Immune Netw.  2017 Apr;17(2):116-120. 10.4110/in.2017.17.2.116.

IL-32-induced Inflammatory Cytokines Are Selectively Suppressed by α1-antitrypsin in Mouse Bone Marrow Cells

• Lee S
• Choi DK
• Kwak A
• Kim S
• Nguyen TT
• Gil G
• Kim E
• Yoo KH
• Kim IA
• Lee Y
• Jhun H
• Chan ED
• Bai X
• Kim H
• Kim YS
• Kim S

• KMID: 2376869
• DOI: http://doi.org/10.4110/in.2017.17.2.116

Abstract

The induction of interleukin (IL)-32 in bone marrow (BM) inflammation is crucial in graft versus host disease (GvHD) that is a common side effect of allogeneic BM transplantation. Clinical trials on α-1 antitrypsin (AAT) in patients with GvHD are based on the preliminary human and mouse studies on AAT reducing the severity of GvHD. Proteinase 3 (PR3) is an IL-32-binding protein that was isolated from human urine. IL-32 primarily induces inflammatory cytokines in myeloid cells, probably due to PR3 expression on the membrane of the myeloid lineage cells. The inhibitory activity of AAT on serine proteinases may explain the anti-inflammatory effect of AAT on GvHD. However, the anti-inflammatory activity of AAT on BM cells remains unclear. Mouse BM cells were treated with IL-32γ and different inflammatory stimuli to investigate the anti-inflammatory activity of AAT. Recombinant AAT-Fc fusion protein inhibited IL-32γ-induced IL-6 expression in BM cells, but failed to suppress that induced by other stimuli. In addition, the binding of IL-32γ to PR3 was abrogated by AAT-Fc. The data suggest that the specific anti-inflammatory effect of AAT in mouse BM cells is due to the blocking of IL-32 binding to membrane PR3.

Keyword

Interleukin-32; Mouse bone marrow cell; Proteinase 3; Interleukin-6; Recombinant AAT-Fc

MeSH Terms

Animals
Bone Marrow Cells*
Bone Marrow*
Cytokines*
Graft vs Host Disease
Humans
Inflammation
Interleukin-6
Interleukins
Membranes
Mice*
Myeloblastin
Myeloid Cells
Serine Proteases
Cytokines
Interleukin-6
Interleukins
Myeloblastin
Serine Proteases

Figure

• 

  Figure 1 The anti-inflammatory effect of recombinant AAT-Fc in mouse BM cells. (A) IL-6 level in the supernatant of mouse BM cells was measured by sandwich ELISA (R&D Systems, Minneapolis MN). (B) The induction of IL-6 by different stimuli was measured without AAT-Fc (open bar) or with pre-incubation of AAT-Fc (closed bar, 2 µg/mL) for 3 h. (C) LPS-, (D) TNFα-, (E) IL-32γ-, and (F) IL-1α-mediated IL-6 level in the supernatant of BM cells isolated from the mice treated with AAT-Fc for 48 h was compared to that in the supernatant of BM cells isolated from the untreated control mice. Data in A are comparisons between untreated control and stimulated mice. Data in B are comparisons between RPMI control and AAT-Fc pre-incubated cells. Data in E are comparisons between BM cells isolated from untreated mice and BM cells isolated from mice treated with AAT-Fc (2 mg/kg) for 48 h. Mean±SEM; *p<0.05; #p<0.001 from three replicates. Representative data from 1 of 3 independent experiments are shown.

• 

  Figure 2 Suppression of inflammatory signaling in recombinant AAT-Fc-pretreated mouse BM cells, AAT-Fc competing with IL-32γ binding to PR3-Fc, and the dogma of AAT anti-inflammatory activity on IL-32-induced inflammation in BM transplantation. (A) The phosphorylation of NF-κB and p38MAPK by IL-32γ in BM cells from 6-week-old C57BL/6 mice was assessed using anti-phospho antibodies. (B) Similar experiments were performed by injecting AAT-Fc (2 mg/kg) into mice for 48 h prior to isolating BM cells. (C) Recombinant PR3-Fc was used for IL-32γ binding assay. IL-32γ was added at the concentration indicated on the bottom, and IL-32γ binds to PR3-Fc in a dose dependent manner. (D) The binding of IL-32γ was blocked with PR3-Fc in a dose dependent manner. Data in C are comparisons between control blank and IL-32γ-treated cells. Data in D are comparisons between control without AAT-Fc and with AAT-Fc. Mean±SEM; *p<0.05; **p<0.01; #p<0.001 from three replicates. Representative data from 1 of 4 independent experiments are shown. (C) Schematic representation of AAT-Fc suppression on IL-32-mediated inflammation in BM transplantation. Allogeneic BM transplantation induces different pro-inflammatory cytokines, such as IL-32, TNFα, IL-1α, and IL-6, resulting in immune reaction causing GvHD and death, while the specific IL-32 blockade AAT-Fc reduces immune reaction increasing survival.

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