IL-4 induces the formation of multinucleated giant cells and expression of β5 integrin in central giant cell lesion

Abstract

Background: It is now well established that IL-4 has a central role in the development of monocytes to multinucleated giant cells (MGCs) by inducing the expression of integrins on the surface of monocytes. The aim of this study was to investigate the potential role of IL-4 in induction of β5 integrin expression in the peripheral blood samples of patients with giant cell granuloma.

Material and methods: Monocytes were isolated from peripheral blood samples of patients with central giant cell granuloma (CGCG) and healthy controls using human Monocyte Isolation Kit II. Isolated monocytes were then cultured in the absence or presence of IL-4 (10 and 20 ng/mL), and following RNA extraction and cDNA synthesis, Real-time PCR was performed to determine the level of β5 integrin expression. The formation of CGCGs and morphological analyses were done under light microscopy. For confirmation of CGCGs, immunocytochemistry technique was also carried out by anti-RANK (receptor-activator of NF-κB ligand) antibody.

Results: In both patient and control groups, β5 levels were significantly enhanced by increasing the IL-4 dose from 10 to 20 ng/mL. In addition, these differences were significant between patient and control groups without IL-4 treatment. On the other hand, the number of cells which expressed RANK and therefore the number of giant cells were significantly higher in the patient group in comparison to controls, as assessed by immunohistochemistry evaluations.

Conclusions: In this study, we showed an elevation in the expression levels of β5 integrin when stimulated by IL-4. It is strongly indicated that this integrin acts as an important mediator during macrophage to macrophage fusion and development of giant cells.

Figure 1

Up-regulation of β5 Integrin mRNA by IL-4. Monocytes were isolated from CGCGs and healthy donors were plated in 25cm2 flasks in the absence or presence of IL-4 (10 and 20 ng/mL). Media and IL-4 were replaced every other day. After 5 days, β5 integrin mRNA levels were examined by Real-time-PCR and relative mRNA expression levels were quantified by the 2 (-ΔΔCt) method. The results represent mean±SD (n=3); * and # p<0.05.

Figure 2

MGC formation is dependent on IL-4 concentration. Monocytes were isolated from CGCGs and plated in 24-well plates in the absence or presence of IL-4 (10 and 20 ng/mL). After 5 days, the cells were fixed and MGC morphology and formation was observed under light microscopy (40×). (A) Absence of IL-4; (B) Cells after IL-4 treatment (10 ng/mL), and (C) Cells after IL-4 treatment (20 ng/mL).

Figure 3

Inductive effect of IL-4 on RANK protein expression and MGC formation. Immunocytochemistry results using anti-RANK Abs in monocytes isolated from CGCGs and plated in 24-well plates in the absence or presence of IL-4 (10 and 20 ng/mL). After 5 days, the cells were stained with DAB. (A) Absence of IL-4; (B) Cells after IL-4 treatment (10 ng/mL), and (C) Cells after IL-4 treatment (20 ng/mL). The intensity of DAB staining (brown stain) shows the RANK protein expression levels.