Induction of Microsomal Prostaglandin E Synthase-1 in Human Gingival Fibroblasts

It is well established that prostaglandin E2 (PGE2) plays an important role in inflammatory diseases including periodontitis. Previously we have reported that the inflammatory mediators interleukin-1beta, (IL-1beta) and tumor necrosis factor alpha (TNFalpha) stimulate PGE2 synthesis by inducing mRNA expression of cyclooxygenase-2 (COX-2) in human gingival fibroblasts. In present study the involvement of microsomal prostaglandin E synthase-1 (mPGES-1) in relation to PGE2 production was investigated. The results showed that IL-1beta as well as TNFalpha induced mPGES-1 mRNA and protein expression accompanied by enhanced PGE2 production in gingival fibroblasts. The anti-inflammatory steroid dexamethasone (DEX) inhibited mPGES-1 mRNA and protein expression as well as PGE2 production induced by IL-1beta or TNFalpha. The COX-2 specific inhibitor, celecoxib, in contrast to the nonspecific COX inhibitor, indomethacin, markedly reduced mPGES-1 expression induced by IL-1beta. The results demonstrate that mPGES-1 regulates PGE2 production in gingival fibroblasts stimulated by inflammatory mediators IL-1beta and TNFa. This novel pathway may be a potential target for treatment strategies of periodontal disease.     

Synoviocytes in chronic synovitis in situ and cytokine stimulated synovial cells in vitro neo-express α1, α3 and α5 chains of β1 integrins

The expression of the 1 integrins was examined immunohistochemically in synoviocytes from normal synovial membrane and from chronic synovitis of different aetiology and intensity. Normal synoviocytes were 61-positive but lacked 1 through 5. In mild inflammation type A synoviocytes neo-expressed 1, 3, and 5 chains. In severe inflammation both type A and B synoviocytes expressed 3, 4, 5, and 6 chains. The effects of inflammatory cytokines, as single agents or in combination, on the 1 integrin expression in cultured normal synoviocytes was determined by immunocytochemistry and flow cytometry. The 1 chain, while absent in unstimulated synoviocytes, was induced by interleukin-1 (IL-1), tumour necrosis factor- (TNF-), and interferon- (INF-). This effect was enhanced by combining IL-1 and TNF-. Expression of the 3 chain was up-regulated by IL-1 and, more intensely, by IFN-. Transforming growth factor (TGF-) inhibited the up-regulating effect of IL-1 and antagonized the effect of IFN- on 3 chain expression. Expression of the 5 chain was up-regulated significantly by co-stimulation through IL-1 together with TGF- or TNF-. Thus, the 1 integrin profile of cytokine activated synoviocytes in vitro resembled that of synoviocytes in synovitis in situ. These data suggest that IL-1, TNF-, IFN-, and TGF- are likely to be among the effectors regulating 1 integrin expression in synoviocytes in vivo.     

Functional adaptation to high PCO2 of apically and basolaterally located Na+/H+ exchange activities in cultured renal cell lines

Cultured renal epithelial cells grown on filter support were examined for functional adaptation of Na⁺/H⁺ exchange activities to respiratory acidaemia, which was mimicked by increasing PCO₂ from 5% to 10% during 24 h or 48 h of cell culture. We have selected proximal tubular cell lines with either dual location of Na⁺/H⁺ exchange activities (MCT cells, RKPC-2 cells), apical location of Na⁺/H⁺ exchange activity (OK/ WOK cells) or a basolateral location of Na⁺/H⁺ exchange activities (LLC-PK₁/clone 4 cells, MDCK cells). Na⁺/H⁺ exchange activity was determined microspectrofluorometrically (using BCECF) in the absence of CO₂/HCO ₃ ^– . Respiratory acidaemia specifically increased apical Na⁺/H⁺ exchange activity (previously classified as amiloride-resistant) in MCT cells, in RKPC-2 cells and in WOK cells; it stimulated basolateral Na⁺/H⁺ exchange activity (previously shown to be amiloride-sensitive) in RKPC-2 cells, in LLC-PK₁/clone 4 cells and in MDCK cells, but did not affect basolateral Na⁺/H⁺ exchange activity in MCT cells. In MCT and in RKPC-2 cells the effect of high PCO₂ on apical Na⁺/H⁺ exchange was prevented by inhibition of protein kinase C. In RKPC-2 cells, activation of basolateral Na⁺/H⁺ exchange by high PCO₂ occurred also when protein kinase C was inhibited. In conclusion, these studies demonstrate stimulation of apical Na⁺/H⁺ exchange, but differential regulation of basolateral Na⁺/H⁺ exchange activities in response to a high-PCO₂-induced acid environment. Protein kinase C activation might be involved in mediating the effect of acidaemia on stimulation of apical Na⁺/H⁺ exchange activity (MCT and RKPC-2 cells).     

Circulating CD4+CD25+ T Regulatory Cells Are Not Altered in Multiple Sclerosis and Unaffected by Disease-Modulating Drugs

Experimental animal models for autoimmunity have demonstrated the existence and crucial role of CD4(+)CD25(+) T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent in vitro functional studies showed that Tr cells are enriched in the CD25(high) cell population among CD4(+) T cells, and that they totally inhibit proliferation and cytokine secretion by CD4(+) T cells. It is not yet known if circulating Tr cells are involved in multiple sclerosis (MS). This study was done firstly to determine whether alterations of the CD4 (+) CD25(high) T cells occur in MS, examining their frequencies. As it was reported that the suppressive activity of CD4(+)CD25(+) Tr cells is mainly through cell surface contact pathway, we secondly analyzed the expression of the functionally important cell surface molecules of CD4(+)CD25(high) Tr cells. Two- or three-colour flow cytometry was used to identify and quantify CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells among blood CD4(+) T cells in MS patients without treatment vs. patients treated with either interferon-beta (IFN-beta) or glatiramer acetate (GA) or IFN-beta + GA in combination vs. healthy controls (HC). Expression of functionally important surface molecules CD45RO, CD69, CD95, HLA-DR, and intracellular CTLA-4 and IL-10 production by CD4(+)CD25(high) Tr cells were investigated. CD4(+)CD25(+) T cells constituted around 6% of CD4(+)T cells in all MS patient groups, and 7% in HC. There were also no changes in the proportions of CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells in a longitudinal follow-up of MS patients before and during IFN-beta treatment. Frequencies of circulating CD4(+)CD25(high)Tr cells among CD4(+) T cells were also similar and their surface or intracellular molecular expression did not vary in MS patients, irrespective of treatment, compared to HC. This study suggests that levels of circulating CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells are not altered in MS, and are unaffected by substances currently used to modulate the disease.     

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha,interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF- production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.     

Mechanisms of Na+-K+-ATPase phosphorylation by PKC in the medullary thick ascending limb of Henle in the rat

Sodium transport correlates with varying Na⁺-K⁺-ATPase activity rates along the nephron. Whether differences in Na⁺-K⁺-ATPase regulation by protein kinase C-dependent phosphorylation are also present has not been tested. We measured the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation by the binding of McK-1 antibody to dephosphorylated Ser-23 and Na⁺-K⁺-ATPase activity in medullary thick ascending limb of Henle (mTAL) and proximal tubules (PCT). The degree of Na⁺-K⁺-ATPase phosphorylation at Ser-23 was lower in mTAL than in PCT (DU 13.43±1.99 versus 2.3±0.20, respectively, P<0.01) while Na⁺-K⁺-ATPase activity was higher in mTAL (3,402±83 vs 711±158 pmol/mm tubule per hour in PCT, P<0.01). PKC inhibitor RO-318220 10^–6 M decreased phosphorylation in PCT to 125±10% (P<0.05). In mTAL, RO-318220 did not modify the phosphorylation degree or the activity of Na⁺-K⁺-ATPase. Both calcineurin inhibitor FK-506 10^–6 M and phorbol 12-myristate 13-acetate (PMA) 10^–6 M increased the degree of Na⁺-K⁺-ATPase phosphorylation (P<0.05) and inhibited Na⁺-K⁺-ATPase activity to 657±152 and 1,448±347 pmol/mm tubule per hour, respectively, in mTAL (P<0.01). Increase in [Na⁺]_i to 30, 50 and 70 mM resulted in no changes in Na⁺-K⁺-ATPase phosphorylation degree or activity in mTAL. Conversely, in PCT increments in [Na⁺]_i were paralleled by decreased phosphorylation (from 120±7 to 160±15% of controls, P<0.05) and increased Na⁺-K⁺-ATPase activity (from 850±139 to 1,874±203 pmol/mm tubule per hour, P<0.01). Dopamine (DA) 10^–6 M decreased both Na⁺-K⁺-ATPase dephosphorylation to 41.85±9.58% (P<0.05) and Na⁺-K⁺-ATPase activity to 2,405±176 pmol/mm tubule per hour in mTAL (P<0.01). RO-318220 reversed DA effects. Data suggest that regulation of the degree of Na⁺-K⁺-ATPase ₁ subunit phosphorylation at Ser-23 and enzyme activity have different mechanisms in mTAL than in PCT, and may help us to understand the physiological heterogeneity of both segments.     

The differential effects of IL-1 and TNF-α on proinflammatory cytokine and matrix metalloproteinase expression in human chondrosarcoma cells

Objective and design:Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and matrix metalloproteinases (MMPs) play important roles in the pathogenesis of osteoarthritis (OA). In the present study, using Affymetrix oligonucleotide array technology and real-time quantitative RT-PCR we have investigated the molecular mechanisms underlying the differential effect of IL-1 and TNF- on gene expression in the human chondrosarcoma cell line, SW1353. Materials and methods:SW1353 cells were stimulated singularly with IL-1, TNF-, Phorbol 12-myristate 13-acetate (PMA), or treated with the combination of cytokine and PMA. Total RNA was collected at multiple time points over a 24-h period followed by biotinylated cRNA target preparation and hybridization onto the Affymetrix HG-U95Av2 array. The differential expression patterns of several cytokine and MMP genes were further confirmed by real time quantitative RT-PCR, Western blot, and ELISA. Results:Our microarray experiments have broadly confirmed previously published data on chondrocyte gene expression regulated by IL-1 and TNF-. The expression pattern of proIL-1, MMP-1, and MMP-13 in chondrocytes is differentially regulated when stimulated with proinflammatory cytokines. IL-1, but not TNF-, can induce IL-6, bone morphogenic protein 2 (BMP-2), and cyclooxygenase (COX-2) expression in SW1353 cells. Additionally, our Western blot results provide the first evidence that IL-1 is produced in the proform in IL-1-activated chondrosarcoma cells and that additional signals are required for its posttranslational processing/activation. Conclusions:IL-1 and TNF- each activate a distinct set of genes in chondrosarcoma cells, and gene expression in these cells is regulated by groups of genes related in part by their function. Chondrocyte IL-1 appears to serve an important role in the pathogenesis OA contributing to joint inflammation and cartilage destruction.Received 15 September 2003; returned for revision 16 October 2003; accepted by J. S. Skotnicki 11 March 2004     

Differential effects of interleukin-1beta and transforming growth factor-beta1 on the expression of the inflammation-associated protein, ADAMTS-1, in human decidual stromal cells in vitro

BACKGROUND: The pro-inflammatory cytokine, interleukin-1 beta (IL-1beta) promotes the proteolytic degradation of the extracellular matrix (ECM) of maternal decidua, a critical step in pregnancy that is counterbalanced by the expression of the anti-inflammatory cytokine, transforming growth factor-beta 1 (TGF-beta1). Recently, the inflammation-associated protein, ADAMTS-1, a member of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin repeats) gene family of metalloproteinases has been assigned a central role in the formation and organization of tissues. In view of these observations, we have hypothesized that ADAMTS-1 contributes to the cytokine-mediated remodelling of decidual ECM. METHODS: The spatiotemporal expression of ADAMTS-1 in human endometrium was examined by immunohistochemistry. A quantitative-competitive (QC)-PCR strategy and western blot analysis was then employed to determine whether IL-1beta and TGF-beta1 regulate ADAMTS-1 mRNA and protein expression levels in primary cultures of stromal cells isolated from first trimester decidua. RESULTS: ADAMTS-1 expression is associated with decidualization of the endometrial stroma in vivo. IL-1beta increased whereas TGF-beta1 decreased ADAMTS-1 mRNA and protein levels in decidual stromal cell cultures in a concentration- and time-dependent manner. These regulatory effects were attenuated by function-perturbing antibodies specific for either cytokine. CONCLUSION: IL-1beta and TGF-beta1 differentially regulate ADAMTS-1 expression in human decidual stromal cells.     

Induction of cyclooxygenase-1 in cultured synovial cells isolated from rheumatoid arthritis patients

Objective and design:The aim of this study was to confirm the involvement of cyclooxygenase (COX)-1 in rheumatoid arthritis (RA).Materials and subjects:Synovial cells isolated from arthritic patients were cultured primarily and consecutively for 8 passages.Treatment:The cultured synovial cells were incubated with 10 ng/ml of interleukin-1 (IL-1) for 6 h.Methods:The effects of either COX-1 or COX-2 selective inhibitor on prostaglandin E₂ (PGE₂) production was estimated by enzyme-linked immunosorbent assay (ELISA) and the expression of COX-1 and COX-2 were determined by Western blotting and immunocytochemistry.Results:IL-1-induced PGE2 production in synovial cells isolated from RA in primary culture was inhibited by mofezolac, a selective inhibitor of COX-1, as well as NS-398, a specific inhibitor of COX-2. The similar inhibitory patterns were obtained in the RA-derived synovial cells within 3 passages. However, COX activity in the RA-derived synovial cells after 5 passages was inhibited by NS-398, but not by mofezolac. In contrast, COX activity in primary and consecutively cultured synovial cells isolated from osteoarthritis (OA) or normal arthritis was inhibited by NS-398, but not by mofezolac. Western blot and immunocytochemical analyses of COX-1 and COX-2 in the synovial cells isolated from RA patients within 3 passages showed an induction in both COX-1 and COX-2 expression by IL-1. The induction of both COX-1 and COX-2 was inhibited by dexamethasone.Conclusions:These experiments demonstrate COX-1 induction in synovial cells isolated from RA patients, suggesting that COX-1 is involved in the progression of RA.Received 12 November 2003; returned for revision 3 December 2003; accepted by M. Katori 5 December 2003     

Integrin-Mediated Preadipocyte Adhesion and Migration on Laminin-1

Cell adhesion and migration are key events in many biological processes and depend on extracellular matrix proteins. Understanding these cellular events in adipogenesis is paramount to elucidating the biological mechanisms underlying preadipocyte-specific aspects of obesity, diabetes, and adipose tissue development for the design of pharmaceutical screening and tissue engineering strategies. We quantitatively investigated preadipocyte adhesion and migration on laminin-1 surfaces and identified candidate cognate preadipocyte receptors for laminin-1. In adhesion studies, we found that preadipocytes readily adhered to laminin-1 as compared with other extracellular matrix proteins. In addition, immunocytochemical analysis demonstrated that an array of integrin molecules was present on the surface of preadipocytes. Preadipocyte adhesion on laminin-1 was quantitatively assessed using a sedimentation adhesion assay, and results suggested that preadipocyte adhesion to laminin-1 was mediated by the ₁₁ integrin. In addition, digital time-lapse microscopy and quantitative cell tracking revealed that inhibition of the ₁₁ integrin resulted in abrogation of preadipocyte migration on laminin-1 surfaces. These results strongly support the hypothesis that preadipocyte adhesion to and migration on laminin-1 substrata are regulated, in part, by integrins. © 2003 Biomedical Engineering Society. PAC2003: 8717Jj, 8715Kg, 8764Rr, 8714Ee     

Differential Regulation of IL-8 by IL-1β and TNFα in Hyaline Membrane Disease

Mechanisms that regulate cytokine-mediated inflammation in the lungs of preterm infants, including factors which regulate production of the chemokine IL-8, remain poorly defined. Sequential bronchoalveolar lavage samples were obtained from preterm newborns with hyaline membrane disease over a 28-day period. Bronchoalveolar lavage cell cytokine relationships were evaluated and the differential regulation of IL-8 by IL-1 and TNF was studied in a short-term culture system. In vivo, IL-8 and IL-l protein levels correlated closely with each other and with macrophage counts. In cell culture, exogenous anti-IL-1 antibody led to a 40% maximum inhibition (approximately) of IL-8 production by lipopolysaccharide stimulated lung inflammatory cells. Comparable amounts of exogenous anti-TNF antibodies achieved a 15% maximum inhibition (approximately) of IL-8 production. Anti-IL-1 and anti-TNF antibodies in combination did not inhibit IL-8 production beyond that achieved by anti-IL-l antibody alone. These results, in preterm newborns, support the concept of lung inflammation mediated in part by a macrophage, IL-1, and IL-8 cell cytokine pathway. The results also suggest that factors other than IL-1 and TNF regulate IL-8 expression in the lungs of preterm infants.     

Expression of insulin-like growth factor mitogenic signals in adult soft-tissue sarcomas: significant correlation with malignant potential

The insulin-like growth factor (IGF) signal transduction system involves receptors, ligands and binding proteins (IGFBPs) that have been shown to have mitogenic and distinct anti-apoptotic effects on malignant cell lines of both epithelial and mesenchymal origin. Expression of the IGF signal system might be a mechanism by which human soft-tissue sarcomas (STS) obtain a proliferative advantage over normal adjacent tissues. IGFBP2, one of at least six different binding proteins identified to date, is secreted by most sarcoma cell lines and appears to be involved in cell proliferation and transformation. Circulating levels of this protein are markedly increased in malignancy. We have assessed 46 adult STS specimens of low, intermediate and high pathological grade of malignancy for the immunohistochemical expression of IGFBP2, IGF1, IGF2, IGF1 receptor- and - (IGF1R/). The protein expression was measured by quantitative color video image analysis and semi-quantitative evaluation, and the measurements correlated well (Spearman, P<0.001). Using both methods, significant differences in expression of IGFBP2 among each of the three grades, expression of IGF2 between intermediate and high grade, and expression of IGF1R between low-intermediate and low-high grade were observed (Dunnett test, P<0.05). Multiple regression analysis for both quantitative and semi-quantitative data confirmed the significance of the relationship and independence of the proteins, except IGF2. We concluded that IGFBP2 and IGF1R are independent predictors of the malignant potential of adult STS.