Rheumatoid arthritis is a chronic destructive disease of joints, characterized by inflammation, synovial hyperplasia, and abnormal cellular and humoral immune responses. Although several types of cells have been proposed to trigger synovial inflammation, the etiologic process and pathogenesis of the disease remain controversial. But, as indicated by a recent study by Lee et al.,1 the mast cell is emerging as a key player in the erosive and inflammatory events leading to joint destruction (Figure 1).The inflammatory processes that result in rheumatoid arthritis are multifactorial, involving complex interactions among the cytokine network, autoantibodies, and the complement cascade. Various mediators are . . .

OBJECTIVES:To determine the effects of histamine on the proliferative rate of human articular chondrocytes (HAC) in vitro, and to demonstrate whether HAC in osteoarthritic (OA) cartilage express histamine and histidine decarboxylase (HDC). METHODS:HAC in vitro were incubated with and without histamine in 96 well culture plates and the extent of cell proliferation was determined using the naphthol blue-black method. Histamine effects were analysed with the histamine H(1) and H(2) receptor antagonists, mepyramine and ranitidine, respectively. Rabbit polyclonal antibodies and alkaline phosphatase conjugated secondary antibodies were used, and histamine and HDC were demonstrated by immunohistochemistry in OA cartilage tissues. RESULTS:Histamine stimulated the proliferation of HAC in culture. This stimulation was blocked by the addition of mepyramine, but not ranitidine, suggesting that the effect is mediated through H(1) histamine receptors. The addition of alpha-fluoromethylhistidine, a specific inhibitor of histidine decarboxylase (the enzyme responsible for histamine production), reduced the rate of proliferation of HAC. Both histamine and histidine decarboxylase were demonstrated in chondrocytes of OA cartilage by immunohistochemistry. CONCLUSION:Changes induced by histamine in the proliferative rate of HAC may contribute to the formation of chondrocyte clusters associated with OA cartilage; an observation supported by the demonstration of histamine and HDC expression by chondrocytes of OA cartilage in situ.

This article describes a novel electrochemical technique for the real-time monitoring of changes in the behaviour of adherent human cells in vitro: i.e., a biosensor that combines a biological recognition mechanism with a physical transduction technique, described collectively as Oncoprobe. Confluent viable cells adherent to gold electrodes (sensors) modify the extracellular microenvironment at the cell:sensor interface to produce a change in the electrochemical potential compared to that measured in the absence of cells. The potential was measured as an open circuit potential (OCP) with respect to a saturated calomel reference in the bulk culture medium. Typical OCP values for confluent cultures of human breast carcinoma cells, 8701-BC, approximated -100 mV compared with cell-free values of approximately -15 mV. The OCP for 8701-BC cells was modified in response to temperature changes over the range 32 to 40 degrees C and also to treatments with phytohemagglutinin (PHA, 25 microg/mL), cycloheximide (30 microM) and interleukin-1 beta (IL-1, 0.5 ng/mL) over 24 h. Cultures of synovial fibroblasts also responded to the same treatments with similar responses, producing negative shifts in the OCP signal with PHA and IL-I, but a positive shift in OCP signal with cycloheximide, all relative to the untreated control cultures. From experimental data and theoretical considerations it is proposed that the cell-derived signals are mixed electrode potentials reflecting a "conditioned," more reducing environment at the cell:sensor interface. Only viable cells caused a negative shift in the OCP signal, this being lost when cells were rendered nonviable by formalin exposure. This technology appears unique in its ability to passively "listen in" on cell surface activities, suggesting numerous applications in the fields of drug discovery, chemotherapy, and cell behaviour.

Objectives: To determine the effects of histamine on matrix metalloproteinase (MMP) production by human articular chondrocytes (HAC) in vitro. Methods: Conditioned culture medium from HAC cultures incubated with and without 20 μM histamine was assayed by enzymne linked immunosorbent assay (ELISA) for MMP-1, MMP-8, MMP-13 (collagenases 1, 2, and 3, respectively) and MMP-3 (stromelysin). Monolayer cultures of HAC were also immunostained for MMP-13 and MMP-3. Results: The HAC cultures showed a significant increase in MMP-13 and MMP-3 production (2.2- and 1.9-fold, respectively) after treatment with 20 μM histamine for 24 hours, but MMP-1 and MMP-8 were unaffected. All cultures showed MMP-13 and MMP-3 detectable by immunolocalisation. MMP-3 was the more prominent enzyme as shown by both ELISA and immunolocalisation techniques. Conclusions: Histamine exposure increased both MMP-13 and MMP-3 production by HAC in vitro, thereby suggesting a pathophysiological role in the chondrocytic phenotype associated with degenerative changes in osteoarthritis.

A novel electrochemical technique which detects and monitors real-time changes in cell behavior in vitro has been used to examine the effects of recognized anticancer drugs on the human ovarian carcinoma cell line A2780 and its adriamycin (A2780adr)- and cisplatin (A2780cispt)-resistant variants. These cells, adherent to gold electrodes or sensors, modify the extracellular microenvironment at the cell:sensor interface, producing an electrochemical potential that is different from that of the bulk culture medium. Confluent, adherent A2780 cells produced an electrochemical signal, measured as an open circuit potential (OCP), of approximately -100 mV compared to a cell-free value of approximately -15 mV. Exposure of A2780 cells to cisplatin (range 10(-4) to 10(-6) M), adriamycin (range 10(-5) to 10(-7) M), and vinblastine (10(-6) M) all produced positive shifts in the OCP signal relative to untreated control cells during 24 h of culture, but Taxotere (range 10(-5) to 10(-7) M) had no effect. These positive shifts in OCP signal were evident well before observations of reduced cellular adhesion and viability after 24 h, as judged in parallel cultures with a plastic substratum and by scanning electron microscopy. By contrast, the same treatments applied to the A2780adr and A2780cispt variants showed that each demonstrated different sensitivities to the same drugs applied to the parental A2780 cells. The effects of the same four anticancer drugs on ovarian carcinoma (A2780) and breast carcinoma (8701-BC) cell lines showed that the former was far more responsive to adriamycin and cisplatin. Such differences in drug sensitivities between the two cell lines were subsequently confirmed using the conventional MTT assay over 5 days. Although this electrochemical technology readily detects changes in cell adhesion and viability, the modified OCP signals recorded within a few hours of anticancer drug treatments are evident well before microscopic morphological changes become apparent. It is proposed that these early changes in OCP signals, relative to control untreated cells, reflect modifications of physiological/behavioral processes manifested at the cell surface.

Eosinophils are a recognized feature of inflammatory bowel disease (IBD), but their tissue distribution and functional importance in Crohn's disease (CD) and ulcerative colitis (UC) remain obscure. This study describes an improved immunohistochemical protocol to identify eosinophils in full thickness bowel wall specimens of IBD (n=40) and their in situ relationships with the chemoattractants eotaxin and RANTES. Eosinophils were identified using immunohistochemistry with a combination of monoclonal antibodies (EG1+EG2+MBP), an ultrasensitive technique superior to other methodologies, and their tissue distributions were related to those for eotaxin, RANTES, mast cells and neutrophils. Increased numbers of eosinophils (up to 400 cells/mm2) were observed in active, fulminant inflammation in both CD and UC, this being related to the severity of inflammation and not the diagnosis of the two disorders. The chemoattractants eotaxin (CCL11) and RANTES (CCL5) were upregulated in IBD tissues showing eosinophilia. Neutrophils and mast cells were commonly associated with eosinophil accumulations. Eosinophil numbers and their in situ activation are increased in active rather than chronic IBD. The observations strongly suggest a pivotal role for the eosinophil and its potent mediators in many pathophysiological symptoms of CD and UC, where it represents the major proportion of all granulocytic cells in active inflammatory bowel disease. Copyright © 2001 John Wiley & Sons, Ltd.

OBJECTIVESTo examine the in situ distributions of vitamin D receptors (VDR) and matrix metalloproteinases (MMPs) in osteoarthritic cartilage for comparison with non-arthritic, normal cartilage; and to assess the in vitro effects of 1alpha,25 dihydroxyvitaminD(3)(1alpha,25(OH)(2)D(3)) on MMPs-1, -3 and -9 and prostaglandin E(2)(PGE(2)) production by cultures of human articular chondrocytes (HAC) shown to be VDR-positive.METHODSUsing immunohistochemistry VDR expression in different specimens of osteoarthritic cartilage (N=11) was compared to that in normal cartilage (N=6), along with the immunodetection of MMPs-1, -3 and -9. The effects of 1alpha25(OH)(2)D(3)on MMP and PGE(2)production by HAC in vitro, with and without stimulation by TNFalpha or phorbol myristate acetate (PMA), was evaluated using ELISA methodology.RESULTSVDR was demonstrated in HAC of all specimens of osteoarthritic cartilage, especially the superficial zone, whereas only two of five normal cartilage specimens were VDR(+)for a minor proportion of HAC. Immunolocalization of MMPs-1, -3 and -9 was often seen in areas where chondrocytes were VDR(+), and dual immunolocalization has demonstrated individual chondrocytes positive for both VDR and MMP-3 in situ. In vitro, 1alpha25(OH)(2)D(3)alone had no effect on MMP-1, -9 and PGE(2)production by HAC, but MMP-3 production was up-regulated by 1alpha25(OH)(2)D(3)either with or without stimulation with TNFalpha or PMA. By contrast the increased production of MMP-9 and PGE(2)induced by PMA was significantly suppressed by concomitant treatment with 1alpha25(OH)(2)D(3).CONCLUSIONSThe demonstration of VDR expression by HAC in osteoarthritic cartilage was often associated with sites where MMP expression was prevalent, observations in contrast to their virtual absence in normal age-matched cartilage. Together with HAC in vitro studies, the data suggests that 1alpha25(OH)(2)D(3)contributes to the regulation of MMP and PGE(2)production by HAC in osteoarthritic cartilage.

OBJECTIVE:To examine by immunohistochemistry the relative distributions of 6 matrix metalloproteinases (MMPs 1, 2, 3, 8, 9, and 13) and the 2 proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in osteoarthritic (OA) cartilage compared with normal, age-matched articular cartilage.METHODS:Articular cartilage samples were obtained from the tibial plateau of OA knees removed at arthroplasty and from normal, nonarthritic, knees obtained at autopsy. Specimens were promptly fixed in Carnoy's fixative, processed, embedded in paraffin, sectioned, and examined by immunohistochemistry for MMP and cytokine production. In addition, human articular chondrocytes (HAC) were treated in vitro with either IL-1beta, TNFalpha, or phorbol myristate acetate (PMA) to assess their potential to produce each of the MMPs, as determined by Western blotting and gelatin zymography.RESULTS:Immunodetection of the collagenases (MMPs 1, 8, and 13) and stromelysin 1 (MMP-3) was demonstrated in a proportion of chondrocytes in the superficial zone of almost all of the OA specimens that had degenerative matrix changes. The gelatinases (MMPs 2 and 9) were also demonstrated by immunohistochemistry but were not so prominent. IL-1beta- and TNFalpha-positive chondrocytes were also observed in a proportion of cells in the superficial zones of OA specimens. Much less immunostaining for MMPs and cytokines was observed in the deep zone of all OA specimens, where the cartilage matrix and chondrocyte morphology appeared normal. In contrast, full-thickness normal cartilage specimens showed virtually no immunostaining for these MMPs or cytokines. Confirmation that chondrocytes can produce these 6 MMPs was obtained from HAC cultures treated with either IL-1beta, TNFalpha, or PMA; conditioned medium from activated HAC contained all the MMPs demonstrated by immunohistochemistry. Dual immunolocalization studies of OA cartilage specimens demonstrated the coexpression of IL-1 with MMP-8 by individual chondrocytes in situ.CONCLUSION:These results indicate that the superficial zone of OA cartilage specimens, which is characterized by fibrillations, chondrocyte clusters, and degenerative matrix changes, contains a variable proportion of cells that immunostain for IL-1beta, TNFalpha, and 6 different MMPs. These observations support the concept that cytokine-MMP associations reflect a modified chondrocyte phenotype and an intrinsic process of cartilage degradation in OA.

The matrix metalloproteinases (MMPs) are considered to have an important role in connective tissue degradation and have been implicated in the mechanisms of tumour invasion and metastatic spread. We have used immunohistochemistry to examine and compare the tissue distributions of collagenase-1 (MMP-1), gelatinase A (MMP-2) and stromelysin-1 (MMP-3) in 18 specimens of malignant melanoma, viz. 10 superficial spreading and 8 nodular melanomas. MMPs-1, -2 and -3 were demonstrated within melanoma and host tissue cells, especially at the periphery of some tumours, but were usually restricted to less than 10% of total melanoma cells. The MMPs were absent from 'normal' skin tissue distant from the tumour. MMP-2 was localised to discrete groups of cells and was especially evident at the epidermal:tumour interface, whereas MMP-3 was mainly confined to the deeper margins of melanoma. No regular pattern of MMP expression was observed for either the superficial spreading or the nodular melanomas. The variable distributions of the MMPs suggested that enzyme expression was subject to local microenvironmental regulation, possibly in response to matrix components and the cellular heterogeneity observed at the tumour margins. These in situ observations add weight to the concept that specific MMPs contribute to the mechanisms of tumour invasion.

The status of bronchial cartilage degeneration in chronic bronchitis is unclear, and little is known about the chondrolytic mechanisms involved. The potential contributions of various inflammatory cells, chondrocytes and cartilage-degrading enzymes to cartilage atrophy have been examined. Bronchial cartilage specimens were obtained at autopsy from lobar secondary bronchi from chronic bronchitics and age-matched controls; each was examined by light microscopy and immunohistology for the distributions of mast cells, macrophages, eosinophils, collagenase 1, collagenase 3, and degradation products of cartilage collagen. Most bronchitic specimens showed hypertrophic chondrocytes, some of which were immunostained for collagenase 3, and occasionally for collagenase 1. Evidence for collagen degradation products was demonstrated around the lacunae of a proportion of chondrocytes, and both collagenases were also observed in the soft inflammatory tissues in close association with the cartilage surface, together with variable distributions of mast cells and macrophages. Such observations were generally absent or very much reduced in the control, non-bronchitic specimens. Degenerative changes, atrophy and loss of bronchial cartilage were common features of most chronic bronchitic specimens, this usually being related to intrinsic changes in the chondrocyte phenotype, including proliferative and matrix-degrading properties. Mast cells and macrophages were often observed in close association with the bronchial cartilage, suggesting that inflammatory cells may also contribute to the mechanisms of bronchial cartilage degradation and loss. These observations of bronchial cartilage degeneration were generally lacking in age-matched non-bronchitic control specimens.

An improved immunohistochemical method has been used to assess neovascularization within the vulnerable 'shoulder' regions of atherosclerotic plaques from carotid arteries. A combination of monoclonal antibodies (CD31, CD34, +/- von Willebrand factor) was shown to be far more effective than conventional techniques in demonstrating extensive vascularizations within the 'shoulder' and cap regions of late-stage plaques. Such sites were shown to be microfocal, often appearing as a plexus of both large and small vessels which occupied a significant proportion of the 'shoulder' area. These regions of marked neovascularization were commonly associated with accumulations of macrophages, mast cells, and T-cells, indicative of local inflammatory reactions. The matrix components elastin and collagen type VI showed variable distributions which suggested extensive tissue remodelling, whereas collagen type IV was recognized as a basement membrane protein of most blood vessels, as well as being associated with 'stellate' smooth muscle cells. Evidence of local microvascular damage within the shoulder regions of some specimens was demonstrated by extravascular red blood cells, macrophages containing haemosiderin, and perivascular fibrin deposition. These local haemorrhages derived from microvessels beneath the lining of the arterial lumen are a further indication of how the microfocal vascularization of the plaque 'shoulder' might contribute to further complications of inflammation and plaque destabilization in late-stage disease.

OBJECTIVESThe active form of vitamin D3, 1α,25 dihydroxyvitamin D3(1,25D3), through its interaction with vitamin D receptors (VDR), is reported to effect a variety of anabolic and catabolic events, especially in bone and cartilage tissues. As cartilage degradation and tissue remodelling are characteristic features of the rheumatoid lesion, the distribution and expression of VDR at sites of cartilage erosion was examined.METHODSImmunolocalisation techniques using a rat monoclonal antibody to VDR and an alkaline phosphatase conjugated avidin/biotin detection system were used to examine VDR in 18 specimens of cartilage-pannus junction, 10 specimens of rheumatoid synovium or cartilage tissue, and four primary cultures of adherent rheumatoid synovial cells (RSC). For comparison, VDR expression was examined in 10 specimens of normal, healthy age matched articular cartilage.RESULTSVDR was demonstrated in 15 of 18 cartilage-pannus junctions either at the interface (8 of 18), within the pannus tissue (12 of 18), and by chondrocytes often close to the erosive lesion (10 of 18). All the rheumatoid synovial tissue and 5 of 10 cartilage specimens showed cells with positive staining, but the extent of this was variable. Negligible VDR staining was observed for normal cartilage. Primary cultures of RSC also showed variability in both the numbers and proportions of macrophages or synovial fibroblasts stained for VDR (range 10–50%), this being more common in cultures with a high proportion of macrophages.CONCLUSIONSVDR expression has been demonstrated by most specimens of cartilage-pannus junction; was associated with various cell types, including chondrocytes, but not exclusively with CD68+ macrophages. The focal nature of VDR expression within the rheumatoid lesion suggests a contributory role for 1α,25D3 in the pathophysiological processes of rheumatoid arthritis.

Neovascularization is a prominent feature of late-stage atherosclerotic lesions and their complications but is generally regarded as an insignificant, undetectable component of the earliest stages of plaque development, probably because of relatively poor histological techniques. Using an improved vascular staining procedure, we have examined the extent of neovascularization in the earliest plaque lesions. Combined monoclonal antibodies to CD31, CD34, and von Willebrand factor have provided an ultrasensitive technique with which to visualize blood vessels in early atherosclerotic lesions (n = 55) of human carotid arteries obtained through surgical procedures. Capillary-like microvessels were shown in very early atherosclerotic lesions (type II), where they were associated with the distribution of macrophages and a few immature mast cells. Neovascularization was more prominent in type III lesions with vessels of variable size, often providing a focus around which local accumulations of macrophages and apolipoproteins A-I and B were visualized. Thickened type III lesions usually showed an intricate network of microvessels, together with numerous mast cells. These studies have shown neovascularization as a prominent feature of early stages of atherosclerotic plaque development. Whereas distribution of apolipoproteins A-I and B were observed in the very earliest stages of the plaque intima, these lipids, together with macrophages, foam cells, and mast cells, were observed as perivascular accumulations in a proportion of type II and III lesions. Such findings indicate that neovascularization is an important feature of early plaque development and may provide an additional or alternative source of leukocyte and lipid accumulations relative to the arterial lumen.

Annals of the New York Academy of SciencesVolume 878, Issue 1 p. 615-618 Effects of 1α,25DihydroxyvitaminD3 on Matrix Metalloproteinase Expression by Rheumatoid Synovial Cells and Articular Chondrocytes in Vitro LYNNE C. TETLOW, LYNNE C. TETLOW University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UKSearch for more papers by this authorDAVID E. WOOLLEY, Corresponding Author DAVID E. WOOLLEY Address for telecommunication: Phone, 0044 0161-276-4240; fax, 0044 0161-274-4833; e-mail, david.wooley@mri.cmht.nwest.nhs.ukSearch for more papers by this author LYNNE C. TETLOW, LYNNE C. TETLOW University Department of Medicine, Manchester Royal Infirmary, Oxford Road, Manchester, M13 9WL, UKSearch for more papers by this authorDAVID E. WOOLLEY, Corresponding Author DAVID E. WOOLLEY Address for telecommunication: Phone, 0044 0161-276-4240; fax, 0044 0161-274-4833; e-mail, david.wooley@mri.cmht.nwest.nhs.ukSearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb07742.xCitations: 2Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume878, Issue1INHIBITION OF MATRIX METALLOPROTEINASES THERAPEUTIC APPLICATIONSJune 1999Pages 615-618 RelatedInformation