophages

• Won KJ
• Park SW
• Lee S
• Kong IK
• Chae JI
• Kim B
• Lee EJ
• Kim DK

• KMID: 2150852
• DOI: http://doi.org/10.4110/in.2015.15.5.232

Abstract

The triggering receptor expressed on myeloid cells (TREM) family, which is abundantly expressed in myeloid lineage cells, plays a pivotal role in innate and adaptive immune response. In this study, we aimed to identify a novel receptor expressed on hematopoietic stem cells (HSCs) by using in silico bioinformatics and to characterize the identified receptor. We thus found the TREM-like transcript (TLT)-6, a new member of TREM family. TLT-6 has a single immunoglobulin domain in the extracellular region and a long cytoplasmic region containing 2 immunoreceptor tyrosine-based inhibitory motif-like domains. TLT-6 transcript was expressed in HSCs, monocytes and macrophages. TLT-6 protein was up-regulated on the surface of bone marrow-derived and peritoneal macrophages by lipopolysaccharide stimulation. TLT-6 exerted anti-proliferative effects in macrophages. Our results demonstrate that TLT-6 may regulate the activation and proliferation of macrophages.

Keyword

TLT-6; ITIM motif; Anti-proliferation; Macrophages; TREM family

MeSH Terms

Adaptive Immunity
Computational Biology
Computer Simulation
Cytoplasm
Hematopoietic Stem Cells
Humans
Immunoglobulins
Macrophages*
Macrophages, Peritoneal
Monocytes
Myeloid Cells*
Immunoglobulins

Figure

• 

  Figure 1 Sequence analysis and character of TLT-6. (A) Predicted amino acid sequences, domains, and motifs of TLT-6. The leader sequence and transmembrane domain are indicated by dotted lines, and the variable type of immunoglobulin-like domain in the extracellular region is denoted by a solid line. The ITIM-like motifs in the intracellular region of TLT-6 are shown in the box. (B) Analysis of deduced molecular weight for Flag-tagged TLT-6 using an in vitro transcription/translation system. (C) Identification of interacting protein with TLT-6 using co-immunoprecipitation. HeLa cells, endogenously expressing SHIP and SHP-2, were used for transfection with HA-tagged TLT-6, immunoprecipitated with anti-Flag antibody, and then immunoblotted with anti-phospho SHIP or SHP-2 monoclonal antibodies. IB, Immunobloting; IP, immunoprecipitation.

• 

  Figure 2 RT-PCR analyses of TLT-6 expression. (A) Organ specific expression of TLT-6 and (B) hematopoietic lineage cells specific expression of TLT-6 purified in the bone marrow, spleen, and peripheral blood, as described in the methods section. cDNA concentration was standardized and quantified using β-actin. (C) TLT-6 expression in HSCs (K+S+L-) and progenitor cells (K+S+L-, K-S+L-, and K-S-L-) isolated from the mouse bone marrow using RT-PCR. For RT-PCR, cDNA concentration was normalized with β-actin. PBL, leukocytes isolated from peripheral blood.

• 

  Figure 3 TLT-6 expressions in hematopoietic stem cells, monocyts and macrophages. (A) Expression patterns of TLT-6 in the surface of hematopoietic lineage cells from the bone marrow of mice analyzed by flow cytometry. Flow cytometry analysis was performed as described in the methods section, on the surface region of CD11b+ cells and HSCs (c-Kit+Sca-1+Lin-cells) from the bone marrow of mice. Expression of TLT-6 on the surface region of peritoneum (B, C)- and bone marrow (D, E)-derived macrophages. Each isolated macrophage sample was stimulated with LPS (10 ng/ml) during the indicated days. After treatment with anti-TLT-6 monoclonal antibody for 2 days, flow cytometry analysis was carried out as described in the methods section. Numbers in the upper parts of histogram curves indicate the positive intensity values of TLT- 6 expression in cells. Dotted line, control; Solid line, LPS-stimulated condition.

• 

  Figure 4 TLT-6 expression and its effect on macrophages. (A, B) TLT-6 expression in activated RAW264.7 cells. The cells were stimulated with each indicated concentration of LPS for 3 days and the positive cells expressing TLT-6 were observed by flow cytometry analysis. Numbers in the upper parts of histogram curves indicate the positive intensity values of TLT-6 expression in cells. Dotted line, control; Solid line, LPS-stimulated condition. (C) Effect of TLT-6 overexpression on RAW264.7 cell proliferation. RAW 264.7 cells were transfected with the TLT-6 cDNA transgene, and cell proliferation was tested using the Cell Counting Kit-8 during the indicated days. (D) Effect of antibody treatment on bone marrow-derived macrophage proliferation. Cells were simultaneously treated with 10 ng/ml M-CSF and 1 µg/ml monoclonal TLT-6 antibody for 2 days and cell proliferation was analyzed using the Cell Counting Kit-8. Data in panel B and C are expressed as mean±standard deviation (SD), respectively.*p<0.05 compared with cells transfected with GFP vector (control in panel B), or with cells treated with IgG isotype (control in panel C).

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