JBJS | Modulation of the Production of Cytokines in Titanium-Stimulated Human Peripheral Blood Monocytes by Pharmacological Agents. The Role of cAMP-Mediated Signaling Mechanisms*†

The Journal of Bone & Joint Surgery, Volume 79, Issue 10

Articles | October 01, 1997

Modulation of the Production of Cytokines in Titanium-Stimulated Human Peripheral Blood Monocytes by Pharmacological Agents. The Role of cAMP-Mediated Signaling Mechanisms*†

THEODORE A. BLAINE, M.D.‡; PAUL F. POLLICE, M.D.‡; RANDY N. ROSIER, M.D., PH.D.‡; PAUL R. REYNOLDS, PH.D.‡; J. EDWARD PUZAS, PH.D.‡; REGIS J. O'KEEFE, M.D.‡, ROCHESTER, NEW YORK

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Investigation performed at the Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, Rochester

The Journal of Bone & Joint Surgery. 1997; 79:1519-28

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Abstract

Cytokines secreted by activated macrophages play a role in the development of osteolysis adjacent to prosthetic joints. To determine whether the synthesis of cytokines can be inhibited by pharmacological agents, we studied the role of the cAMP-protein kinase A signal transduction pathway in the synthesis of interleukin-6 and tumor necrosis factor-a and examined the effect of potential pharmacological regulators of this pathway in human peripheral blood monocytes stimulated with titanium particles. Dibutyryl cAMP enhanced the synthesis of interleukin-6 by titanium-stimulated monocytes and resulted in a marked increase (maximum, seventyfold) in the synthesis of interleukin-6 even in the absence of titanium particles. However, the active analogs (agonists) of cAMP, dibutyryl cAMP and Sp cAMP, inhibited the production of tumor necrosis factor-a by titanium-stimulated monocytes (the maximum effects resulted in complete inhibition), while the cAMP antagonist, Rp cAMP, enhanced the production of tumor necrosis factor-a. Additional agents that alter the intracellular levels of cAMP were examined for their effects on the synthesis of cytokines. Prostaglandins E₁ and E₂ were potent inhibitors of the synthesis of tumor necrosis factor-a but stimulated the synthesis of interleukin-6. In contrast, indomethacin enhanced the stimulatory effects of titanium particles on tumor necrosis factor-a, resulting in a more than threefold increase in the maximum levels of tumor necrosis factor-a. Phosphodiesterase inhibitors, such as isobutyryl methylxanthine and pentoxifylline, which increase intracellular levels of cAMP, caused a decrease in the production of tumor necrosis factor-a and an increase in the production of interleukin-6. In contrast, the fluoroquinolone antibiotic ciprofloxacin, which is also a phosphodiesterase inhibitor, caused a dose-dependent inhibition of the synthesis of both tumor necrosis factor-a and interleukin-6 by titanium-stimulated monocytes, suggesting that ciprofloxacin suppresses the synthesis of interleukin-6 through a mechanism that is independent of cAMP.CLINICAL RELEVANCE: Pharmacological agents, in combination with efforts at reducing the generation of wear debris, may lead to novel therapeutic strategies to prevent the loosening of implants. Alteration of the synthesis of cytokines in response to pharmacological manipulation of the cAMP-protein kinase A signaling pathway suggests that these events are regulated by normal physiological mechanisms in titanium-stimulated human peripheral blood monocytes rather than by an irreversible toxic response. The differential response of tumor necrosis factor-a and interleukin-6 to agents that alter intracellular levels of cAMP indicates that these two cytokines are independently regulated. Thus, pharmacological strategies to inhibit the release of cytokines may need to involve multiple agents to inhibit diverse regulatory pathways. Basic-science studies identifying the pathways involved in titanium-mediated synthesis of cytokines are essential for the efficient design and selection of inhibitory agents.

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Introduction

Introduction | Materials and Methods | Results | Discussion | References

Despite improvements in the design and manufacture of prosthetic implants, osteolysis and loosening remain major complications of joint replacement. In one recent study²⁷ osteolysis was found adjacent to 57 per cent (thirty-nine) of sixty-nine femoral implants within six years postoperatively, and in another study³ extensive osteolysis was found adjacent to 16 per cent (thirty-one) of 199 acetabular cups that had been implanted without cement within five years previously. Other authors have demonstrated that microscopic debris, including particles of polyethylene and metal, is released from implants and incites an inflammatory response that results in bone resorption^7,12,30. Central to this process is the tissue macrophage, which responds to the phagocytosis of particulate debris with the synthesis and secretion of cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor-a, which mediate the inflammatory process and stimulate bone resorption^13,21.

A recent study demonstrated that the phagocytosis of particulate material is associated with the expression of the major histocompatibility complex HLA-DR on macrophages²⁰. HLA-DR is an immunoglobulin-like molecule that is involved in the presentation of antigen to T lymphocytes. Since the expression of HLA-DR is part of the normal physiological response of immunologically activated macrophages, its presence in macrophages after phagocytosis of particulate debris suggests that the expression of cytokines in these cells results from physiological activation rather than from a non-specific, toxic response. This theory is further supported by evidence indicating that the secretion of tumor necrosis factor-a and interleukin-6 by titanium-stimulated monocytes requires the novel synthesis of both messenger RNA (mRNA) and protein⁶.

One possible method for preventing aseptic loosening of total joint replacements is the pharmacological inhibition of activated macrophages. Since physiological responses are typically mediated through the activation of signaling pathways within the cell, pharmacological agents designed to alter intracellular signals may affect the production of cytokines by macrophages. The two major signaling pathways include the protein kinase A and the protein kinase C-mediated pathways. Activation of these pathways results in a cascade of intracellular events that ultimately leads to either enhancement or suppression of gene expression^2,36.

Cyclic AMP, produced intracellularly by adenylate cyclase, binds to protein kinase A and initiates intracellular signals mediated by this enzyme^36,37. In its inactive state, protein kinase A is part of a holoenzyme complex composed of two catalytic subunits bound to a dimeric regulatory subunit^8,22. The binding of four cAMP molecules to the regulatory subunit dimer releases both catalytic subunits, enabling them to phosphorylate their substrates^8,9,22. Active analogs (agonists) of cAMP, including dibutyryl cAMP and Sp cAMP, bind to the regulatory subunits and stimulate release of the catalytic subunits from the holoenzyme⁹. In contrast, the cAMP antagonist Rp cAMP binds to the holoenzyme but prevents dissociation and activation of the catalytic subunit⁹.

The dissociated catalytic subunits mediate a series of protein phosphorylations, resulting in the suppression or enhancement of specific genes^8,22. Although the proteins regulating the expression of cAMP-protein kinase A target genes are not completely known, both positive (cAMP-responsive element binding protein) and negative (cAMP-responsive element modulator) regulators of gene expression have been identified^8,22. Thus, it has been found that the expression of certain genes, such as interleukin-6, leukemia inhibitory factor, and c-fos, are stimulated by cAMP^8,14, while the expression of tumor necrosis factor-a, similar to that of other genes, is suppressed by agents that increase the intracellular levels of cAMP^{4,15,16,26,39,43,44}. Since elevated levels of tumor necrosis factor-a are found in a variety of disease states, including septic shock, tumor and acquired immunodeficiency syndrome-related cachexia, and hypercalcemia associated with malignant tumors, clinical trials have been initiated¹⁰ to examine the effects of pharmacological agents designed to alter the intracellular levels of cAMP.

Tumor necrosis factor-a may also play a pivotal role in the stimulation of osteolysis associated with prosthetic loosening. Tumor necrosis factor-a is the first cytokine to be induced by particles of debris⁶, and it may directly stimulate the osteoclast to resorb bone^1,5,13,26,31. We therefore studied the mechanism of signal transduction involved in the synthesis of tumor necrosis factor-a in titanium particle-stimulated cultures of human peripheral blood monocytes as well as the degree to which tumor necrosis factor-a can be inhibited by pharmacological agents that affect signal transduction. These agents were found to decrease the synthesis of tumor necrosis factor-a but to increase the synthesis of interleukin-6, indicating the complexity of the release of cytokines in wear-debris-stimulated monocytes.

*One or more of the authors may receive benefits for personal or professional use from a commercial party related directly or indirectly to the subject of this article. Funds were received in total or partial support of the research or clinical study presented in this article. The funding sources were Orthopaedic Research and Education Foundation Grant 94-002 (T. A. B.), Orthopaedic Research and Education Foundation Career Development Award (R. J. O'K.), and National Institutes of Health Grant R29 44220 (R. J. O'K.).

†Read in part at the Annual Meeting of the Orthopaedic Research Society, Atlanta, Georgia, February 19, 1996. This paper received a 1997 American Orthopaedic Association-Zimmer Resident Research Award (T. A. B.).

‡Department of Orthopaedics, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 665, Rochester, New York 14642. The e-mail address for Dr. O'Keefe is rokeefe@orthomail.ortho.rochester.edu.

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Fig. 1-A Graphs showing the effect of dibutyryl cAMP on the release of tumor necrosis factor-a (TNF-a) (Fig. 1-A) and interleukin-6 (IL-6) (Fig. 1-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of dibutyryl cAMP in the presence and absence of titanium particles, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated and titanium-free cultures and the respective dibutyryl cAMP-free controls. The symbols in the boxes denote the p value for the comparisons between the titanium-free cultures and the titanium-stimulated cultures for each dose of dibutyryl cAMP. § = p < 0.0001, * = p < 0.005, + = p < 0.01, # = p < 0.001, and ? = p < 0.05. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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Fig. 1-B Graphs showing the effect of dibutyryl cAMP on the release of tumor necrosis factor-a (TNF-a) (Fig. 1-A) and interleukin-6 (IL-6) (Fig. 1-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of dibutyryl cAMP in the presence and absence of titanium particles, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated and titanium-free cultures and the respective dibutyryl cAMP-free controls. The symbols in the boxes denote the p value for the comparisons between the titanium-free cultures and the titanium-stimulated cultures for each dose of dibutyryl cAMP. § = p < 0.0001, * = p < 0.005, + = p < 0.01, # = p < 0.001, and ? = p < 0.05. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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Fig. 2 Graph showing the effect of Sp cAMP and Rp cAMP on the release of tumor necrosis factor-a (TNF-a) by human peripheral blood monocytes. Isolated monocytes were treated with one-millimolar Sp cAMP or Rp cAMP in the presence or absence of titanium particles. The mean concentration of tumor necrosis factor-a was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated and titanium-free cultures and the respective cAMP-free controls. The symbols in the boxes denote the p value for the comparisons between the titanium-free cultures and the titanium-stimulated cultures for each type of treatment. § = p < 0.0001, + = p < 0.01, and * = p < 0.005. The I-bars represent the standard error of the mean.

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Fig. 3-A Graphs showing the effect of prostaglandin E2 on the release of tumor necrosis factor-a (TNF-a) (Fig. 3-A) and interleukin-6 (IL-6) (Fig. 3-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of prostaglandin E2 in the presence of titanium particles, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated cultures and the prostaglandin E2-free control. The symbols in the boxes denote the p value for the comparisons between the titanium-free control cultures and the titanium-stimulated control cultures. § = p < 0.0001, @ = p < 0.025, and * = p < 0.005. The I-bars represent the standard error of the mean.

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Fig. 3-B Graphs showing the effect of prostaglandin E2 on the release of tumor necrosis factor-a (TNF-a) (Fig. 3-A) and interleukin-6 (IL-6) (Fig. 3-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of prostaglandin E2 in the presence of titanium particles, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated cultures and the prostaglandin E2-free control. The symbols in the boxes denote the p value for the comparisons between the titanium-free control cultures and the titanium-stimulated control cultures. § = p < 0.0001, @ = p < 0.025, and * = p < 0.005. The I-bars represent the standard error of the mean.

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Fig. 4-A Graphs showing the effect of indomethacin on the release of tumor necrosis factor-a (TNF-a) (Fig. 4-A) and interleukin-6 (IL-6) (Fig. 4-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of indomethacin, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated and titanium-free cultures and their respective indomethacin-free control. The symbols in the boxes denote the p value for the comparisons between the titanium-free cultures and the titanium-stimulated cultures for each dose of indomethacin. § = p < 0.0001, @ = p < 0.025, # = p < 0.001, ? = p < 0.05, + = p < 0.01, and * = p < 0.005. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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Fig. 4-B Graphs showing the effect of indomethacin on the release of tumor necrosis factor-a (TNF-a) (Fig. 4-A) and interleukin-6 (IL-6) (Fig. 4-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of indomethacin, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured with enzyme-linked immunosorbent assay. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated and titanium-free cultures and their respective indomethacin-free control. The symbols in the boxes denote the p value for the comparisons between the titanium-free cultures and the titanium-stimulated cultures for each dose of indomethacin. § = p < 0.0001, @ = p < 0.025, # = p < 0.001, ? = p < 0.05, + = p < 0.01, and * = p < 0.005. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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Fig. 5-A Graphs showing the effect of ciprofloxacin and pentoxifylline on the release of tumor necrosis factor-a (TNF-a) (Fig. 5-A) and interleukin-6 (IL-6) (Fig. 5-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of ciprofloxacin or pentoxifylline, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated cultures and the titanium-stimulated ciprofloxacin or pentoxifylline-free controls. The symbols in the boxes denote the p value for the comparisons between the titanium-free control cultures and the titanium-stimulated control cultures. * = p < 0.005, ? = p < 0.05, @ = p < 0.025, and + = p < 0.01. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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Fig. 5-B Graphs showing the effect of ciprofloxacin and pentoxifylline on the release of tumor necrosis factor-a (TNF-a) (Fig. 5-A) and interleukin-6 (IL-6) (Fig. 5-B) by human peripheral blood monocytes. Isolated monocytes were treated with various concentrations of ciprofloxacin or pentoxifylline, and the mean concentration of tumor necrosis factor-a or interleukin-6 was measured. The symbols at the top of the bars denote the p value for the comparisons between the titanium-stimulated cultures and the titanium-stimulated ciprofloxacin or pentoxifylline-free controls. The symbols in the boxes denote the p value for the comparisons between the titanium-free control cultures and the titanium-stimulated control cultures. * = p < 0.005, ? = p < 0.05, @ = p < 0.025, and + = p < 0.01. The I-bars represent the standard error of the mean; in some cases, the standard error is too small to be observed on the graphs.

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TABLE I EFFECT OF PROSTAGLANDINS ON THE RELEASE OF TUMOR NECROSIS FACTOR-a BY HUMAN PERIPHERAL BLOOD MONOCYTES

*P < 0.0001 for the comparison with the control cultures.†P < 0.005 for the comparison with the control cultures.‡P < 0.0001 for the comparison with the titanium-stimulated cultures without prostaglandin.§P < 0.005 for the comparison with the titanium-stimulated cultures without prostaglandin.

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)
Control	153 ± 6
Titanium	1394 ± 47*
10^-7-M prostaglandin E2 with titanium	220 ± 7†‡
10^-7-M prostaglandin E1 with titanium	329 ± 26†‡
10^-7-M prostaglandin F2a with titanium	777 ± 76†§

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TABLE I EFFECT OF PROSTAGLANDINS ON THE RELEASE OF TUMOR NECROSIS FACTOR-a BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)
Control	153 ± 6
Titanium	1394 ± 47*
10^-7-M prostaglandin E2 with titanium	220 ± 7†‡
10^-7-M prostaglandin E1 with titanium	329 ± 26†‡
10^-7-M prostaglandin F2a with titanium	777 ± 76†§

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TABLE II EFFECT OF ISOBUTYRYL METHYLXANTHINE ON THE RELEASE OF TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-6 BY HUMAN PERIPHERAL BLOOD MONOCYTES

* P < 0.005 for the comparison with the titanium-free controls.†P < 0.0001 for the comparison with the titanium-free controls.‡P < 0.005 for the comparison with the titanium-stimulated cultures without isobutyryl methylxanthine. There was no significant difference compared with the titanium-free control cultures.§Not significant compared with the titanium-stimulated cultures without isobutyryl methylxanthine.

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)	Concentration of Interleukin-6 (pg/ml)
Control	87 ± 16	719 ± 127
Titanium	865 ± 119†	10,020 ± 440‡
Titanium with 100-µM isobutyryl methylxanthine	76 ± 8 ‡	10,175 ± 508†§

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TABLE II EFFECT OF ISOBUTYRYL METHYLXANTHINE ON THE RELEASE OF TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-6 BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)	Concentration of Interleukin-6 (pg/ml)
Control	87 ± 16	719 ± 127
Titanium	865 ± 119†	10,020 ± 440‡
Titanium with 100-µM isobutyryl methylxanthine	76 ± 8 ‡	10,175 ± 508†§

Materials and Methods

Introduction | Materials and Methods | Results | Discussion | References

Metal Particles and Pharmacological Agents

Titanium particles (one to three micrometers in diameter) were purchased from Johnson Matthey Chemicals (Ward Hill, Massachusetts). The particles were characterized and used in monocyte culture studies, as previously described⁶. More than 90 per cent of the particles were less than five micrometers in diameter, a size consistent with phagocytosis and activation of macrophages²¹. Particle suspensions were proved to be free of endotoxin by Limulus assay (Biowhittaker, Gaithersburg, Maryland).

Ciprofloxacin was provided by Bayer (Pharmaceuticals Division, West Haven, Connecticut). Pentoxifylline, isobutyryl methylxanthine, dibutyryl cAMP, indomethacin, and the prostaglandin compounds were obtained from Sigma Chemical (St. Louis, Missouri). The adenosine 3',5'-cyclic monophosphorothioate isomers, the Sp diastereomer of adenosine 3',5'-phosphorothioate (Sp cAMP) and the Rp diastereomer of adenosine 3',5'-phosphorothioate (Rp cAMP), were from Ruth Langhorst (La Jolla, California).

Isolation and Culture of Monocytes

Human monocytes and lymphocytes were isolated from the buffy-coat layer obtained from blood donated to the American Red Cross, as previously described⁶. The monocytes were separated from the lymphocytes by adherence in a thirty-milliliter tissue-culture flask for one hour at 37 degrees and 7.5 per cent CO₂. After an additional fifteen-minute incubation in three milliliters of phosphate-buffered saline solution containing one-millimolar EDTA and 10 per cent fetal bovine serum, the monocytes were gently scraped from the flask. The monocytes were washed twice with sterile phosphate-buffered saline solution and were resuspended in RPMI-1640 medium (Sigma Chemical) (5 x 10⁶ cells per milliliter) containing 10 per cent fetal bovine serum. One milliliter of medium was then added to each well in twenty-four-well tissue-culture plates, and the cells were permitted to adhere for thirty to sixty minutes before the experiments were initiated. For the experiments in which the effect of pharmacological agents was examined, the cultures were pretreated for thirty minutes with the appropriate pharmacological agent before stimulation with a concentration of 10⁷ particles of titanium per milliliter. This concentration was chosen as it has been shown previously to maximally stimulate the secretion of cytokines in this culture system⁶. Cells were examined with light microscopy and the trypan-blue exclusion method to verify phagocytosis of the particles and cell viability.

Assay for Cytokines

After eighteen hours of incubation, the titanium particles were removed from the conditioned medium by centrifugation. Supernatants were stored at -70 degrees Celsius and thawed once for measurement of the cytokines. The levels of interleukin-6 and tumor necrosis factor-a were measured with commercially available enzyme-linked immunosorbent assay kits (Biosource International, Camarillo, California, and Pharmingen, San Diego, California) with spectroscopy, as previously described⁶, on an automated microELISA plate reader (model MRS80, Dynatech Labs, Alexandria, Virginia). The absorbance (the results on optical densitometry) was compared with the absorbance of standard concentrations of the appropriate cytokine (Biosource International).

Statistical Analysis

Statistical comparisons were performed with the Student t test for experiments consisting of only one control and one treatment group, and they were performed with analysis of variance for experiments involving multiple groups. A p value of less than 0.05 was considered significant. All of the experiments consisted of groups of four cultures.

Results

Introduction | Materials and Methods | Results | Discussion | References

Effect of cAMP on Production of Cytokines

Monocytes in monolayer culture were treated with a maximally stimulatory concentration of titanium particles⁶ (10⁷ particles per milliliter) for eighteen hours in the presence or absence of dibutyryl cAMP. In the absence of dibutyryl cAMP, the titanium particles caused an approximately fivefold increase in the levels of tumor necrosis factor-a (Fig. 1-A). In the presence of dibutyryl cAMP, there was a dose-dependent inhibition of the production of tumor necrosis factor-a in the titanium-stimulated cultures (Fig. 1-A). Maximum inhibition (a 66 per cent decrease) of the levels of tumor necrosis factor-a occurred at a concentration of 0.3-millimolar dibutyryl cAMP in the titanium-stimulated cultures. The basal level of production of tumor necrosis factor-a in the unstimulated cultures was slightly elevated at intermediate concentrations of dibutyryl cAMP (0.03 and 0.1 millimolar), although the highest concentration of dibutyryl cAMP that was examined (0.3 millimolar) did not alter the basal level of production of tumor necrosis factor-a.

In contrast, dibutyryl cAMP increased the basal level of interleukin-6 in monocyte cultures in a dose-dependent manner (Fig. 1-B). In the absence of titanium particles, a concentration of 0.3-millimolar dibutyryl cAMP per milliliter increased the levels of interleukin-6 maximally (seventy times). At low concentrations of dibutyryl cAMP the effect of titanium particles on the production of interleukin-6 was enhanced, while at higher concentrations the effect was blunted and similar levels of interleukin-6 were observed in the presence or absence of titanium particles. At high concentrations of dibutyryl cAMP, the levels of interleukin-6 were considerably greater than those observed in titanium-stimulated control cultures, suggesting that the markedly stimulatory effect of dibutyryl cAMP masked the effect of the titanium particles.

The experiments demonstrate the responsiveness of human peripheral blood monocytes to pharmacological manipulation and suggest that the effects of titanium on the production of tumor necrosis factor-a and interleukin-6 may be differentially modulated by agents that alter intracellular levels of cAMP.

To further confirm the role of cAMP in the modulation of synthesis of cytokines by human peripheral blood monocytes, we examined the effects of one-millimolar concentrations of the cAMP isomers Sp cAMP (an agonist) and Rp cAMP (an antagonist) on levels of tumor necrosis factor-a and interleukin-6. Sp cAMP, similar to dibutyryl cAMP, completely inhibited the production of tumor necrosis factor-a elicited by titanium particles. In contrast, Rp cAMP, which interferes with protein kinase A signaling, resulted in an approximately 50 per cent increase in basal and titanium-stimulated levels of tumor necrosis factor-a in human peripheral blood monocyte cultures (Fig. 2). Similar to dibutyryl cAMP, Sp cAMP substantially increased the basal level of interleukin-6 as well as enhanced the synthesis of interleukin-6 by titanium-stimulated monocytes. Basal levels of interleukin-6 and the response of monocytes to titanium particles were not altered by Rp cAMP.

Collectively, these data demonstrate that cAMP inhibits the secretion of tumor necrosis factor-a and stimulates the secretion of interleukin-6 by titanium-stimulated monocytes.

Prostaglandins and Production of Cytokines

Prostaglandins, in part, mediate their effects through modulation of intracellular levels of cAMP. Prostaglandins E₁ and E₂ are potent stimulators of intracellular cAMP production, whereas the effects of prostaglandin F_2a are primarily mediated through activation of protein kinase C^25,32,42. While equivalent (10^-7-molar) doses of prostaglandin E₁ and prostaglandin E₂ resulted in a 76 and 84 per cent reduction, respectively, in the production of tumor necrosis factor-a by titanium-stimulated monocytes, prostaglandin F_2a resulted in only a moderate (44 per cent) reduction in the levels of tumor necrosis factor-a (Table 1).

The effect of prostaglandin E₂ on titanium-stimulated monocytes was dose-dependent (Fig. 3-A). Maximum inhibition of the production of tumor necrosis factor-a occurred at a 10^-6-molar concentration, resulting in an 88 per cent reduction in the levels compared with the levels produced by cells stimulated with titanium alone. In contrast, the levels of interleukin-6 produced by titanium-stimulated monocytes were increased in cultures treated with prostaglandin E₂ (Fig. 3-B); this finding is consistent with the observed increases in the levels of interleukin-6 in cultures treated with dibutyryl cAMP. The effect was first observed at a 10^-9-molar concentration of prostaglandin E₂ and was maximum at a 10^-7-molar concentration, at which point the level of interleukin-6 was increased 60 per cent compared with that in the titanium-stimulated cultures in the absence of prostaglandin E₂.

Prostaglandin Inhibitors and Production of Cytokines

Indomethacin and other non-steroidal anti-inflammatory agents indirectly inhibit intracellular levels of cAMP by blocking the production of prostaglandins through inhibition of cyclo-oxygenase activity³⁸. The presence of indomethacin enhanced the stimulatory effect of titanium particles on the production of tumor necrosis factor-a by peripheral blood monocytes. The effect was dose-dependent and occurred at typical pharmacological concentrations of this agent (Fig. 4-A). The secretion of tumor necrosis factor-a increased substantially at a one-micromolar concentration of indomethacin, and it increased the most (more than threefold compared with the production in control cultures stimulated with titanium alone) at a ten-micromolar concentration.

Indomethacin had a biphasic effect on the levels of interleukin-6 in titanium-stimulated monocyte cultures (Fig. 4-B). At low (0.001, 0.01, and 0.1-micromolar) concentrations of indomethacin, the levels of interleukin-6 were decreased, while higher concentrations of indomethacin resulted in an increase in the levels of interleukin-6 in titanium-stimulated monocytes. Thus, the effect of indomethacin on the synthesis of interleukin-6 appears to be multifactorial and dependent on the concentration of the drug. Since indomethacin would be expected to decrease the synthesis of interleukin-6 by decreasing the synthesis of prostaglandin, the elevation of the levels of interleukin-6 at high concentrations of indomethacin suggests another mechanism of action, independent of the synthesis of prostaglandin and the intracellular levels of cAMP.

In unstimulated monocytes, indomethacin did not alter the basal level of tumor necrosis factor-a and it decreased the level of interleukin-6 only slightly (Figs. 4-A and 4-B), suggesting a minor role for prostaglandin metabolism in the regulation of the synthesis of cytokines in the absence of titanium particles.

Phosphodiesterase Inhibitors and Production of Cytokines

Phosphodiesterase inhibitors increase concentrations of cAMP by preventing its metabolism to its inactive monophosphate form. At a 100-micromolar concentration, the phosphodiesterase inhibitor isobutyryl methylxanthine completely blocked the stimulation of production of tumor necrosis factor-a by human peripheral blood monocytes treated with titanium particles. In contrast, isobutyryl methylxanthine did not alter the synthesis of interleukin-6 by peripheral blood monocytes treated with titanium particles (Table II).

Other agents that possess phosphodiesterase inhibitory activity and are frequently used clinically include pentoxifylline and ciprofloxacin. Pentoxifylline resulted in a marked, dose-dependent decrease in the production of tumor necrosis factor-a by titanium-stimulated monocytes (Fig. 5-A). Substantial inhibition occurred at a concentration of thirty micrograms per milliliter and complete inhibition occurred at a concentration of 1000 micrograms per milliliter. The levels of tumor necrosis factor-a in these cultures were lower than those observed in the control cultures. Pentoxifylline resulted in a dose-dependent increase in the synthesis of interleukin-6 in cultures of titanium-stimulated monocytes (Fig. 5-B), a finding that is consistent with the observed effects of elevated levels of cAMP on the synthesis of interleukin-6.

Ciprofloxacin also resulted in a marked, dose-dependent decrease in the production of tumor necrosis factor-a in titanium-stimulated cultures (Fig. 5-A). Similar to the effects observed with pentoxifylline, the maximum inhibition resulted in levels of tumor necrosis factor-a that were lower than those in the control cultures. However, unlike pentoxifylline, ciprofloxacin also resulted in a dose-dependent decrease in the production of interleukin-6 in titanium-stimulated cultures (Fig. 5-B). Significant inhibition first occurred at a concentration of 100 micrograms per milliliter, and the maximum effects resulted in complete inhibition of the titanium-stimulated increase in the levels of interleukin-6. The effects of ciprofloxacin or pentoxifylline were not due to cell toxicity as the cells remained viable when examined with the trypan-blue exclusion method at all of the concentrations examined.

Discussion

Introduction | Materials and Methods | Results | Discussion | References

The results of the present study demonstrate that the synthesis of interleukin-6 and tumor necrosis factor-a by peripheral blood monocytes is regulated by the cAMP-mediated intracellular signaling pathway. The binding of cAMP releases the catalytic subunit from the protein kinase A holoenzyme, leading to the phosphorylation of target proteins and resulting in the enhancement or suppression of specific genes^8,22. Active analogs (agonists) of cAMP inhibited the synthesis of tumor necrosis factor-a and increased the synthesis of interleukin-6. Furthermore, agents that alter the intracellular levels of cAMP, including the non-steroidal anti-inflammatory agent indomethacin, prostaglandins, and phosphodiesterase inhibitors, all regulated the synthesis of cytokines by monocytes activated by titanium particles. These data confirm the role of physiological signaling mechanisms in the synthesis and secretion of cytokines by wear-debris-stimulated monocytes. Although other important cytokines and growth factors, including interleukin-1, were not examined in this study, it is likely that they also respond to agents that modulate intracellular signaling events.

Although marked improvements in the design of prostheses have limited the release of wear debris^11,12,23,29, osteolysis secondary to particles of material continues to be a major complication of joint replacement. High concentrations (10⁹ particles per milliliter) of wear debris, including polyethylene, polymethylmethacrylate, and metal, have been found in the periprosthetic membrane³⁰. Although the mechanisms by which particles of debris ultimately lead to bone resorption around joint prostheses remain unclear, cytokines produced locally by macrophages in the periprosthetic membrane are thought to be the primary stimulus^{7,13,17,18,21,24,34}. As demonstrated in the present study, the release of cytokines by monocytes occurs in the absence of lymphocytes, fibroblasts, osteoblasts, and the mechanical stimulation caused by a loose implant. Despite recognition of the role of wear-debris-induced release of cytokines in periprosthetic bone resorption, the design of pharmacological agents to interfere with this process has been limited because of the paucity of information regarding the basic mechanisms involved.

Previously the mechanisms of synthesis of cytokines by monocytes exposed to external stimuli were investigated in the model of septic shock with the focus on tumor necrosis factor-a. In lipopolysaccharide-stimulated peripheral blood monocytes, phosphodiesterase inhibitors have been shown to inhibit the production of tumor necrosis factor-a, presumably through their ability to increase levels of cAMP^{16,33,35,41,43,44}. We similarly observed a marked inhibition of titanium-induced production of tumor necrosis factor-a in the presence of agents designed to increase intracellular concentrations of cAMP. Two cAMP active analogs, dibutyryl cAMP and Sp cAMP, markedly inhibited the secretion of tumor necrosis factor-a. In contrast, Rp cAMP, a competitive inhibitor (antagonist) of cAMP that binds to and inactivates protein kinase A, increased both the basal and the titanium-stimulated synthesis of tumor necrosis factor-a, further confirming the role of this signaling pathway in the regulation of secretion of tumor necrosis factor-a by titanium-stimulated monocytes.

The prostaglandins also were potent inhibitors of the accumulation of tumor necrosis factor-a in monocyte cultures treated with titanium particles. The effect was dose-dependent and corresponded with the capacity of the various prostaglandin compounds to increase the intracellular levels of cAMP, with prostaglandin E₁ and prostaglandin E₂ being more potent inhibitors than prostaglandin F_2a. Prostaglandins E₁ and E₂ are potent stimulators of adenylate cyclase, whereas the effects of prostaglandin F_2a are primarily mediated through activation of protein kinase C^25,32,42. Since activated monocytes synthesize prostaglandins^28,40, the effect of indomethacin, a non-steroidal anti-inflammatory agent and a potent cyclo-oxygenase inhibitor, was examined in cultures of human peripheral blood monocytes stimulated with titanium particles. Indomethacin increased the production of tumor necrosis factor-a more than threefold, suggesting that prostaglandins may act as autocrine regulators of the synthesis of cytokines by peripheral blood monocytes.

In contrast to the effects on tumor necrosis factor-a, dibutyryl cAMP and Sp cAMP increased both basal and titanium-stimulated secretion of interleukin-6 by peripheral blood monocytes, suggesting that the activation of protein kinase A enhances the synthesis of interleukin-6 even in the absence of monocyte stimulation with titanium particles. Although dibutyryl cAMP enhanced the release of interleukin-6 in monocyte cultures treated with titanium particles, this was observed only at low concentrations of dibutyryl cAMP. At higher concentrations, the pronounced elevation of levels of interleukin-6 by dibutyryl cAMP alone obscured the effect of titanium particles on the release of this cytokine. The production of interleukin-6 in response to titanium particles was also enhanced in cultures treated with prostaglandin E₂ and the phosphodiesterase inhibitor pentoxifylline, further suggesting a role for cAMP in the enhancement of the monocyte response to titanium particles. These findings are consistent with those of a recent study that demonstrated enhanced synthesis of interleukin-6 by osteoblasts treated with cAMP-elevating agents¹⁴. The data demonstrate that the biosynthesis and secretion of tumor necrosis factor-a and interleukin-6 are differentially regulated by agents that alter intracellular levels of cAMP.

All of the agents tested consistently implicated a cAMP-dependent mechanism for the regulation of secretion of tumor necrosis factor-a, but the findings with regard to interleukin-6 were less uniform. The levels of interleukin-6 in culture were not decreased by Rp cAMP, an agent designed to inhibit cAMP-mediated signaling. Similarly, while all three phosphodiesterase inhibitors examined had an identical effect on tumor necrosis factor-a, isobutyryl methylxanthine did not alter the synthesis of interleukin-6 and ciprofloxacin decreased the synthesis of interleukin-6. Only pentoxifylline increased the synthesis of interleukin-6, a finding that is consistent with the hypothesis that elevated levels of cAMP enhance the synthesis of interleukin-6. Finally, indomethacin had a biphasic effect on the release of interleukin-6 in titanium-stimulated cultures, inhibiting the production of interleukin-6 only at low-to-intermediate concentrations. The inconsistencies of these findings are likely multifactorial. They probably reflect the complexity of regulation of interleukin-6, with convergence of multiple regulatory factors and events, as well as the likelihood that the various pharmacological agents have multiple effects on cellular physiology. While alterations in the levels of cAMP also affect the synthesis of interleukin-6, the role of this signaling pathway must be interpreted more cautiously.

While prostaglandin E₂ may function as a primary stimulator of bone resorption in periprosthetic osteolysis^19,21, the results of the present study suggest that prostaglandins may also have an important role as autocrine regulators of the production of cytokines by monocytes. Thus, the addition of prostaglandin inhibitors, such as indomethacin, may result in complex biochemical changes and an increase in the synthesis of cytokines. This may partly explain the inability of non-steroidal anti-inflammatory agents to prevent periprosthetic osteolysis in a recent study of aseptic loosening¹⁹. However, the role of non-steroidal anti-inflammatory agents in periprosthetic bone resorption is dependent on the effect of these agents on a variety of cells in a complex biological tissue and requires additional investigation.

The present findings demonstrate that two commonly used pharmacological agents with phosphodiesterase activity, pentoxifylline and ciprofloxacin, are potent inhibitors of the release of tumor necrosis factor-a by titanium-stimulated human peripheral blood monocytes. Pentoxifylline, a hemorheological agent used to treat peripheral vascular disease, inhibited the titanium-stimulated production of tumor necrosis factor-a by 65 per cent at a concentration of 100 micrograms per milliliter. A previous study in humans, examining the in vivo effects of orally administered pentoxifylline, demonstrated a marked reduction in the secretion of tumor necrosis factor-a by lipopolysaccharide-stimulated peripheral blood monocytes³³. Ciprofloxacin, a commonly used fluoroquinolone antibiotic, inhibited the production of tumor necrosis factor-a by monocytes stimulated with titanium particles, although the effect was less marked at concentrations in the therapeutic range (as much as five micrograms per milliliter in serum and as much as tenfold more in other tissues). While pentoxifylline increased the release of interleukin-6 by titanium-stimulated monocytes, presumably through elevation of intracellular levels of cAMP, surprisingly ciprofloxacin decreased the levels of interleukin-6 in monocyte cultures treated with titanium particles. Neither of these effects was due to cell toxicity, as demonstrated by the trypan-blue exclusion method in cultures treated with these agents. The data suggest that ciprofloxacin may alter the synthesis of interleukin-6 through some undetermined mechanism. While the potential clinical utility of these and other pharmacological agents designed to prevent aseptic loosening requires additional study, our results demonstrate the influence of commonly used drugs on the synthesis of cytokines by titanium-activated monocytes.

The results of the present study also demonstrate the responsiveness of wear-debris-stimulated monocytes to pharmacological manipulation and substantiate the potential of biological inhibitors of prosthetic loosening. Because of the separate and conflicting regulation, attempts to inhibit the secretion of cytokines pharmacologically will likely require multiagent therapy designed to inhibit the important intracellular signals associated with the release of specific cytokines in response to the phagocytosis of wear debris. Thus, additional study of the biological mechanisms involved in wear-debris-induced synthesis of cytokines is an essential part of the strategy to develop and design inhibitors of osteolysis.

References

Introduction | Materials and Methods | Results | Discussion | References

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TABLE I EFFECT OF PROSTAGLANDINS ON THE RELEASE OF TUMOR NECROSIS FACTOR-a BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)
Control	153 ± 6
Titanium	1394 ± 47*
10^-7-M prostaglandin E2 with titanium	220 ± 7†‡
10^-7-M prostaglandin E1 with titanium	329 ± 26†‡
10^-7-M prostaglandin F2a with titanium	777 ± 76†§

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TABLE I EFFECT OF PROSTAGLANDINS ON THE RELEASE OF TUMOR NECROSIS FACTOR-a BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)
Control	153 ± 6
Titanium	1394 ± 47*
10^-7-M prostaglandin E2 with titanium	220 ± 7†‡
10^-7-M prostaglandin E1 with titanium	329 ± 26†‡
10^-7-M prostaglandin F2a with titanium	777 ± 76†§

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TABLE II EFFECT OF ISOBUTYRYL METHYLXANTHINE ON THE RELEASE OF TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-6 BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)	Concentration of Interleukin-6 (pg/ml)
Control	87 ± 16	719 ± 127
Titanium	865 ± 119†	10,020 ± 440‡
Titanium with 100-µM isobutyryl methylxanthine	76 ± 8 ‡	10,175 ± 508†§

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TABLE II EFFECT OF ISOBUTYRYL METHYLXANTHINE ON THE RELEASE OF TUMOR NECROSIS FACTOR-a AND INTERLEUKIN-6 BY HUMAN PERIPHERAL BLOOD MONOCYTES

Treatment	Concentration of Tumor Necrosis Factor-a (pg/ml)	Concentration of Interleukin-6 (pg/ml)
Control	87 ± 16	719 ± 127
Titanium	865 ± 119†	10,020 ± 440‡
Titanium with 100-µM isobutyryl methylxanthine	76 ± 8 ‡	10,175 ± 508†§