Comparison of the roles of IL-1, IL-6, and TNFalpha in cell culture and murine models of aseptic loosening

Abstract

Pro-inflammatory cytokines, such as IL-1, IL-6, and TNF, are considered to be major mediators of osteolysis and ultimately aseptic loosening. This study demonstrated that synergistic interactions among these cytokines are required for the in vitro stimulation of osteoclast differentiation by titanium particles. In contrast, genetic knock out of these cytokines or their receptors does not protect murine calvaria from osteolysis induced by titanium particles. Thus, the extent of osteolysis was not substantially altered in single knock out mice lacking either the IL-1 receptor or IL-6. Osteolysis also was not substantially altered in double knock out mice lacking both the IL-1 receptor and IL-6 or in double knock out mice lacking both TNF receptor-1 and TNF receptor-2. The differences between the in vivo and the cell culture results make it difficult to conclude whether the pro-inflammatory cytokines contribute to aseptic loosening. One alternative is that in vivo experiments are more physiological and that therefore the current results do not support a role for the pro-inflammatory cytokines in aseptic loosening. We however favor the alternative that, in this case, the cell culture experiments can be more informative. We favor this alternative because the role of the pro-inflammatory cytokines may be obscured in vivo by compensation by other cytokines or by the low signal to noise ratio found in measurements of particle-induced osteolysis.

Figure 1

Neutralizing antibodies that target IL-1α (A), IL-1β (B), IL-6 (C), and TNFα (D) significantly inhibit osteoclast differentiation induced in vitro by titanium particles. Control cultures were incubated with non-immune goat IgG (A) or non-immune rat IgG1 (B). Osteoclast differentiation was assessed by measuring the number of TRAP-positive multinuclear cells/cm². N = 5–6 for all groups.

Figure 2

Recombinant IL-1 receptor antagonist significantly inhibits osteoclast differentiation induced in vitro by titanium particles. Osteoclast differentiation was assessed by measuring the number of TRAP-positive multinuclear cells/cm². N = 5–6 for all groups.

Figure 3

IL-1 receptor single knock out mice are not protected from titanium particle-induced osteolysis. Osteolysis was measured in the entire parietal bones (A) and in the suture regions (B). Data are presented as box plots showing the 10^th, 25^th, 50^th, 75^th, and 90^th, percentiles. In A, n = 16–18 for first and second groups, n = 48 for the third and fifth groups, and n = 34 for the fourth and sixth groups. In B, n = 8–9 for the first and second groups, n = 24 for the third and fifth groups, and n = 17 for the fourth and sixth groups.

Figure 4

IL-6 single knock out mice are not protected from titanium particle-induced osteolysis. Osteolysis was measured in the entire parietal bones (A) and in the suture regions (B). Data are presented as box plots showing the 10^th, 25^th, 50^th, 75^th, and 90^th, percentiles. In A, n = 22–28 for all groups. In B, n = 11–14 for all groups.

Figure 5

IL-1 receptor/IL-6 double knock out mice are not protected from titanium particle-induced osteolysis. Osteolysis was measured in the entire parietal bones (A) and in the suture regions (B). Data are presented as box plots showing the 10^th, 25^th, 50^th, 75^th, and 90^th, percentiles. In A, n = 26–36 for all groups. In B, n = 13–18 for all groups. Panel C shows genotyping of the IL-1 receptor/IL-6 double knock out mice. PCR amplicons from three representative wild type and three representative double knock mice are shown. Tail clip DNA was amplified with primers specific for the IL-1 receptor gene, the IL-6 gene, and the neomycin cassette, respectively.

Figure 6

TNF receptor 1/TNF receptor 2 double knock out mice are not protected from titanium particle-induced osteolysis. Osteolysis was measured in the entire parietal bones (A) and in the suture regions (B). Data are presented as box plots showing the 10^th, 25^th, 50^th, 75^th, and 90^th, percentiles. In A, n = 12 for the first and second groups and n = 24–26 for the third, fourth, fifth, and sixth groups. In B, n = 6 for the first and second groups and n = 12–13 for the third, fourth, fifth, and sixth groups.

Figure 7

Represenative examples of titanium-induced osteolysis in wild type and IL-1 receptor/IL-6 double knock out. The images are of the skull with the per cent osteolysis in the suture region that is closest to the median for each group. Arrows indicate some of the osteolytic areas.