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Interleukin-4 and Interleukin-13 Potentiate Interleukin-1 Induced Secretion of Interleukin-6 in Human Osteoblast-Like Cells

Posted on: Saturday, 4 September 2004, 06:00 CDT

Abstract

Formation of interleukin-6 (IL-6) in osteoblasts and bone marrow stromal cells is believed to regulate osteoclast recruitment. The anti-inflammatory cytokines interleukin-4 and -13 (IL-4 and IL-13) stimulate IL-6 production in human osteoblasts. We investigated the relative potencies, and synergistic effects, between IL-4, IL-13 and interleukin-1 (IL-1) on IL-6 formation in human osteoblast-like cells.

Isolated human osteoblast-like cells were incubated for 72 h in the presence of various concentrations of IL-4, IL-13 and IL-1, and IL-6 secretion was measured by ELISA. All cytokines stimulated the secretion of IL-6. The rank order of potency was IL1 [much greater than] IL-4> IL-13. There were no additive or synergistic effects between IL-4 and IL-13. However, co-stimulation with IL-1 and IL-4 resulted in a marked synergistic effect on IL-6 secretion. Co- stimulation with IL-1 and IL-13 gave a minor synergistic effect.

In conclusion, IL-4/13 synergistically potentiates IL-1 induced secretion of IL-6 in human osteoblast-like cells.

2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.

Keywords: IL-1; IL-4; IL-6; IL-13; Osteoblasts

Introduction

The process of bone remodeling constituted by ostcoclastic bonc- rcsorption and osteoblastic bone-formation involves a complex interplay between cells in, or adjacent to bone. Apart from endocrine systemic factors many of the cell interactions occur at a local level in a paracrinc or autocrinc manner [7], Both pro- and antiinflammatory cytokines derived from hematopoetic cells as well as mesenchymal cells within the bone are involved [18]. Apart from their role in normal mature bone turnover the cytokines are believed to interact in different pathological conditions as osteoporosis, Paget's disease, bone malignancies [23,28,36], fracturehealing [3] and inflammatory diseases e.g. rheumatoid arthritis [25]. Most studies in the literature concerning bone-tissue have focused on the effect of pro-inflammatory cytokines.

Interleukin-6 (IL-6), a multifunctional cytokine of the LIF/IL-6- family, was discovered and characterized in the late 1980s. IL-6 is produced by a variety of tissue cells and also many tumors [17,39]. Osteoblasts produce IL-6 [8,13], and also express IL-6 receptors indicating a possible autocrine function [5,38]. Osteotropic factors, systemic like PTH [13] and 1,25-(OH)2 Vit-D3 and local like TNF and IL-I, stimulate the production of IL-6 in osteoblasts [35]. The role of IL-6 in bone is predominantly ostcoclastogenic [35], where the activity seems to be dependent on either IL-6 receptors on osteoblasts or a complex of IL-6 and a soluble IL-6 receptor [2O]. However there is also evidence for IL-6 receptors expressed by osteoclasts [16].

Interleukin-1 (IL-I) together with tumor necrosis factor (TNF) are two of the most potent pro-inflammatory cytokines, both mainly derived from activated macrophages. They are markedly active in different inflammatory diseases, typically rheumatoid arthritis. While TNF appears to act more on a systemic level, IL-I seems to exert its effects more locally. IL-I has the capacity to induce bone resorption either by direct stimulation of osteoclasts or acting via osteoblasts stimulating expression of RANKL and IL-6 [2,10, 11,18].

The two cytokines interleukin-4 and interleukin-13 (IL-4 and IL- 13), both derive from Th2-CeIIs. They share many immunoregulatory effects and have been assigned as anti-inflammatory because of their ability to suppress pro-inflammatory interleukins, e.g. TNF, IL-I and IL-6 [12,24]. Previous studies have shown that they play a role in bone metabolism affecting both osteoblasts and osteoclasts. Osteoblastic effects includes inhibition of proliferation in osteoblastic cell cultures [15,33] and up regulation of IL-6 transcription and secretion in human osteoblasts [14]. Concerning osteoclast effects most data indicates an inhibitory effect on osteoclastogenesis [1,26,31], through different mechanisms including influence on the important RANK/RANKL pathway [27,4O]. However some studies has shown a positive [19] or bi-directional [37] effect on osteoclast formation. In vivo murine experiments has shown that recombinant IL-4 cause a decrease in bone turnover without affecting bone volume [32] while Lewis et al. already in 1993 reported severe osteoporosis in transgenic mice over expressing IL-4 [22].

In the present study we have investigated the relative potencies, and possible synergistic effects, between pro(IL-l/TNF) and anti- (IL-4/13) inflammatory cytokines on IL-6 secretion in human osteoblast-like cells.

Materials and methods

Materials

Alpha modification of Eagle's medium ([alpha]-MEM) and human recombinant IL-I, IL-4, 1L-13 and TNF-[alpha] and [beta] were purchased from Sigma Chemical Co., St. Louis, MO, USA. Penicillin, streptomycin, Lglutamine, trypsin-ethylenediamine tetraacetate (trypsin-EDTA), phosphate buffered saline (PBS) and fetal calf serum (FCS) were purchased from SVA, Uppsala, Sweden. The IL-6 ELISA kit was purchased from Pall Filtron AB, Solna, Sweden.

Isolation of human oateoblast-like cells

Trabecular bone was obtained from the proximal femoral shaft of patients undergoing total hip replacement (Table 1). The specimens were cul into small fragments, l 2 mm in diameter, thoroughly rinsed with PBS, and cultured in 75 cm^sup 2^ tissue culture flasks containing [alpha]-MEM supplemented with PEST (100 U/ml of penicillin, 100 mg/ml of streptomycin), amphotericin B (0.5 mg/ml), i.-glutaininc (2 mmol/1) and 10% FCS. After 4 weeks, the culture dishes were confluent with cells that had migrated from the trabecular bone. The cells were detached with trypsin-EDTA and seeded in multiwell culture dishes in which the subsequent experiments were performed as described below. Only first passage cells were used in these experiments. The project was approved by the hospitals local ethics committee.

Table 1

Donor data

Measurement of inlcrleiikin-6 secretion

Osteoblast-like cells were seeded in 24-wcll culture plates at a density of 30 000 cells/well and left to adhere in a-MEM supplemented with 10% FCS and antibiotics for 72 h. Then the cells were washed in PBS and the medium was changed to a-MEM with 0.1% FCS. Cylokines were added, except in control wells, in quadruple series for each sample. After 72 h of incubation the culture media were harvested and IL-6 levels analyzed by ELISA.

Text for enchtoxin

Control of the test media for lipopolysaccharide (LPS) contamination was performed using the limulus amebocyle lysate assay "E-Toxate" (SIGMA, Stockholm, Sweden) with a sensitivity of 0.03 EU/ ml according to the manufacturer. No traces of LPS were detected in our test media's.

Statistics

Student's Mest was used for statistical analysis and differences were considered significant when p-values were less than 0.05.

Results

In this investigation isolated human osteoblast-like cells were stimulated with various concentrations of interleukins (IL-4, IL- 13, IL-I and TNF) for 72 h and the IL-6 secretion was measured by ELISA in the culture medium. The anti-inflammatory cytokines, IL-4 and IL-13, as well as the pro-inflammatory cylokine IL-I stimulated the secretion of IL-6 in a dose-dependent manner. The stimulation effect with IL-I clearly exceeded that of the two anti-inflammatory cytokines (Fig. 1). To investigate possible interactions we performed costimulation experiments. In these a cytokine concentration level of 10 pM was chosen. This dose represents the approximate EC^sub 50^ concentration for each cytokine concerning stimulation of IL-6 secretion (data not shown). No additive or synergistic effect was detected on IL-6 secretion in human osteoblast-like cells when co-stimulating with IL-4 and IL-13 (Fig. 2a). However, co-stimulation with IL-I together with IL-4 resulted in a significant synergistic effect on IL-6 secretion (Fig. 2b). Co- stimulation with IL-I together with IL-13 also resulted in a synergistic effect on IL-6 secretion. This effect was not as pronounced as that exerted by IL-4, and was in some experiments not significantly evident (Fig. 2c). To investigate the specificity in the synergistic effect of IL-4/13 and IL-I we also performed co- stimulatory experiments with TNF. Also in these experiments there was a clear synergistic potcntiation of IL-6 secretion by IL-4/13 (Table 2). With these results we have demonstrated that pro- and anti-inflammatory cytokines, despite other contra-acting functions, synergistically enhance IL-6 secretion in human osteoblast-likc cells.

Discussion

Communication between osteoblasts and osteoclasts regulating normal bone turnover is referred to as the coupling mechanism. IL-6 has been a prime candidate in this communication system. Studies have pointed out IL6 as an important osteoclast activating factor in different conditions with pathological osteoclastic bone resorption, e.g. arthritis and bone malignancies [4,21]. The importance of the RAN K/RANKL axis in bone metabolism has been elucidated and is undisputable. Although it is very tempting to seek the answer in a single molecule or chain reaction of molecules biology is often more complicated. Like many other homeostatic systems bone remodeling is dependenton a variety of factors. Hence, it is known that IL-6 as well as RANK/RANKL are regulated by both pro- and anti-inflammatory cytokines. Furthermore there are data indicating interactions between IL-6 and the powerful RANK/RANKL pathway enhancing bone resorption in diseases like Paget's and possibly bone metastasis's or Myeloma [34],

Table 2

lL-4/13 + TNF-[alpha]/[beta]

In the present study we have focused on the action of, as well as the interaction between, pro- and antiinflammatory cytokines affecting IL-6 secretion in osteoblasts. As it is almost impossible to study absolute events, single or combined, in vivo we have chosen a well evaluated in vitro system of bone producing human osteoblast- like cells. Our results show that there is a clear difference in potency between the pro-inflammatory cytokine IL-I and the two anti- inflammatory cytokines IL-4 and IL-13 enhancing IL-6 secretion. When co-stimulating human osteoblast-like cells with IL-4 and IL-13 we found no synergistic or additive effects on IL-6 secretion. This can be explained by competition between the two anti-inflammatory cytokines sharing subunits on the receptor level.

Receptors for IL-4 and IL-13 have been investigated in different cell-systems and at least four different receptor subunits have been elucidated which in different constellations are believed to activate the JAK/STAT intracellular signaling pathway [6,9,3O]. There is evidence that IL-4 and IL-13 share these receptor subunits [29]. In human osteoblasts expression of IL-4R, IL-13R and IL-ISRa, but not IL-2Ryc, has been demonstrated and apparently the subunit IL- 4R plays a role in the IL4 and IL-13 induced 1L-6 formation [14].

Pro- and anti-inflammatory cytokines are proposed to work antagonistically. However co-stimulation by IL-4/13 together with IL- I, or TNF, clearly cause a synergistic stimulation of IL-6 secretion. The synergistic effect of IL-I and IL-13 in combination was repeated but not consistently significant. We know from earlier experiments that, although IL-4 and IL-13 share activation mechanisms through the same receptor subunits, there is a difference in potency where the effect of IL-4 is approximately 10 times that of IL-13. This can explain the difference in effect also in the present investigation and could be explained on the receptor level in terms of affinity. It might be tempting to look at pro- and antiinflammatory cytokines as two entities always counteracting each other. However in this study we show that the anti-inflammatory interleukins IL-4 and IL-13, otherwise known to suppress pro- inflammatory cytokines, here work synergistically with IL-I and TNF, promoting IL-6 secretion in human osteoblast-like cells.

We propose that IL-4 and IL-13 have the capacity to affect osteoclastogenesis not merely by promoting IL-6 secretion, but maybe more importantly by facilitating IL-6 secretion from human osteoblasts stimulated with other bone resorbing cytokines such as IL-I and TNF.

Acknowledgements

This work was supported by grants from the Swedish Cancer Society, the Swedish Rheumatism Association, and the Swedish Medical Research Council. We are grateful to Anna-Lena Johansson and Tomas Bjrkman for skilful technical support.

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Carl-Johan Silfverswrd a,*, Anders Frost a, Helena Brndstrm b, Olle Nilsson a, Osten Ljunggren b

a Departm\ent of Surgical Sciences, University of Uppsala, S-751 85 Uppsala, Sweden

b Department of Medical Sciences, University of Uppsala, S-751 85 Uppsala, Sweden

Received 15 May 2003; accepted 10 February 2004

* Corresponding author. Tel.: +46-18-611-0000; fax: +46-18-553- 601.

E-mail address: carl-johaii.silfversward@surgsci.uu.se (C.-J. SiIfverswrd).

Copyright Journal of Bone and Joint Surgery, Inc. Sep 2004

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