Interleukin - 10이 interleukin-1β로 유도되는 골흡수에 미치는 효과

Abstract

[한글]

골흡수는 조골세포에서 유리되는 cytokine에 의하여 일어나며, 이때 cytosine 온 파골세포 전구세포의 분화를 활성화시켜 골흡수에 관여한다. 이 과정에서IL-1β(interleukin-1β)에 의한 골흡수는 조골세포로부터 생성되는 파골세포분화 촉진인자인 granulocyte ma

crophage-colony stimulating factor (GM-CSF), interleukin-6 (IL-6) 및 tumor necrosis factor-α (TNF-α)에 의하여 매개된다. 그러나 이들 cytoklne (GM-CSF, IL-6 및 TNF-α)은 조골세포 이외에 단핵세포에서도 생성될 뿐 아니라, interleukin-10 (IL-10)이 단핵

세포로부터 이들 cytosine의 생성을 억제하는 것으로 알려져 있으므로, IL-10이 조골세포에서 유리되는 GM-CSF, IL-6 및 TNF-α에도 영향을 미쳐 IL-1β에 의한 골흡수를 조절할 수 있을 것으로 생각된다. 이에 IL-10이 골흡수에 미치는 영향을 규명하고자 본 실험을 시행하였다.

(45)**Ca 를 임신 16-17일 된 마우스에 주사하고 1일 후에 회생시켜 태자 마우스 두개골을 분리하였다. 이렇게 분리한 (45)Ca로 표시된 마우스 두개골에서 1) lipopolysaccahride (LPS), TNF-α, IL-8, IL-lβ 및 IL-lα에 의한 골흡수 정도를 비교하였으며, 2) IL-10과 interferon-γ (IFN-γ)가 IL-1β에 의한 마우스 두개골 흡수에 미치는 영향 그리고 3) IL-10에 의한 IL-1β로 유도된 골수세포희의 파골세포형성 형 골세포 (bone cell)의 IL-6, TNF-α, GM-CSF 생성의 변화를 평가하였다. 골흡수 정도는 대조군 (cytoklne 비처치군)애 대한 실험군 (cytokine 처치군)의 (45)**Ca 유리올로 나타내었다. 또한 골수세포를 배양하여 형성되는 파골세포는 tartrate 저항성 산성인산분해효소를 염색하여 확인하였고, 각 cytokine의 농도는 효소면역흡착법 (enzyme linked immunosorbent method)으로 측정하여 다음과 같은 결과를 얻었다.

1. LPS 1 μg/ml에서 (45)**Ca 유리올은 1.14±0.07로서 이는 골흡수를 유도하였으며, 이의 농도에 따라 마우스 두개골로부터 (45)**Ca 유리올이 증가하였다.

2. IL-lα, IL-lβ 및 TNF-α 1 ng/ml에서 (45)**Ca 유리올은 각각 1.61±0.26, 1.77±0.30 및 1.20±0.15로서 이들 cytekine은 골흡수를 촉진하였으나, IL-8에 의한 (45)**Ca 유리올은 0.93±0.12로 이는 골흡수를 일으키지 않았다.

3. IL-10 (400 ng/ml) 및 IFN-γ (100 ng/ml)의 (45)**Ca 유리올은 각각 1.24±0.12 및 1.08±0.04로서 이들 cytosine은 IL-lβ (10 ng/ml)에 의하여 유도된 골흡수를 감소시켰다 ((45)**Ca 유리올 1.65±0.24).

4. IL-lβ (10 ng/ml)는 골수세포에서 tartrate 저항성 산성인산분해효소 양성 다핵세포의 수를 증가시켰으나 (20±11 개), IL-10 (400 ng/ml)을 IL-lβ (10 ng/ml)와 함께 처치한 경우에는 이들 세포의 수가 감소하였다 (2±2개).

5. IL-10 (400 ng/ml)은 IL-lβ (10 ng/ml)로 자극한 마우스 두개골세포에서 생성되는 IL-6, GM-CSF 및 TNF-α양에 영향을 주지 않았다.

이상의 실험결과로 볼 때 IL-10은 IL-1β에 의한 파골세포의 분화를 차단하여 골흡수를 억제하는 것으로 생각되며, 이러한 현상은 적어도 IL-10에 의한 조골세포로부터의 IL-6, GM-CSF 및 TNF-α의 생성 억제가 아닌 다른 기전에 의한 것으로 생각된다.

Effect of interleukin-10 on the bone resorption induced by interleukin- lβ

Yun-Jung Yu

Department of Dental Science, Graduate School, Yonsei University

(Directed by Syng-Ill Lee, D.D.S., Pf.D)

The cytokines released by osteoblasts induce bone resorption via the

differentiation of osteoclast precursors. In this process, interleukin-lβ(

IL-1β)-induced bone resorption is mediated by granulocyte macrophage-colony

stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor

α(TNF-α) released from osteoblasts. Since these cytokines (GM-CSF, IL-6, TNF-α)

are produced by not only osteoblasts but also monocytes, and interleukln-10(IL-10)

inhibits the secretion of these cytokines from monocytes, it may be speculated that

IL-10 could modulate the production of GM-CSF, IL-6, and TNF-α by osteoblasts,

then control IL-1β-induced bone resorption. Therefore, the aims of the present

study were to examine the effects of IL-10 on bone resorption.

The sixteen or seventeen-day pregnant ICR mice were injected with (45)**Ca and

sacrificed one day after injection. Then fetal mouse calvaria prelabeled with

(45)**Ca were dissected out. In order to confirm the degree of bone resorption,

mouse calvaria were treated with Lipopolysaccharide (LPS), TNF-α, IL-8, IL-1β,

and IL-lα. Then, IL-10 and interferon-γ (IFN-γ) were added to calvarial medium,

in an attempt to evacuate the effect of IL-1β-induced bone resorption. In

addition, osteoclasts formation in bone marrow cell cultures, and the concentration

of IL-6, TNF-α, and GM-CSF produced from mouse calvarial cells were investigated

in response to IL-1β alone and simultaneously adding of IL-1β and IL-10.

The degree of bone resorption was expressed as the ratio of (45)**Ca release (the

treated/the control). The osteoclasts in bone marrow cultures were identified by

tartrate resistant acid phosphatase (TRAP) stain and the concentration of the

cytokines was quantified using enzyme 1 inked immunosorbent method.

As results of these studios, bone resorption was induced by LPS (1 ng/ml : the

ratio of (45)**Ca release, 1.14±0.07). Also IL-1β (1 ng/ml), IL-1α (1 ng/71),

and TNF-α (1 ng/ml ) resulted in bone resorption (the ratios of (45)**Ca release,

1.61±0.26, 1.77±0.30, 1.20±0.15 respectively), but IL-8 did not (the ratio of

(45)**Ca release, 0.93±0.12). The ratios of (45)**Ca release in response to IL-10

(400 ng/ml) and IFN-γ (100 ng/ml) were 1.24±0.12 and 1.08±0.04 respectively,

hence these cytokines inhibited IL-lβ (1 ng/ml )-induced bone resorption (the

ratio of (45)**Ca release 1.65±0.24). While IL-lβ (1 ng/ml )increased the number

of TRAP positive multinulcleated cells in bone marrow cultured (20±11),

simultaneously adding IL-lβ (1 ng/ml) and IL-10 (400 ng/ml ) decreased the number

of these cells (2 ±7: 2). Nevertheless, IL-10 (400 ng/ml ) did not affect the

IL-6, GM-CSF, and TNF-α secretion from IL-1β (1 ng/ml )-activated mouse calvarial

cells.

From the above results, it may be suggested that IL-10 inhibites IL-1β-induced

osteoclast differentiation and bone resorption. However, the inhibitory effect of

IL-10 on the osteoclast formation seems to be mediated not by the reduction of

IL-6, GM-CSF, and TNF-α production, but by other mechanisms.

[영문]

The cytokines released by osteoblasts induce bone resorption via the differentiation of osteoclast precursors. In this process, interleukin-lβ(IL-1β)-induced bone resorption is mediated by granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-6 (IL-6), and tumor necrosis factor α(TNF-α) released from osteoblasts. Since these cytokines (GM-CSF, IL-6, TNF-α) are produced by not only osteoblasts but also monocytes, and interleukln-10(IL-10) inhibits the secretion of these cytokines from monocytes, it may be speculated that IL-10 could modulate the production of GM-CSF, IL-6, and TNF-α by osteoblasts, then control IL-1β-induced bone resorption. Therefore, the aims of the present study were to examine the effects of IL-10 on bone resorption.

The sixteen or seventeen-day pregnant ICR mice were injected with (45)**Ca and sacrificed one day after injection. Then fetal mouse calvaria prelabeled with (45)**Ca were dissected out. In order to confirm the degree of bone resorption, mouse calvaria were treated with Lipopolysaccharide (LPS), TNF-α, IL-8, IL-1β,

and IL-lα. Then, IL-10 and interferon-γ (IFN-γ) were added to calvarial medium, in an attempt to evacuate the effect of IL-1β-induced bone resorption. In addition, osteoclasts formation in bone marrow cell cultures, and the concentration of IL-6, TNF-α, and GM-CSF produced from mouse calvarial cells were investigated

in response to IL-1β alone and simultaneously adding of IL-1β and IL-10.

The degree of bone resorption was expressed as the ratio of (45)**Ca release (the treated/the control). The osteoclasts in bone marrow cultures were identified by tartrate resistant acid phosphatase (TRAP) stain and the concentration of the cytokines was quantified using enzyme 1 inked immunosorbent method.

As results of these studios, bone resorption was induced by LPS (1 ng/ml : the ratio of (45)**Ca release, 1.14±0.07). Also IL-1β (1 ng/ml), IL-1α (1 ng/71), and TNF-α (1 ng/ml ) resulted in bone resorption (the ratios of (45)**Ca release, 1.61±0.26, 1.77±0.30, 1.20±0.15 respectively), but IL-8 did not (the ratio of (45)**Ca release, 0.93±0.12). The ratios of (45)**Ca release in response to IL-10 (400 ng/ml) and IFN-γ (100 ng/ml) were 1.24±0.12 and 1.08±0.04 respectively, hence these cytokines inhibited IL-lβ (1 ng/ml )-induced bone resorption (the

ratio of (45)**Ca release 1.65±0.24). While IL-lβ (1 ng/ml )increased the number of TRAP positive multinulcleated cells in bone marrow cultured (20±11), simultaneously adding IL-lβ (1 ng/ml) and IL-10 (400 ng/ml ) decreased the number of these cells (2 ±7: 2). Nevertheless, IL-10 (400 ng/ml ) did not affect the

IL-6, GM-CSF, and TNF-α secretion from IL-1β (1 ng/ml )-activated mouse calvarial cells.

From the above results, it may be suggested that IL-10 inhibites IL-1β-induced osteoclast differentiation and bone resorption. However, the inhibitory effect of IL-10 on the osteoclast formation seems to be mediated not by the reduction of

IL-6, GM-CSF, and TNF-α production, but by other mechanisms.