Immune Netw.  2010 Oct;10(5):173-180. 10.4110/in.2010.10.5.173.

Cell to Cell Interaction Can Activate Membrane-bound APRIL Which Are Expressed on Inflammatory Macrophages

• Lee SM
• Kim WJ
• Suk K
• Lee WH

• KMID: 1456221
• DOI: http://doi.org/10.4110/in.2010.10.5.173

Abstract

BACKGROUND
APRIL, originally known as a cytokine involved in B cell survival, is now known to regulate the inflammatory activation of macrophages. Although the signal initiated from APRIL has been demonstrated, its role in cellular activation is still not clear due to the presence of BAFF, a closely related member of TNF superfamily, which share same receptors (TACI and BCMA) with APRIL.
METHODS
Through transfection of siRNA, BAFF-deficient THP-1 cells (human macrophage-like cells) were generated and APRIL-mediated inflammatory activities were tested. The expression patterns of APRIL were also tested in vivo.
RESULTS
BAFF-deficient THP-1 cells responded to APRIL-stimulating agents such as monoclonal antibody against APRIL and soluble form of TACI or BCMA. Furthermore, co-incubation of the siBAFF-deficient THP-1 cells with a human B cell line (Ramos) resulted in an activation of THP-1 cells which was dependent on interactions between APRIL and TACI/BCMA. Immunohistochemical analysis of human pathologic samples detected the expression of both APRIL and TACI in macrophage-rich areas. Additionally, human macrophage primary culture expressed APRIL on the cell surface.
CONCLUSION
These observations indicate that APRIL, which is expressed on macrophages in pathologic tissues with chronic inflammation, may mediate activation signals through its interaction with its counterparts via cell-to-cell interaction.

Keyword

Macrophage; APRIL; TNFSF; Inflammation; Signaling transduction

MeSH Terms

Cell Communication
Cell Line
Cell Survival
Diphenylamine
Humans
Inflammation
Macrophages
RNA, Small Interfering
Transfection
Diphenylamine
RNA, Small Interfering

Figure

• 

  Figure 1 Transfection of BAFF-specific siRNA resulted in a significant reduction of BAFF expression levels in THP-1 cells. (A) THP-1 cells transfected with control siRNA (Control) or BAFF specific siRNA (siBAFF) were stained with anti-APRIL or anti-BAFF mAb. Histograms from specific staining (open area) and background staining (filled area, stained with mouse IgG1) are compared. (B) RT-PCR analysis was performed in order to detect the presence of ARPIL, BAFF, or GAPDH mRNA in cells transfected with control siRNA (Control) or BAFF specific siRNA (siBAFF). PCR product sizes for APRIL, BAFF, and GAPDH are 394, 370, and 391 bp, respectively. The results are representatives for three independent experiments.

• 

  Figure 2 THP-1 cells transfected with BAFF-specific siRNA were stimulated with agents that can interact with APRIL. (A) THP-1 cells transfected with control siRNA (Control) or BAFF specific siRNA (siBAFF) were stimulated with 1µg/ml of LPS, TACI:Fc (TF), BCMA:Fc (BF), human IgG (H), anti-APIRL mAb (A), anti-BAFF mAb (B), or mouse IgG. Culture supernatants were collected 24 hr after the activation and the concentration of IL-8 was measured using double sandwich ELISA. IL-8 concentrations were compared with anti-APRIL stimulated samples which were set to 100%. (B) THP-1 cells transfected with siBAFF was stimulated with 1µg/ml LPS or 1, 3, 10µg/ml of BCMA:Fc for 24 hr. Culture supernatants were then collected for the measurement of IL-8 concentration using ELISA and for the analysis of MMP-9 activity through gelatin zymogram. Data points are represented as mean±SD of triplicate measurements. These experiments were repeated three times with essentially the same results. *p<0.001 when compared with negative control (the first lane). †p<0.001 when compared with the value obtained without T/B pre-incubation.

• 

  Figure 3 Ramos cells stimulated THP-1 cells through interaction between APRIL and its counterparts. (A) Ramos cells were stained with mAb against TACI, BCMA, or APRIL. Histograms from specific staining (open area) and background staining (filled area, stained with mouse IgG1) are compared. (B) SiBAFF-transfected THP-1 cells were stimulated with either 1µg/ml of anti-APRIL mAb (A) or co-incubation with Ramos cells. For the blocking, Ramos cells were pre-incubated with 1µg/ml of anti-TACI/anti-BCMA (T/B) mixture or mouse IgG (M) for 20 min. After washing out the antibodies, cells were then incubated with siBAFF-transfected THP-1 cells. Data points are represented as mean±SD of triplicate measurements. These experiments were repeated twice with essentially the same results. *p<0.001 when compared with negative control (the first lane). †p<0.001 when compared with the value obtained without T/B pre-incubation.

• 

  Figure 4 Macrophages in human atherosclerotic plaques express both APRIL and TACI. Consecutive sections of an atherosclerotic plaque were stained with mAbs against CD68 (a marker for macrophages), APRIL, or TACI. Isotype-matching mouse immunoglobulins (mIgG) were used for staining as a negative control. Magnification: ×100. The results are representative for three independent analyses.

• 

  Figure 5 Expression of both APRIL and TACI was in macrophages from human arthritic synovial tissues. Consecutive sections of an atherosclerotic plaque RA-synovial tissue (A) or osteoarthritis tissues (B) were stained with mAbs against CD68, APRIL, or TACI. Isotype-matching mouse immunoglobulins (mIgG) were used for staining as a negative control. Magnification: ×100. The results are representative for three independent analyses.

• 

  Figure 6 Primary macrophage culture expresses APRIL and TACI on the cell surface. Primary macrophages derived from RA patient peripheral blood monocytes were stained with mAbs against CD68, APRIL, or TACI. In case APPRIL and TACI, specific mAbs were added before fixation and mounting in order to detect the expression of these molecules on the cell surface. Isotype-matching mouse immunoglobulins (mIgG) were used for staining as a negative control. Magnification: ×400. The experiment was repeated twice with essentially the same results.

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