Proinflammatory Activation of Macrophages by Basic Calcium Phosphate Crystals via Protein Kinase C and MAP Kinase Pathways
http://www.100md.com
循环研究杂志 2005年第6期
The British Heart Foundation Cardiovascular Medicine Unit (I.M., J.C.M., P.P., O.F., C.D.W.S., R.C.L., D.O.H.), Eric Bywaters Centre for Vascular Inflammation, Faculty of Medicine, Imperial College London, Hammersmith Hospital, London; and the Department of Clinical Pharmacology (G.M.M.), The Royal College of Surgeons, Dublin. The current address of R.C.L. is the Edmund Cohen Laboratory for Vascular Research, Chronic Disease Research Centre, Tropical Medicine Research Institute, UWI, Barbados, West Indies.
Abstract
Basic calcium phosphate (BCP) crystal deposition underlies the development of arterial calcification. Inflammatory macrophages colocalize with BCP deposits in developing atherosclerotic lesions and in vitro can promote calcification through the release of TNF alpha. Here we have investigated whether BCP crystals can elicit a proinflammatory response from monocyte-macrophages. BCP microcrystals were internalized into vacuoles of human monocyte-derived macrophages in vitro. This was associated with secretion of proinflammatory cytokines (TNF, IL-1 and IL-8) capable of activating cultured endothelial cells and promoting capture of flowing leukocytes under shear flow. Critical roles for PKC, ERK1/2, JNK, but not p38 intracellular signaling pathways were identified in the secretion of TNF alpha, with activation of ERK1/2 but not JNK being dependent on upstream activation of PKC. Using confocal microscopy and adenoviral transfection approaches, we determined a specific role for the PKC-alpha isozyme. The response of macrophages to BCP crystals suggests that pathological calcification is not merely a passive consequence of chronic inflammatory disease but may lead to a positive feed-back loop of calcification and inflammation driving disease progression.
Key Words: atherosclerosis pathophysiology cell biology calcification inflammation macrophage
Introduction
Coronary artery calcification occurs as part of the atherosclerotic process, and is due to the deposition in the arterial intima of basic calcium phosphate (BCP) crystals, consisting mainly of calcium hydroyapatite (Ca10(PO4)6(OH)2) and similar to those that mineralize bone.1eC6 Using electron beam computed tomography, the amount of vessel calcification has been shown to closely correlate with the extent of plaque burden and may predict future coronary events.7eC10 However, although arterial calcification is now accepted to be an active and highly regulated process similar to that of bone ossification,11eC14 it has tended to be seen merely as a surrogate marker for the burden of atherosclerotic disease rather than a contributor to disease progression.
Growing evidence from the study of degenerative arthritis, in which BCP crystals can be found in the majority of affected joints and closely correlate with the extent of joint destruction, suggests a pathogenic role in driving disease.15,16 Thus, BCP crystals have been shown to activate synovial fibroblasts, inducing cellular proliferation and matrix metalloproteinase (MMP) secretion through a variety of intracellular signaling pathways, including protein kinase C (PKC), ERK1/2 MAP kinase, NFB, and AP-1.17eC19 Limited studies using murine cells have also demonstrated the ability of macrophages to interact with BCP crystals in vitro, resulting in DNA synthesis and cytokine production.20,21
Cells of the monocyte/macrophage lineage form an important part of the innate immune system and play a key role in atherogenesis.22 Inflammatory mediators derived from monocyte-macrophages, including TNF, are thought to contribute to the calcification process through the promotion of osteoblastic differentiation and calcification of vascular smooth muscle cells.23eC25 Furthermore, a link between the activated macrophage and the deposition of calcification may help to explain why histological studies have consistently shown plaque macrophages to colocalize with BCP crystal deposits.26,27 As yet, however, the ability of BCP crystals to directly activate macrophages in the calcified atherosclerotic plaque remains a relatively unexplored possibility. In this study we have shown for the first time that BCP crystals can interact with and activate human macrophages in a proinflammatory manner via mechanisms involving protein kinase C (PKC) and ERK1/2 MAP kinase.
Materials and Methods
Antibodies and Other Reagents
Anti-E/P-selectin (1.2B6), VCAM-1 (IE5), and ICAM-1 (6.5B5) antibodies were generated within our group. Anti-PKC, PKCI, PKCII, PKC, PKC, and PKC antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Anti-PKC and anti-PKC antibodies were from Transduction Laboratories (Lexington, Ky). Anti-ERK1/2, JNK, and phospho-specific ERK1/2, and JNK antibodies were from Cell Signaling Technology (Beverly, Mass). Secondary antibodies included swine antirabbit-HRP (DAKO, Glostrup, Denmark), sheep antimouse-HRP (Amersham Life Science, Bucks, UK), and goat antirabbit Alexa Fluor-488 (Molecular Probes, Eugene, Ore). GF109203X, SB203580, JNKII, and U0126 inhibitors were purchased from Merk-Biosciences (Nottingham, UK). LY379196 was a gift from Dr. K. Ways (Eli Lilly, Indianapolis, Ind). Myristoylated PKC/ inhibitor peptide (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) was purchased from Promega (Madision, Wis). Human recombinant macrophage colony-stimulating factor (M-CSF) was purchased from Peprotech EC (London, UK). All other reagents were from Sigma-Aldrich (Poole, UK) unless otherwise stated.
BCP Crystal Synthesis and Preparation
BCP crystals were synthesized by a modification of a previously published method involving alkaline hydrolysis of brushite, CaHPO4x2H2O.17,28 Mineral prepared by this method contains 90% hydroxyapatite and 10% octacalcium phosphate, and has a composite calcium/phosphate molar ratio of 1.3 to 1.45. The mean particle size was <1 e蘭. Crystals were baked for 2 hours at 200°C before use. Endotoxin levels were quantified by the QCL-1000 Chromogenic LAL test kit (BioWhittaker) and shown to be less than 20 pg/mL (<0.2 EU/mL) in a 5.7 mg/mL stock solution. Crystals were suspended in Hanks’ buffered salt solution and sonicated for 1 minute before being added to cell cultures using a 10-fold dilution of this stock solution.
Monocyte/Macrophage Isolation and Cell Culture
Human monocytes were isolated from the freshly drawn venous blood of healthy volunteers and differentiated in vitro into macrophages over a period of 7 days as previously described.29 Cells were stimulated with BCP crystals or vehicle media control for 20 hours at 37°C in humidified atmosphere with 5% CO2. Supernatants were collected, centrifuged at 350 x g to remove particulate debris and stored at eC70°C before analysis.
Transmission Electron Microscopy and X-Ray Elemental Microanalysis
Transmission electron microscopy and X-Ray elemental microanalysis were performed as detailed in the online data supplement available at http://circres.ahajournals.org.
Enzyme-Linked Immunosorbent Assays
TNF, IL-1, IL-8, and MCP-1 levels in macrophage-conditioned media were determined by enzyme-linked immunosorbent assays (R&D Systems). All samples were measured in duplicate, with the results expressed as the mean±SEM cytokine concentration.
Quantitative Real Time Polymerase Chain Reaction (RT-PCR)
Monocytes were differentiated over 7 days into macrophages on a 6 well plate as previously described, followed by stimulation with BCP crystals (300 e蘥/cm2). Cells were then rinsed once with ice-cold PBS to remove any unbound BCP crystals, after which total RNA was extracted. Quantitative reverse-transcription PCR was then performed, as detailed in the online data supplement. Steady-state mRNA levels were expressed as the ratio of mRNA levels of TNF (relative to GAPDH) in stimulated cells as compared with that of resting cells.
Endothelial Cell Flow Cytometry and Hydrodynamic Shear Flow Adhesion Assay
Human umbilical vein endothelial cells (HUVEC) were obtained from fresh umbilical cords and cultured as previously described.30 Endothelial cell flow cytometric analysis and hydrodynamic shear flow adhesion assays were performed as detailed in the online data supplement.
Intracellular Signaling Inhibition
Macrophage cell culture medium was replaced with fresh medium containing the specific signaling inhibitor or vehicle control one hour before stimulation. Following 20 hours stimulation, conditioned media or whole-cell lysates were collected and stored at eC70°C for later analysis. None of the inhibitors or adenoviruses used at the specified doses had any adverse effects on cell viability as judged by trypan blue exclusion and a MTT cell viability assay (Cell Titer 96 Aqueous One Stop solution, Promega).
Western Blot Analysis
Western blot analysis was performed by SDS-PAGE and semidry transfer onto a PDVF membrane as previously described (see online data supplement).30
Confocal Scanning Fluorescent Microscopy
Stimulated cells were fixed with 4% paraformaldehyde for 30 minutes followed by blocking for 30 minutes at 4°C with 1% BSA in HBSS. Cells were permeabilised by sequential treatment with 100% methanol for 10 minutes at 4°C and HBSS containing 0.1% Triton X100 and 5% rabbit serum (DAKO) for 50 minutes at 4°C. Immunostaining was performed using the primary anti-PKC antibody (10 e蘥/mL) for 1 hour at room temperature followed by a secondary Alexa-Fluor-488 conjugated antibody (8 e蘥/mL) for a further 1.5 hours at room temperature in the dark. Fluorescence was analyzed using a Carl Zeiss LSM5-PASCAL laser-scanning microscope (Herts).
Adenovirus Infection of Macrophages
Details of adenovirus amplification, purification, titration, and infection of macrophages are given in the online data supplement.
Statistical Analysis
All data were expressed as the mean of individual experiments±standard error of the mean (SEM). Data were analyzed using a two-tailed paired t test or a one-way analysis of variance (ANOVA) with Dunnett’s correction (GraphPad Prism software).
Results
Inflammatory Cytokine Secretion by BCP-Treated Macrophages
Transmission electron microscopy of macrophages treated with BCP crystals for 20 hours showed vacuoles containing structures similar in appearance to previously published images of hydoxyapatite within cells31,32 (Figure 1). Furthermore, X-ray elemental microanalysis of macrophages that had been cultured with BCP and then washed showed the presence of a new calcium peak, consistent with internalized crystals (online Figure I).
Analysis of the effects of BCP crystal uptake by macrophages from 10 different donors demonstrated concentration-dependent release of TNFa (312.9±64.9 pg/mL, P<0.01) and IL-1 (59.74±29.54 pg/mL, P<0.05). TNF and IL-1 release were dose-dependent with a maximal response at 300 e蘥/cm2. We also found evidence for release of IL-8 (13.75±3.34 ng/mL, P=0.0034) but not MCP-1, when tested at a single concentration of BCP crystals (300 mg/mL2) (Figure 2). A time course revealed slow kinetics of TNF release with detectable levels first seen after 6 hours, with a continual rise over 48 hours (Figure 3A). TNF release was dependent on de novo gene transcription and protein translation, as shown by abrogation in the presence of actinomycin D and cycloheximide (both P<0.01) (Figure 3B). RT-PCR confirmed that steady-state mRNA levels were raised as early as 1 hour (299.3±116.3) with a continuous rise over 6 hours (1165±141.4, P<0.01)(Figure 3C). Activation of macrophages by BCP crystals was not secondary to dissolution of BCP crystals raising the calcium and phosphate concentrations, since (i) we found no detectable change in calcium or phosphate concentrations in supernatants of macrophages incubated with BCP crystals for 48 hours; (ii) BCP crystals cultured with macrophages on different sides of a semi-permeable membrane did not induce release of TNF (online Figure II); and (iii) centrifuged supernatants from macrophages cocultured with BCP crystals for 20 hours failed to induce an increase in TNF mRNA when transferred to fresh macrophage cultures (online Figure III). Unlike TNF secretion in response to LPS (not shown), the release of TNF was abrogated by cytochalasin D (P<0.01) (Figure 3B), suggesting the requirement for an intact cytoskeleton.
To establish whether the proinflammatory response of macrophages to BCP was sufficient to activate endothelial cells, HUVEC were coincubated with macrophage-conditioned media and analyzed by flow cytometry for induction of adhesion molecule expression (online Figure IVA). The common epitope on E- and P-selectin recognized by mAb 1.2B6 was upregulated 9-fold after 4 hours and VCAM-1 and ICAM-1 5 and 6-fold respectively after 16 hours. To demonstrate that activated HUVEC were able to support the capture of leukocytes under shear flow, lawns of HUVEC were established in a parallel-plate flow chamber and treated with macrophage-conditioned media for 16 hours before superfusion with mononuclear cells. Conditioned media from BCP treated macrophages promoted a 2.9-fold increase in rolling and a 13.1-fold increase in mononuclear cell arrest at a hydrodynamic shear force of 1.5 dynes/cm2 (online Figure IVB).
Intracellular Signaling Pathways Stimulated by BCP
Pretreatment with increasing doses of the broad spectrum PKC isozyme inhibitor GF109203X abrogated TNF production in a dose dependent manner in response to BCP crystals (Figure 4A). At 10 e蘭ol/L of GF109203X, TNF synthesis was 100% inhibited (272.0±59.4 pg/mL versus 46.7±39.3 pg/mL, P<0.01). Next, the 3 main MAP kinase-signaling pathways, ERK1/2, JNK, and p38, were investigated. Pretreatment with the ERK1/2 inhibitor, U0126, reduced TNF secretion in a dose-dependent manner, with maximum inhibition of 74% seen at 2.5 e蘭ol/L (487.8±169.2 pg/mL versus 182.3±52.5 pg/mL, P<0.01) (Figure 4B). The role of JNK was demonstrated by dose-dependent inhibition of up to 74% with the JNK inhibitor JNK-II (382.4±86.5 pg/mL versus 128.0±58.9 pg/mL, P<0.01) (Figure 4C). In contrast, inhibition of p38 MAP kinase with concentrations of SB203580 up to 25 e蘭ol/L had no significant inhibitory effect on TNF production (Figure 4D).
PKC Activation by BCP
Since the production of TNF in response to BCP was completely dependent on PKC signaling, we investigated which isozymes(s) of PKC might be involved. In agreement with the previous reports,33,34 Western blot analysis of unstimulated macrophages indicated the presence of the classical PKC, 1, 2 but not isozymes (Figure 5A). Similarly the atypical PKC, the novel PKC, and isozymes, but not , were detected. As a positive control, a HUVEC lysate was blotted for the presence of PKC and , revealing 80 and 79 kDa bands respectively (not shown). As GF109203X inhibits PKC, , , , isozymes, we further investigated the roles of individual isozymes in the macrophage response to BCP. The involvement of PKC or PKC in BCP-mediated TNF release was first established using a selective and specific myristoylated peptide PKC/ inhibitor (Figure 5B). Pretreatment with increasing doses of peptide had a partial inhibitory effect of up to 60% with 100 e蘭ol/L of peptide (1225±466.0pg/mL reduced to 503.1±193.0 pg/mL, P<0.01). Similar results were achieved with G6976, another PKC/ isozyme pharmacological antagonist (data not shown). A role for PKC was eliminated by the lack of effect with the PKC specific inhibitor LY379196 (Figure 5C). Activation of PKC was investigated by confocal scanning microscopy to examine the subcellular distribution of this isozyme at various time points following treatment with BCP crystals (Figure 5D). This showed that PKC translocated from the cytoplasm to a membrane-associated distribution within 5 minutes of stimulation. By 20 minutes, PKC partitioned to numerous discrete vacuoles. Taken together, these data suggest that TNF secretion involves the PKC isozyme.
The specific involvement of PKC was further confirmed by using a PKC dominant negative (DN) kinase expressed in an adenovirus. Maximal expression of PKC was seen with a multiplicity of infection (MOI) for the PKC DN adenovirus of between 200 and 400, as shown by Western blotting (Figure 6A). There was no significant difference in TNF expression by control GFP-infected versus uninfected cells (Figure 6B). However, infection with the PKC specific DN adenovirus construct resulted in a significant 72% inhibition of TNF release (542.5±94.7 pg/mL versus 234.7±42.0 pg/mL, P<0.01). These results demonstrate unequivocally that TNF release involves the classical PKC isozyme.
ERK1/2 and JNK MAP Kinase Activation by BCP
The involvement of the ERK1/2 and JNK MAP kinase pathways were further investigated by assessing phosphorylation of these proteins. Phosphorylation of ERK1/2 was shown by Western blotting to take place within 10 minutes of BCP crystal treatment (Figure 7A). Similarly, JNK was phosphorylated within 10 minutes, but unlike ERK1/2, was downregulated within 30 minutes (Figure 7B). As the phosphorylation of both these proteins appeared to be maximal at 10 minutes, this time point was investigated in experiments aimed at linking ERK1/2 and JNK signaling with prior PKC activation. Pretreatment with PKC inhibitor GF109203X inhibited ERK1/2 phosphorylation (Figure 8A), whereas no effect on the phosphorylation of JNK was seen (Figure 8B).
Discussion
The presence of arterial calcification has tended to been viewed either as a passive bystander phenomenon or as a useful clinical marker of atherosclerotic disease progression. Here we clearly demonstrate an active inflammatory response by macrophages to BCP crystals with implications for atherosclerosis disease pathogenesis and treatment. We have shown that human macrophages interact with BCP crystals, apparently taking them into vacuoles. In response, macrophages secrete biologically significant quantities of the proinflammatory cytokines TNF, IL-1, and IL-8, which are capable of stimulating the activation of endothelial cells and recruitment of mononuclear cells in an in vitro hemodynamic flow assay. Since TNF can promote osteoblastic differentiation and calcification of vascular cells,23eC25 our data suggest a positive feedback loop predisposing to chronic inflammatory pathology. The model we propose may be of more relevance to intimal than medial calcification, in which the proximity of calcific deposits to macrophages is less clear and in which there may be separate calcification mechanisms.35
Scanning electron microscopy of BCP particles extracted from atherosclerotic arteries has revealed marked heterogeneity of shape and size,2 and it remains to be determined whether these physical characteristics influence macrophage inflammatory responsiveness. In preliminary experiments we have found that the macrophage activating potential of BCP particles is inversely correlated with their size, suggesting that small isolated calcific particles in early disease may be more proinflammatory than larger deposits resembling bone seen in established lesions.26,36 If so, it is possible that the inflammatory calcification paradigm we propose is of particular relevance to the progression of early atherosclerosis.
Although the amount of arterial calcification may predict coronary events, it is still unclear whether calcified plaques are more prone to rupture.7eC10 Nevertheless, the biological response to BCP crystals could promote plaque instability. Thus, several studies have demonstrated that BCP is capable of inducing fibroblast MMP production via PKC and ERK1/2 signaling mechanisms.19,37 In preliminary experiments we have not been able to show increased expression of MMP-2 or -9 in BCP crystal stimulated macrophages. Nevertheless, BCP crystals might lead to increased tissue proteinase expression and activation by acting on macrophages under conditions so far untested, or indirectly via the effects of macrophage-derived TNF on vascular smooth muscle cells (VSMC).38 Furthermore, besides contributing to leukocyte recruitment, macrophage-derived TNF might also destabilise plaque by contributing to VSMC apoptosis.39
It is increasingly recognized that macrophages are not always proinflammatory but may play a role in resolution and repair.40 Thus we and others have demonstrated a more antiinflammatory profile of cytokine expression in response to other particulate stimuli, such as apoptotic neutrophils, monosodium-urate crystals, and hemoglobin-haptoglobin complexes.41eC43 The differences in the receptor and signaling mechanisms responsible for the variable response of macrophages to these particles requires further study.
A surface receptor responsible for the uptake of BCP crystals by cells has not yet been identified, and it is possible that signal transduction may be mediated by proteins opsonised by crystals rather than by the crystal surface per se. The effects we have observed, however, appear not to be secondary to dissolution of BCP crystals raising the calcium and phosphate concentrations, since we found no detectable change in calcium or phosphate concentrations in supernatants of BCP-macrophage cocultures, and centrifuged supernatants from macrophages cocultured with BCP crystals count not reproduce the effects. Judging from an experiment culturing BCP crystals with macrophages on different sides of a semipermeable membrane, contact between the particles and macrophages is required. However contact is not sufficient, since TNF release was blocked by inhibiting the cytoskeleton with cytochalasin-D.
We have unequivocally identified PKC as a key regulator of BCP-induced TNF release. Several studies have demonstrated an active role for this isoenzyme in the development of phagolysosomes by macrophages in response to microorganisms and other stumili.44eC47 Similarly, our confocal microscopy data demonstrate that BCP crystals stimulate the rapid translocation of PKC, first to the plasma membrane and then to vacuoles, consistent with PKCa activation being a critical link for downstream signaling. It remains to be determined whether these vacuoles are truly intracellular, or similar to the surface-connected compartments that take up aggregated LDL.48
Our data show that both the ERK1/2 and the JNK kinase pathways are involved in BCP-induced TNF release, but that only ERK1/2 is dependent on upstream PKC activation. These data are consistent with several similar observations in human monocytic cells in response to other agonists.49eC51 Interestingly, there appears to be cell specificity in the macrophage response to BCP, as BCP crystal-induced activation of ERK1/2 in fibroblasts has been found to be dependent on PKCe?rather than a calcium-dependent PKC.19,52 A recent study in human macrophages described the release of TNF in response to phorbol ester or LPS, mediated through ERK1/2 via the upstream activation of one of the conventional PKC isozymes, or .53 Importantly, that study demonstrated differential effects on proinflammatory versus antiinflammatory cytokine secretion depending on whether conventional PKC ( or ) or atypical PKC isozymes were activated. Based on the conclusions of this study, therapeutic blockade of PKC isozyme might result in blockade of proinflammatory cytokine production but also a switch to antiinflammatory cytokine production.
In conclusion, through investigation of the mechanisms regulating TNF production, we have identified key roles for PKC, ERK1/2, and JNK in downstream signaling in response to BCP crystals. This raises the possibility that specific targeting of such upstream signaling pathways may represent a more effective strategy than blocking TNF, a downstream product of the inflammatory cascade. Specifically, the identification of PKC as a proximal signaling molecule makes this a possible therapeutic target for the development of novel treatments against arterial calcification and other calcification-related chronic inflammatory diseases.
Acknowledgments
The study was funded by the British Heart Foundation.
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Abstract
Basic calcium phosphate (BCP) crystal deposition underlies the development of arterial calcification. Inflammatory macrophages colocalize with BCP deposits in developing atherosclerotic lesions and in vitro can promote calcification through the release of TNF alpha. Here we have investigated whether BCP crystals can elicit a proinflammatory response from monocyte-macrophages. BCP microcrystals were internalized into vacuoles of human monocyte-derived macrophages in vitro. This was associated with secretion of proinflammatory cytokines (TNF, IL-1 and IL-8) capable of activating cultured endothelial cells and promoting capture of flowing leukocytes under shear flow. Critical roles for PKC, ERK1/2, JNK, but not p38 intracellular signaling pathways were identified in the secretion of TNF alpha, with activation of ERK1/2 but not JNK being dependent on upstream activation of PKC. Using confocal microscopy and adenoviral transfection approaches, we determined a specific role for the PKC-alpha isozyme. The response of macrophages to BCP crystals suggests that pathological calcification is not merely a passive consequence of chronic inflammatory disease but may lead to a positive feed-back loop of calcification and inflammation driving disease progression.
Key Words: atherosclerosis pathophysiology cell biology calcification inflammation macrophage
Introduction
Coronary artery calcification occurs as part of the atherosclerotic process, and is due to the deposition in the arterial intima of basic calcium phosphate (BCP) crystals, consisting mainly of calcium hydroyapatite (Ca10(PO4)6(OH)2) and similar to those that mineralize bone.1eC6 Using electron beam computed tomography, the amount of vessel calcification has been shown to closely correlate with the extent of plaque burden and may predict future coronary events.7eC10 However, although arterial calcification is now accepted to be an active and highly regulated process similar to that of bone ossification,11eC14 it has tended to be seen merely as a surrogate marker for the burden of atherosclerotic disease rather than a contributor to disease progression.
Growing evidence from the study of degenerative arthritis, in which BCP crystals can be found in the majority of affected joints and closely correlate with the extent of joint destruction, suggests a pathogenic role in driving disease.15,16 Thus, BCP crystals have been shown to activate synovial fibroblasts, inducing cellular proliferation and matrix metalloproteinase (MMP) secretion through a variety of intracellular signaling pathways, including protein kinase C (PKC), ERK1/2 MAP kinase, NFB, and AP-1.17eC19 Limited studies using murine cells have also demonstrated the ability of macrophages to interact with BCP crystals in vitro, resulting in DNA synthesis and cytokine production.20,21
Cells of the monocyte/macrophage lineage form an important part of the innate immune system and play a key role in atherogenesis.22 Inflammatory mediators derived from monocyte-macrophages, including TNF, are thought to contribute to the calcification process through the promotion of osteoblastic differentiation and calcification of vascular smooth muscle cells.23eC25 Furthermore, a link between the activated macrophage and the deposition of calcification may help to explain why histological studies have consistently shown plaque macrophages to colocalize with BCP crystal deposits.26,27 As yet, however, the ability of BCP crystals to directly activate macrophages in the calcified atherosclerotic plaque remains a relatively unexplored possibility. In this study we have shown for the first time that BCP crystals can interact with and activate human macrophages in a proinflammatory manner via mechanisms involving protein kinase C (PKC) and ERK1/2 MAP kinase.
Materials and Methods
Antibodies and Other Reagents
Anti-E/P-selectin (1.2B6), VCAM-1 (IE5), and ICAM-1 (6.5B5) antibodies were generated within our group. Anti-PKC, PKCI, PKCII, PKC, PKC, and PKC antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif). Anti-PKC and anti-PKC antibodies were from Transduction Laboratories (Lexington, Ky). Anti-ERK1/2, JNK, and phospho-specific ERK1/2, and JNK antibodies were from Cell Signaling Technology (Beverly, Mass). Secondary antibodies included swine antirabbit-HRP (DAKO, Glostrup, Denmark), sheep antimouse-HRP (Amersham Life Science, Bucks, UK), and goat antirabbit Alexa Fluor-488 (Molecular Probes, Eugene, Ore). GF109203X, SB203580, JNKII, and U0126 inhibitors were purchased from Merk-Biosciences (Nottingham, UK). LY379196 was a gift from Dr. K. Ways (Eli Lilly, Indianapolis, Ind). Myristoylated PKC/ inhibitor peptide (Myr-Arg-Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln-Lys-Asn-Val) was purchased from Promega (Madision, Wis). Human recombinant macrophage colony-stimulating factor (M-CSF) was purchased from Peprotech EC (London, UK). All other reagents were from Sigma-Aldrich (Poole, UK) unless otherwise stated.
BCP Crystal Synthesis and Preparation
BCP crystals were synthesized by a modification of a previously published method involving alkaline hydrolysis of brushite, CaHPO4x2H2O.17,28 Mineral prepared by this method contains 90% hydroxyapatite and 10% octacalcium phosphate, and has a composite calcium/phosphate molar ratio of 1.3 to 1.45. The mean particle size was <1 e蘭. Crystals were baked for 2 hours at 200°C before use. Endotoxin levels were quantified by the QCL-1000 Chromogenic LAL test kit (BioWhittaker) and shown to be less than 20 pg/mL (<0.2 EU/mL) in a 5.7 mg/mL stock solution. Crystals were suspended in Hanks’ buffered salt solution and sonicated for 1 minute before being added to cell cultures using a 10-fold dilution of this stock solution.
Monocyte/Macrophage Isolation and Cell Culture
Human monocytes were isolated from the freshly drawn venous blood of healthy volunteers and differentiated in vitro into macrophages over a period of 7 days as previously described.29 Cells were stimulated with BCP crystals or vehicle media control for 20 hours at 37°C in humidified atmosphere with 5% CO2. Supernatants were collected, centrifuged at 350 x g to remove particulate debris and stored at eC70°C before analysis.
Transmission Electron Microscopy and X-Ray Elemental Microanalysis
Transmission electron microscopy and X-Ray elemental microanalysis were performed as detailed in the online data supplement available at http://circres.ahajournals.org.
Enzyme-Linked Immunosorbent Assays
TNF, IL-1, IL-8, and MCP-1 levels in macrophage-conditioned media were determined by enzyme-linked immunosorbent assays (R&D Systems). All samples were measured in duplicate, with the results expressed as the mean±SEM cytokine concentration.
Quantitative Real Time Polymerase Chain Reaction (RT-PCR)
Monocytes were differentiated over 7 days into macrophages on a 6 well plate as previously described, followed by stimulation with BCP crystals (300 e蘥/cm2). Cells were then rinsed once with ice-cold PBS to remove any unbound BCP crystals, after which total RNA was extracted. Quantitative reverse-transcription PCR was then performed, as detailed in the online data supplement. Steady-state mRNA levels were expressed as the ratio of mRNA levels of TNF (relative to GAPDH) in stimulated cells as compared with that of resting cells.
Endothelial Cell Flow Cytometry and Hydrodynamic Shear Flow Adhesion Assay
Human umbilical vein endothelial cells (HUVEC) were obtained from fresh umbilical cords and cultured as previously described.30 Endothelial cell flow cytometric analysis and hydrodynamic shear flow adhesion assays were performed as detailed in the online data supplement.
Intracellular Signaling Inhibition
Macrophage cell culture medium was replaced with fresh medium containing the specific signaling inhibitor or vehicle control one hour before stimulation. Following 20 hours stimulation, conditioned media or whole-cell lysates were collected and stored at eC70°C for later analysis. None of the inhibitors or adenoviruses used at the specified doses had any adverse effects on cell viability as judged by trypan blue exclusion and a MTT cell viability assay (Cell Titer 96 Aqueous One Stop solution, Promega).
Western Blot Analysis
Western blot analysis was performed by SDS-PAGE and semidry transfer onto a PDVF membrane as previously described (see online data supplement).30
Confocal Scanning Fluorescent Microscopy
Stimulated cells were fixed with 4% paraformaldehyde for 30 minutes followed by blocking for 30 minutes at 4°C with 1% BSA in HBSS. Cells were permeabilised by sequential treatment with 100% methanol for 10 minutes at 4°C and HBSS containing 0.1% Triton X100 and 5% rabbit serum (DAKO) for 50 minutes at 4°C. Immunostaining was performed using the primary anti-PKC antibody (10 e蘥/mL) for 1 hour at room temperature followed by a secondary Alexa-Fluor-488 conjugated antibody (8 e蘥/mL) for a further 1.5 hours at room temperature in the dark. Fluorescence was analyzed using a Carl Zeiss LSM5-PASCAL laser-scanning microscope (Herts).
Adenovirus Infection of Macrophages
Details of adenovirus amplification, purification, titration, and infection of macrophages are given in the online data supplement.
Statistical Analysis
All data were expressed as the mean of individual experiments±standard error of the mean (SEM). Data were analyzed using a two-tailed paired t test or a one-way analysis of variance (ANOVA) with Dunnett’s correction (GraphPad Prism software).
Results
Inflammatory Cytokine Secretion by BCP-Treated Macrophages
Transmission electron microscopy of macrophages treated with BCP crystals for 20 hours showed vacuoles containing structures similar in appearance to previously published images of hydoxyapatite within cells31,32 (Figure 1). Furthermore, X-ray elemental microanalysis of macrophages that had been cultured with BCP and then washed showed the presence of a new calcium peak, consistent with internalized crystals (online Figure I).
Analysis of the effects of BCP crystal uptake by macrophages from 10 different donors demonstrated concentration-dependent release of TNFa (312.9±64.9 pg/mL, P<0.01) and IL-1 (59.74±29.54 pg/mL, P<0.05). TNF and IL-1 release were dose-dependent with a maximal response at 300 e蘥/cm2. We also found evidence for release of IL-8 (13.75±3.34 ng/mL, P=0.0034) but not MCP-1, when tested at a single concentration of BCP crystals (300 mg/mL2) (Figure 2). A time course revealed slow kinetics of TNF release with detectable levels first seen after 6 hours, with a continual rise over 48 hours (Figure 3A). TNF release was dependent on de novo gene transcription and protein translation, as shown by abrogation in the presence of actinomycin D and cycloheximide (both P<0.01) (Figure 3B). RT-PCR confirmed that steady-state mRNA levels were raised as early as 1 hour (299.3±116.3) with a continuous rise over 6 hours (1165±141.4, P<0.01)(Figure 3C). Activation of macrophages by BCP crystals was not secondary to dissolution of BCP crystals raising the calcium and phosphate concentrations, since (i) we found no detectable change in calcium or phosphate concentrations in supernatants of macrophages incubated with BCP crystals for 48 hours; (ii) BCP crystals cultured with macrophages on different sides of a semi-permeable membrane did not induce release of TNF (online Figure II); and (iii) centrifuged supernatants from macrophages cocultured with BCP crystals for 20 hours failed to induce an increase in TNF mRNA when transferred to fresh macrophage cultures (online Figure III). Unlike TNF secretion in response to LPS (not shown), the release of TNF was abrogated by cytochalasin D (P<0.01) (Figure 3B), suggesting the requirement for an intact cytoskeleton.
To establish whether the proinflammatory response of macrophages to BCP was sufficient to activate endothelial cells, HUVEC were coincubated with macrophage-conditioned media and analyzed by flow cytometry for induction of adhesion molecule expression (online Figure IVA). The common epitope on E- and P-selectin recognized by mAb 1.2B6 was upregulated 9-fold after 4 hours and VCAM-1 and ICAM-1 5 and 6-fold respectively after 16 hours. To demonstrate that activated HUVEC were able to support the capture of leukocytes under shear flow, lawns of HUVEC were established in a parallel-plate flow chamber and treated with macrophage-conditioned media for 16 hours before superfusion with mononuclear cells. Conditioned media from BCP treated macrophages promoted a 2.9-fold increase in rolling and a 13.1-fold increase in mononuclear cell arrest at a hydrodynamic shear force of 1.5 dynes/cm2 (online Figure IVB).
Intracellular Signaling Pathways Stimulated by BCP
Pretreatment with increasing doses of the broad spectrum PKC isozyme inhibitor GF109203X abrogated TNF production in a dose dependent manner in response to BCP crystals (Figure 4A). At 10 e蘭ol/L of GF109203X, TNF synthesis was 100% inhibited (272.0±59.4 pg/mL versus 46.7±39.3 pg/mL, P<0.01). Next, the 3 main MAP kinase-signaling pathways, ERK1/2, JNK, and p38, were investigated. Pretreatment with the ERK1/2 inhibitor, U0126, reduced TNF secretion in a dose-dependent manner, with maximum inhibition of 74% seen at 2.5 e蘭ol/L (487.8±169.2 pg/mL versus 182.3±52.5 pg/mL, P<0.01) (Figure 4B). The role of JNK was demonstrated by dose-dependent inhibition of up to 74% with the JNK inhibitor JNK-II (382.4±86.5 pg/mL versus 128.0±58.9 pg/mL, P<0.01) (Figure 4C). In contrast, inhibition of p38 MAP kinase with concentrations of SB203580 up to 25 e蘭ol/L had no significant inhibitory effect on TNF production (Figure 4D).
PKC Activation by BCP
Since the production of TNF in response to BCP was completely dependent on PKC signaling, we investigated which isozymes(s) of PKC might be involved. In agreement with the previous reports,33,34 Western blot analysis of unstimulated macrophages indicated the presence of the classical PKC, 1, 2 but not isozymes (Figure 5A). Similarly the atypical PKC, the novel PKC, and isozymes, but not , were detected. As a positive control, a HUVEC lysate was blotted for the presence of PKC and , revealing 80 and 79 kDa bands respectively (not shown). As GF109203X inhibits PKC, , , , isozymes, we further investigated the roles of individual isozymes in the macrophage response to BCP. The involvement of PKC or PKC in BCP-mediated TNF release was first established using a selective and specific myristoylated peptide PKC/ inhibitor (Figure 5B). Pretreatment with increasing doses of peptide had a partial inhibitory effect of up to 60% with 100 e蘭ol/L of peptide (1225±466.0pg/mL reduced to 503.1±193.0 pg/mL, P<0.01). Similar results were achieved with G6976, another PKC/ isozyme pharmacological antagonist (data not shown). A role for PKC was eliminated by the lack of effect with the PKC specific inhibitor LY379196 (Figure 5C). Activation of PKC was investigated by confocal scanning microscopy to examine the subcellular distribution of this isozyme at various time points following treatment with BCP crystals (Figure 5D). This showed that PKC translocated from the cytoplasm to a membrane-associated distribution within 5 minutes of stimulation. By 20 minutes, PKC partitioned to numerous discrete vacuoles. Taken together, these data suggest that TNF secretion involves the PKC isozyme.
The specific involvement of PKC was further confirmed by using a PKC dominant negative (DN) kinase expressed in an adenovirus. Maximal expression of PKC was seen with a multiplicity of infection (MOI) for the PKC DN adenovirus of between 200 and 400, as shown by Western blotting (Figure 6A). There was no significant difference in TNF expression by control GFP-infected versus uninfected cells (Figure 6B). However, infection with the PKC specific DN adenovirus construct resulted in a significant 72% inhibition of TNF release (542.5±94.7 pg/mL versus 234.7±42.0 pg/mL, P<0.01). These results demonstrate unequivocally that TNF release involves the classical PKC isozyme.
ERK1/2 and JNK MAP Kinase Activation by BCP
The involvement of the ERK1/2 and JNK MAP kinase pathways were further investigated by assessing phosphorylation of these proteins. Phosphorylation of ERK1/2 was shown by Western blotting to take place within 10 minutes of BCP crystal treatment (Figure 7A). Similarly, JNK was phosphorylated within 10 minutes, but unlike ERK1/2, was downregulated within 30 minutes (Figure 7B). As the phosphorylation of both these proteins appeared to be maximal at 10 minutes, this time point was investigated in experiments aimed at linking ERK1/2 and JNK signaling with prior PKC activation. Pretreatment with PKC inhibitor GF109203X inhibited ERK1/2 phosphorylation (Figure 8A), whereas no effect on the phosphorylation of JNK was seen (Figure 8B).
Discussion
The presence of arterial calcification has tended to been viewed either as a passive bystander phenomenon or as a useful clinical marker of atherosclerotic disease progression. Here we clearly demonstrate an active inflammatory response by macrophages to BCP crystals with implications for atherosclerosis disease pathogenesis and treatment. We have shown that human macrophages interact with BCP crystals, apparently taking them into vacuoles. In response, macrophages secrete biologically significant quantities of the proinflammatory cytokines TNF, IL-1, and IL-8, which are capable of stimulating the activation of endothelial cells and recruitment of mononuclear cells in an in vitro hemodynamic flow assay. Since TNF can promote osteoblastic differentiation and calcification of vascular cells,23eC25 our data suggest a positive feedback loop predisposing to chronic inflammatory pathology. The model we propose may be of more relevance to intimal than medial calcification, in which the proximity of calcific deposits to macrophages is less clear and in which there may be separate calcification mechanisms.35
Scanning electron microscopy of BCP particles extracted from atherosclerotic arteries has revealed marked heterogeneity of shape and size,2 and it remains to be determined whether these physical characteristics influence macrophage inflammatory responsiveness. In preliminary experiments we have found that the macrophage activating potential of BCP particles is inversely correlated with their size, suggesting that small isolated calcific particles in early disease may be more proinflammatory than larger deposits resembling bone seen in established lesions.26,36 If so, it is possible that the inflammatory calcification paradigm we propose is of particular relevance to the progression of early atherosclerosis.
Although the amount of arterial calcification may predict coronary events, it is still unclear whether calcified plaques are more prone to rupture.7eC10 Nevertheless, the biological response to BCP crystals could promote plaque instability. Thus, several studies have demonstrated that BCP is capable of inducing fibroblast MMP production via PKC and ERK1/2 signaling mechanisms.19,37 In preliminary experiments we have not been able to show increased expression of MMP-2 or -9 in BCP crystal stimulated macrophages. Nevertheless, BCP crystals might lead to increased tissue proteinase expression and activation by acting on macrophages under conditions so far untested, or indirectly via the effects of macrophage-derived TNF on vascular smooth muscle cells (VSMC).38 Furthermore, besides contributing to leukocyte recruitment, macrophage-derived TNF might also destabilise plaque by contributing to VSMC apoptosis.39
It is increasingly recognized that macrophages are not always proinflammatory but may play a role in resolution and repair.40 Thus we and others have demonstrated a more antiinflammatory profile of cytokine expression in response to other particulate stimuli, such as apoptotic neutrophils, monosodium-urate crystals, and hemoglobin-haptoglobin complexes.41eC43 The differences in the receptor and signaling mechanisms responsible for the variable response of macrophages to these particles requires further study.
A surface receptor responsible for the uptake of BCP crystals by cells has not yet been identified, and it is possible that signal transduction may be mediated by proteins opsonised by crystals rather than by the crystal surface per se. The effects we have observed, however, appear not to be secondary to dissolution of BCP crystals raising the calcium and phosphate concentrations, since we found no detectable change in calcium or phosphate concentrations in supernatants of BCP-macrophage cocultures, and centrifuged supernatants from macrophages cocultured with BCP crystals count not reproduce the effects. Judging from an experiment culturing BCP crystals with macrophages on different sides of a semipermeable membrane, contact between the particles and macrophages is required. However contact is not sufficient, since TNF release was blocked by inhibiting the cytoskeleton with cytochalasin-D.
We have unequivocally identified PKC as a key regulator of BCP-induced TNF release. Several studies have demonstrated an active role for this isoenzyme in the development of phagolysosomes by macrophages in response to microorganisms and other stumili.44eC47 Similarly, our confocal microscopy data demonstrate that BCP crystals stimulate the rapid translocation of PKC, first to the plasma membrane and then to vacuoles, consistent with PKCa activation being a critical link for downstream signaling. It remains to be determined whether these vacuoles are truly intracellular, or similar to the surface-connected compartments that take up aggregated LDL.48
Our data show that both the ERK1/2 and the JNK kinase pathways are involved in BCP-induced TNF release, but that only ERK1/2 is dependent on upstream PKC activation. These data are consistent with several similar observations in human monocytic cells in response to other agonists.49eC51 Interestingly, there appears to be cell specificity in the macrophage response to BCP, as BCP crystal-induced activation of ERK1/2 in fibroblasts has been found to be dependent on PKCe?rather than a calcium-dependent PKC.19,52 A recent study in human macrophages described the release of TNF in response to phorbol ester or LPS, mediated through ERK1/2 via the upstream activation of one of the conventional PKC isozymes, or .53 Importantly, that study demonstrated differential effects on proinflammatory versus antiinflammatory cytokine secretion depending on whether conventional PKC ( or ) or atypical PKC isozymes were activated. Based on the conclusions of this study, therapeutic blockade of PKC isozyme might result in blockade of proinflammatory cytokine production but also a switch to antiinflammatory cytokine production.
In conclusion, through investigation of the mechanisms regulating TNF production, we have identified key roles for PKC, ERK1/2, and JNK in downstream signaling in response to BCP crystals. This raises the possibility that specific targeting of such upstream signaling pathways may represent a more effective strategy than blocking TNF, a downstream product of the inflammatory cascade. Specifically, the identification of PKC as a proximal signaling molecule makes this a possible therapeutic target for the development of novel treatments against arterial calcification and other calcification-related chronic inflammatory diseases.
Acknowledgments
The study was funded by the British Heart Foundation.
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