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 Ⅲ cell biology Ⅲ calcification Ⅲ inflammation Ⅲ macrophage C oronary 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 (Ca 10 (PO 4 ) 6 (OH) 2 ) and similar to those that mineralize bone. [1][2][3][4][5][6] 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. [7][8][9][10] However, although arterial calcification is now accepted to be an active and highly regulated process similar to that of bone ossification, [11][12][13][14] 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. [17][18][19] 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 monocy...

Objective. We have hypothesized that the process of monocyte to macrophage differentiation may alter the inflammatory response of mononuclear phagocytes to the uptake of monosodium urate monohydrate (MSU) crystals.Methods. Eight mouse monocyte/macrophage cell lines were arranged in increasing order of differentiation, as judged by expression of the macrophage markers F4/80 and BM 8 and by phagocytic capacity. Secretion of tumor necrosis factor ␣ (TNF␣) in response to MSU was measured by enzyme-linked immunosorbent assay.Results. The panel of monocyte/macrophage cell lines revealed a close linkage between the state of differentiation and the capacity of the cells to ingest MSU crystals. TNF␣ production, however, was not linked to phagocytic ability. Peak TNF␣ levels were synthesized by cells at an intermediate state of differentiation (3.2-14.1 ng/ml), whereas mature macrophages, which efficiently phagocytosed crystals, did not secrete TNF␣. Mature cell lines produced TNF␣ when stimulated with zymosan (5.9-6.2 ng/ml), but this was abolished by coincubation with MSU crystals. Suppression of the zymosan response was not due to apoptosis or steric hindrance by MSU crystals. Culture supernatants from mature macrophages did not stimulate endothelial cell activation, in contrast to MSU-treated cells at an earlier stage of differentiation, which stimulated intercellular adhesion molecule 1 expression on sEND endothelioma cells through the release of TNF␣ (inhibited 80.6% by anti-TNF␣).Conclusion. We demonstrated that phagocytosis and TNF␣ production are distinct events in the response of mononuclear phagocytes to urate crystals, and these events can be distinguished at the level of macrophage differentiation. The noninflammatory removal of urate crystals by mature macrophages defines a new pathway that may be important in controlling the development of acute gout in patients with hyperuricemia.Acute gouty arthritis is a self-limiting inflammatory response to the intraarticular deposition of monosodium urate monohydrate (MSU) microcrystals. Previous studies investigating the effect of MSU crystals in mononuclear phagocytes have focused primarily on the ability of peripheral blood monocytes or monocytic cell lines to secrete proinflammatory cytokines such as tumor necrosis factor ␣ (TNF␣), interleukin-1 (IL-1), . Investigators in our group have further shown that TNF␣ and IL-1 released by peripheral blood monocytes can activate vascular endothelial cell expression of E-selectin, intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1, thereby stimulating both the recruitment of leukocytes to the site of crystal deposition and the amplification of the inflammatory response (6-8).Hyperuricemia and precipitation of inflammatory microcrystals in synovial fluid per se are not always predictive of acute synovitis, as the severity of disease Supported by grants from the British Heart Foundation and Glaxo Wellcome PLC.

Human apocrine and sebaceous glands function to secrete lipids, predominantly triglycerides, fatty acids, cholesterol and its esters, and, in the sebaceous gland, squalene. The enzymes that catalyze the important regulatory steps in cholesterol and fatty acid biosyntheses, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acetyl-CoA carboxylase, respectively, were therefore studied in isolated human skin appendages, and their relevant kinetic parameters determined. The enzyme activities that were observed can account for previously described rates of incorporation of radiolabeled substrates into the appropriate lipids by glands in vitro. Reduced enzyme activities following homogenization in the presence of fluoride indicated that both of these enzymes in skin appendages are inactivated by phosphorylation. The activity of the enzyme known to catalyze this phosphorylation, the AMP-activated protein kinase, was also measured. Compactin was shown to inhibit HMG-CoA reductase in homogenates of these appendages. Conversely, incubation of whole sebaceous glands with compactin resulted in the stimulation of enzyme activity, which suggests that these appendages can respond to diminishing cholesterol levels. The effect of exogenous low density lipoprotein and 25-hydroxycholesterol on HMG-CoA reductase activity from skin appendages was investigated. HMG-CoA reductase activity in both apocrine and sebaceous glands was reduced following incubation with either low density lipoprotein or 25-hydroxycholesterol. Low density lipoprotein receptor and lipoprotein lipase mRNA expression was also detected in skin appendages. These results indicate that apocrine and sebaceous glands have the capacity to sequester dietary cholesterol and fatty acids that may have important implications for the understanding of both acne and axillary odor.

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