Production and distribution of interleukin 15 and its receptors (IL-15Ralpha and IL-R2beta) in the implant interface tissues obtained during revision of failed total joint replacement

Abstract

Failure of total joint replacement (TJR) is a major problem and it is estimated that 15-20% of TJR will fail within 5-10 years after implantation. Most TJR is attributed to aseptic loosening of the implants in association with resorption of related bone due to the release of bone-associated cytokines. IL-15 is a cytokine that activates T cells and natural killer (NK) cells. IL-15 protein is ubiquitous and is expressed in many tissues and cell types. Using immunohistochemical techniques, we demonstrated the expression of IL-15 and its receptors IL-15Ralpha and IL-2Rbeta in the interface tissues obtained from revision surgery. Both IL-15 protein and IL-15Ralpha were observed in macrophages, multinucleated giant cells and endothelial cells around blood vessels. Both the SDS-PAGE and western blot revealed multiple bands and after stages of glycosylation, this resulted in a band at 13 KDa which corresponds to the IL-15 protein. Again RT-PCR results demonstrated a band of 420 bp corresponding to the IL-15 protein. In addition, using U937 cells, the expression of both IL-15 protein and IL-15Ralpha were considerably up-regulated when challenged with retrieved metal particles. Our results illustrated the IL-15 to be an intact protein and that it is stored in the cytoplasm. A dye exclusion cell viability test displayed an increase in toxicity with an increase in the amount of metal particles added. There was a discrepancy between abundant IL-15 mRNA, intracellularly detectable IL-15 protein and apparently inefficient secretion. This suggests that IL-15 protein production is predominantly regulated post-transcriptionally and this is indicated by its strict regulation, especially at cell trafficking. Finally, unlike IL-2, IL-15 plays a certain role in bone resorption that leads to failed joint prostheses. It is apparent that this cytokine is an important T cell mediated immune response which needs further research.

Figure 1

In routine preparation of histological sections, heavy deposits of black metal wear debris are demonstrated in interface tissue obtained from failed joint prosthesis, × 200. M = metal wear debris.

Figure 2

Immunohistochemical staining of IL-15 protein in interface tissue obtained from failed joint prosthesis. The binding of specific antibody to IL-15 was detected by alkaline phosphate-streptavidin method, × 200.

Figure 3

Immunohistochemical staining of IL-15 protein expressed by multinucleated giant cell containing a large piece of polyethylene debris (arrow), viewed by compensated polarization microscopy. The binding of specific antibody to IL-15 was detected by an alkaline phosphate-streptavidin method, × 250.

Figure 4

Immunohistochemical staining of T lymphocytes in interface tissue. The binding of specific antibody to CD3 (arrows) was detected by an alkaline phosphate-streptavidin method, × 200.

Figure 5

Immunohistochemical staining of IL-15Rα in interface tissue. The binding of specific antibody to IL-15Rα (arrows) was detected by an alkaline phosphate-streptavidin method, × 200.

Figure 6

Immunohistochemical staining of IL-15Rα of endothelial cell (arrow) and giant cell (arrow). The binding of specific antibody to IL-15Rα was detected by an alkaline phosphate-streptavidin method, × 250.

Figure 7

Immunohistochemical staining of IL-2Rβ in interface tissue. The binding of specific antibody to IL-2Rβ (arrows) in lymphocytes was detected by an alkaline phosphate-streptavidin method, × 250.

Figure 8

SDS-PAGE results of precursor IL-15 protein (a) and western blotting of IL-15 protein extracted from interface tissues (b). The arrows indicate the position of the extracted IL-15 protein and the rhIL-15 protein used as positive control. Randomly chosen cases from patient's interface tissues obtained from revision surgery are specified by numbers.

Figure 9

SDS-PAGE results of IL-15 protein after subjecting to enzymatic glycosylation (a) and western blotting of IL-15 protein extracted from interface tissues (b). The arrows indicate the position of the extracted IL-15 protein and the rhIL-15 protein used as the positive control. Randomly chosen cases from patient's interface tissues obtained from revision surgery are specified by numbers.

Figure 10

RT-PCR analysis of mRNA from β–Actin (10a). RT-PCR analysis of mRNA extracted from interface tissue (10b), cases 1–6 are randomly chosen interface tissues obtained from revision surgery. RT-PCR analysis of mRNA extracted from U937 cell lysates (10c) of untreated and treated with LPS = lipopolysaccharide, Ti = titanium, Ss = stainless steel, PMA = phorbol 12-myristate 13-acetate.

Figure 11

The expression of IL-15 by U937 cells showing both untreated and treated with stimulatory agents. Data were calculated as the mean ± SD for four replicates and expressed as a percentage of the controls. P < 0.05. LPS = lipopolysaccharide, Ti = titanium, Ss = stainless steel, PMA = phorbol 12-myristate 13-acetate.

Figure 12

The expression of IL-15Rα by U937 cells showing both untreated and treated with stimulatory agents. Data were calculated as the mean ± SD for four replicates and expressed as a percentage of the controls. P < 0.05. LPS = lipopolysaccharide, Ti = titanium, Ss = stainless steel, PMA = phorbol 12-myristate 13-acetate.