Expression of major histocompatibility complex antigens and intercellular adhesion molecule-1 (ICAM-1) on renal cell cancer. De novo expression and modulation by cytokines on renal cell cancer cell lines]

Major histocompatibility complex (MHC) antigens and intercellular adhesion molecule-1 (ICAM-1) play important roles in immune response. In order to investigate the association between renal cell cancer (RCC) and host's immune system, expression of MHC antigens and ICAM-1 was examined on RCC. Immunohistochemical analysis revealed a positive correlation between the expression of MHC antigens and ICAM-1. In general, tumor with higher degree of mononuclear cell infiltration expressed MHC antigens and ICAM-1 more frequently and intensely. Among cytokines which were reported to be potent inducers of ICAM-1 on malignant melanoma cell lines, interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha augmented the expression of ICAM-1 on ACHN cells whereas ICAM-1 and class I antigens on KRC/Y cells. IFN-alpha enhanced MHC class I antigens but not ICAM-1. Class II antigen expression of both cell lines was augmented by only IFN-gamma. These results suggest that cytokines which could be produced by tumor-infiltrating mononuclear cells, especially IFN-gamma and TNF-alpha, might modulate expression of MHC antigens and ICAM-1, and influence host immune response against RCC.     

Interleukin‐17 synergizes with IFNγ or TNFα to promote inflammatory mediator release and intercellular adhesion molecule‐1 (ICAM‐1) expression in human intervertebral disc cells

Interleukin‐17 (IL‐17) is a cytokine recently shown to be elevated, along with interferon‐γ (IFNγ) and tumor necrosis factor (TNFα), in degenerated and herniated intervertebral disc (IVD) tissues, suggesting a role for these cytokines in intervertebral disc disease. The objective of our study was to investigate the involvement of IL‐17 and costimulants IFNγ and TNFα in intervertebral disc pathology. Cells were isolated from anulus fibrosus and nucleus pulposus tissues of patients undergoing surgery for intervertebral disc degeneration or scoliosis. The production of inflammatory mediators, nitric oxide (NOx), prostaglandin E2 (PGE2) and interleukin‐6 (IL‐6), as well as intercellular adhesion molecule (ICAM‐1) expression, were quantified for cultured cells following exposure to IL‐17, IFNγ, and TNFα. Intervertebral disc cells exposed to IL‐17, IFNγ, or TNFα showed a remarkable increase in inflammatory mediator release and ICAM‐1 expression (GLM and ANOVA, p < 0.05). Addition of IFNγ or TNFα to IL‐17 demonstrated a synergistic increase in inflammatory mediator release, and a marked increase in ICAM‐1 expression. These findings suggest that IVD cells not only respond with a catabolic phenotype to IL‐17 and costimulants IFNγ and TNFα, but also express surface ligands with consequent potential to recruit additional lymphocytes and immune cells to the IVD microenvironment. IL‐17 may be an important regulator of inflammation in the IVD pathologies. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29:1–7, 2011     

Mesangial cell accessory functions: mediation by intercellular adhesion molecule-1

Mesangial cell (MC) proliferation is an early pathologic alteration characteristic of many forms of immune mediated glomerulonephritis. The intracapillary position, contractile capacity, and production of cytokines and other inflammatory molecules place MC in a pivotal position to initiate, mediate, and direct glomerular damage. We as well as others have noted increased levels of cytokines including IFN gamma, TNF, and IL-1 and the cell surface MHC class II and ICAM-1 molecules in the kidneys of mice with lupus nephritis. MHC class II and ICAM-1 molecules are central to the interaction of T cells with antigen presenting cells (APC). Since cytokines can increase both MHC class II and ICAM-1 molecules, we investigated whether mesangial cells could function as APC or accessory cells after cytokine stimulation. For these studies we established a permanent MC line through transformation with origin-deficient SV40 DNA. Surface expression of ICAM-1 was similar in untransformed MC as well as SV40 transformed MC from normal mice and in untransformed cells from mice with lupus nephritis. Basal expression of ICAM-1 was upregulated rapidly by IFN gamma, TNF, and IL-1. MHC class II expression could not be induced with TNF or IL-1 alone but required prolonged stimulation with IFN gamma. MC adhered and presented antigen to an antigen specific IaK restricted T cell hybridoma. Anti-ICAM-1 mAb decreased adhesion and antigen presentation of cytokine stimulated MC. By comparison, MHC class II mAb abrogated antigen presentation by MC bearing MHC class II but did not block adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dominance of chemokine ligand 2 and matrix metalloproteinase-2 and -9 and suppression of pro-inflammatory cytokines in the epidural compartment after intervertebral disc extrusion in a canine model

Background contextIn canine intervertebral disc (IVD) disease, a useful animal model, only little is known about the inflammatory response in the epidural space.PurposeTo determine messenger RNA (mRNA) expressions of selected cytokines, chemokines, and matrix metalloproteinases (MMPs) qualitatively and semiquantitatively over the course of the disease and to correlate results to neurologic status and outcome.Study design/settingProspective study using extruded IVD material of dogs with thoracolumbar IVD extrusion.Patient sampleSeventy affected and 13 control (24 samples) dogs.Outcome measuresDuration of neurologic signs, pretreatment, neurologic grade, severity of pain, and outcome were recorded. After diagnostic imaging, decompressive surgery was performed.MethodsMessenger RNA expressions of interleukin (IL)-1β, IL-2, IL-4, IL-6, IL-8, IL-10, tumor necrosis factor (TNF), interferon (IFN)γ, MMP-2, MMP-9, chemokine ligand (CCL)2, CCL3, and three housekeeping genes was determined in the collected epidural material by Panomics 2.0 QuantiGene Plex technology. Relative mRNA expression and fold changes were calculated. Relative mRNA expression was correlated statistically to clinical parameters.ResultsFold changes of TNF, IL-1β, IL-2, IL-4, IL-6, IL-10, IFNγ, and CCL3 were clearly downregulated in all stages of the disease. MMP-9 was downregulated in the acute stage and upregulated in the subacute and chronic phase. Interleukin-8 was upregulated in acute cases. MMP-2 showed mild and CCL2 strong upregulation over the whole course of the disease. In dogs with severe pain, CCL3 and IFNγ were significantly higher compared with dogs without pain (p=.017/.020). Dogs pretreated with nonsteroidal anti-inflammatory drugs revealed significantly lower mRNA expression of IL-8 (p=.017).ConclusionsThe high CCL2 levels and upregulated MMPs combined with downregulated T-cell cytokines and suppressed pro-inflammatory genes in extruded canine disc material indicate that the epidural reaction is dominated by infiltrating monocytes differentiating into macrophages with tissue remodeling functions. These results will help to understand the pathogenic processes representing the basis for novel therapeutic approaches. The canine IVD disease model will be rewarding in this process.     

Characterization of the Cytokine Pattern of Porcine Bone Marrow‐Derived Cells Treated with 1α,25(OH)2D3

The biologically active form of vitamine D₃ [1α,25(OH)₂D₃] has recently been described not only to influence bone metabolism but also to exert immunomodulating activities, which may have an impact on bone formation/resorption as well. In this study, we analysed the effects of 1α,25(OH)₂D₃ on the cytokine pattern of porcine bone marrow‐derived cells from piglets aged 1–3 weeks. After culture for 1 week, the number of osteoclasts was determined, with tartrate‐resistant acid phosphatase (TRAP)‐positive, multinucleated cells being considered osteoclasts. Cultured bone marrow cell‐derived mRNA was subjected to semiquantitative RT‐PCR specific for a panel of porcine cytokines (IL‐1α, IL‐6, IL‐8, IL‐10, and TNF‐α). In addition, an immunofluorescence analysis using anti‐porcine mAbs specific for IL‐1β, IL‐2, IL‐4, IL‐6, IL‐12, TNF‐α, and IFN‐γ was performed. In order to prove the existence of a porcine homologue of the receptor activator of NF‐κB ligand (RANKL) bone marrow cell‐ as well as porcine white blood cell‐derived mRNA was investigated by RT‐PCR using primer pairs specific for murine RANKL. Cell culture supernatant was analysed for soluble RANKL by means of an ELISA designed for quantification of human RANKL. By means of RT‐PCR, expression of IL‐1α, IL‐6, IL‐8, IL‐10 and TNF‐α mRNA could be found in cells cultured with and without 1α,25(OH)₂D₃. Immunofluorescence analysis revealed that IL‐1, IL‐6, and TNF‐α were produced by both stromal cells and osteoclasts. Besides its known osteoclastogenic effects, 1α,25(OH)₂D₃ tended to downregulate the respective cytokines, but significantly upregulated RANKL expression. The homology between the porcine RANKL‐specific sequence and the corresponding human RANKL sequence was 79%. The data found support the idea that porcine bone marrow cell cultures may provide a suitable alternative to murine systems in human osteological research.     

Expression of cell adhesion molecules in an established and characterized new human renal cell cancer line,CCF-RC7

In order to investigate the importance of cell adhesion molecules (CAMs) in renal cell carcinoma (RCC), a cell line, designated as CCF-RC7, was established from a human RCC of the clear cell type. CCF-RC7 was passaged over 50 times in vitro for 31/2 years. The cell line has an epithelial morphology and a doubling time of 30 h, forming colonies in soft agar with an average efficiency of 10.4% and producing clear cell tumors in athymic nude mice. CCF-RC7 cells have an aneuploid-hypotetraploid karyotype with a modal chromosome number of 82 and rearrangements in chromosomes 9, 12 and 14. Immunohistochemical and flow immunocytometric analyses revealed high expression of ICAM-1 (CD54), and Hermes antigen (CD44), which was significantly upregulated by cytokine and PMA treatment. VLA-4 was expressed on approximately 20% of tumor cells and could not be altered by cytokine or PMA stimulation. High expression of sialyl Lewis X was also demonstrated by immunohistological examination. This newly characterized cell line will serve as a useful model for the study of CAMs during hematogenous metastasis and host defense mechanisms in human RCC.     

Porcine IL‐6, IL‐1β, and TNF‐α regulate the expression of pro‐inflammatory‐related genes and tissue factor in human umbilical vein endothelial cells

Whether porcine cytokines are induced after pig‐to‐primate xenotransplantation and activate human cells remains unknown. First, we investigated the regulation of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using an in vitro model in which porcine aortic endothelial cells (PAECs) and porcine peripheral blood mononuclear cells (PBMCs) were stimulated with human serum. Downstream cytokines/chemokines were monitored. Pro‐inflammatory cytokines (IL‐6, IFN‐γ, and IL‐1β) and chemokines (IL‐8, MCP‐1, and CXCL2) were upregulated in the both cell types. TNF‐α was induced 10‐fold in PAECs, but not in PBMCs. Then, we assessed the role of porcine IL‐6, IFN‐γ, IL‐1β, and TNF‐α in xenotransplantation using western blotting and real‐time PCR. Human umbilical vein endothelial cells (HUVECs) were selected as the target cells. Signaling pathways and downstream genes, such as those related to adhesion, inflammation, and coagulation, and chemokines were investigated. Porcine IL‐1β and TNF‐α significantly activated NF‐κB and P38, and STAT3 was activated by porcine IL‐6 in HUVECs. The adhesion genes (E‐selectin, VCAM‐1, and ICAM‐1), inflammatory cytokines (IL‐6, IL‐1β, and TNF‐α), chemokines (MCP‐1 and IL‐8), and the pro‐coagulation gene (tissue factor) were upregulated by porcine IL‐1β and TNF‐α. Porcine IL‐6 increased the expression of ICAM‐1, IL‐6, MCP‐1, and tissue factor, but decreased IL‐8 expression slightly. Surprisingly, porcine IFN‐γ could not activate STAT1 or regulate the expression of any of the above genes in HUVECs. In conclusion, these findings suggest that porcine IL‐6, IL‐1β, and TNF‐α activate HUVECs and regulate downstream genes expression, which may promote inflammation and coagulation response after xenotransplantation.     

Immunosuppressive effects of surgery assessed by flow cytometry in nonhuman primates after nephrectomy

Despite previous studies suggesting that surgery cause immune suppression, the underlying biologic mechanisms have not been studied using advanced immune function assays. Unilateral nephrectomy was performed in nonhuman primates. Blood was collected before surgery and at different time‐points through 14 days after surgery. Lymphocyte proliferation (expression of proliferating cell nuclear antigen in cells in S/G₂M‐phase), production of intracellular cytokines [interleukin (IL)‐2, interferon (IFN)‐γ, tumor necrosis factor (TNF)‐α] and expression of surface‐activation antigens (CD25, CD71) on T‐lymphocytes were assessed in whole blood using flow cytometry. Results were compared with nonoperated control animals. The procedure caused a decrease of 25% in absolute lymphocyte count on postoperative day 3. Inhibition of lymphocyte proliferation was maximal on postoperative day 1 (55% normalized to preoperative values) and was detectable until postoperative day 7, when it was 25%. Expression of T‐cell activation antigens was decreased during the first postoperative week with a maximum on postoperative day 1 for CD71 (29%) and on postoperative day 3 for CD25 (49%). Intracellular production of cytokines by T cells was decreased only on postoperative day 1 (50% for IL‐2, 29% for IFN‐γ and 22% for TNF‐α). Immune functions returned to presurgery values by day 14. A major surgical procedure severely inhibits lymphocyte proliferation and various T‐cell functions up to 1 week postoperatively.     

Cell surface expression and serum levels of intercellular adhesion molecule-1 in renal cell carcinoma

Intercellular adhesion molecule-1 (ICAM-1) is the natural ligand of the T-lymphocyte adhesion molecule LFA-1 (lymphocyte-function-associated antigen-1). ICAM-1 is involed in target cell recognition by T-lymphocytes, LAK cells and natural killer cells. The molecule has also been detected on a variety of normal cells and on human tumors. Renal cell carcinoma (RCC) is one of the few tumors that respond to immunotherapy, but clinical results are generally disappointing. We therefore analyzed, by immunohistochemistry, the expression of ICAM-1 in pairs of normal kidneys, RCC, and RCC metastases. Moreover, serum ICAM-1 was determined in RCC patients and compared with surface expression of cell-bound ICAM-1. Strong glomerular expression of ICAM-1 was observed in all specimens of normal kidney examined. Proximal tubuli were weakly stained in the majority of specimens. Of the tumors, 80% stained positive for ICAM-1. Although ICAM-1 was detected on the majority of extrarenal tumor specimens examined, staining was generally weaker in the metastases. Patients without metastases at initial presentation more frequently expressed ICAM-1 in their primary tumors than did patients with metastases. Levels of serum ICAM-1 (sICAM-1) were significantly higher in RCC patients than in controls with non-malignant renal diseases. Patients with an unfavorable prognosis, e.g. with advanced tumor stage or metastasis at initial presentation, had higher levels of sICAM-1 than patients with low-grade and/or low-stage tumors. An inverse correlation was observed between expression of ICAM-1 in tumors and levels of sICAM-1. On the basis of our data we suggest that cell-bound or soluble ICAM-1 is correlated with tumor-host interaction in RCC.     

利多卡因抑制内皮细胞粘附因子表达进而抑制肝癌细胞粘附脐带静脉内皮细胞

【摘要】 目的 探讨利多卡因对血管内皮细胞粘附因子表达的影响。方法 采用不同浓度利多卡因预处理脐带静脉内皮细胞（HUVECs）30 min后,加入肿瘤坏死因子α（TNFα）进行刺激。通过实时荧光定量聚合酶链反应检测选择素E（CD62E）、血管细胞粘附分子-1（VCAM-1）和细胞间粘附分子-1（ICAM-1）表达量,蛋白免疫印迹分析NF-Kappa B（NF-κB）通路蛋白的改变,并通过细胞粘附实验评估利多卡因预处理对肝癌细胞（HepG2）粘附于HUVECs的影响。结果 利多卡因预处理可以明显抑制p65并抑制HepG2粘附于HUVECs。qRT-PCR结果表明利多卡因预处理可明显抑制TNF-α刺激后的CD62E、VCAM-1和ICAM-1表达水平的增高。结论 利多卡因可能通过抑制NF-κB通路进而抑制细胞粘附因子表达并抑制结肝癌细胞粘附于血管内皮细胞。     

Cytokine regulation of ICAM-1 expression on human renal tubular epithelial cells in vitro.

Regulation of the intercellular adhesion molecule-1 (ICAM-1) expression on human renal tubular epithelial cells in culture (hKEC-1) was investigated. A large proportion of hKEC-1 cells from the primary cultures expressed the ICAM-1 antigen. Supernatants from mixed lymphocyte reaction (MLR) of both specific and third-party combinations augmented the expression of the ICAM-1 antigen, in a dose-dependent manner. A kinetic study revealed maximal augmentation by MLR supernatant on the first day, with a gradual decrease thereafter. Among several recombinant human cytokines tested, i.e., interferon-gamma, tumor necrosis factor-alpha, interleukin 1 alpha and beta, and IL-4, IFN-gamma, TNF-alpha, and IL-1 alpha/beta were shown to augment the expression of ICAM-1. MLR supernatants and IFN-gamma were more effective in augmenting ICAM expression than TNF-alpha and IL-1 alpha/beta. IFN-gamma upregulated ICAM-1 expression in a dose-dependent manner, and maximal augmentation was achieved on the first day. The MLR supernatants were shown to contain IFN-gamma and TNF-alpha, and the activity of the MLR supernatant was partially inhibited by neutralizing antibody against IFN-gamma. These data suggest that cytokines, especially IFN-gamma, TNF-alpha, and IL-1 alpha/beta, released by T cells and antigen-presenting cells upon recognition of alloantigens upregulate ICAM-1 expression on renal tubular epithelial cells. This may result in an increase in the attachment of graft-infiltrating T cells to the renal tubular cells, by the ICAM-1-LFA-1 interaction.     

Sequential mRNA expression for immediate early genes, cytokines, and neurotrophins in spinal cord injury

In this communication, we demonstrate the sequential expression of endogenous molecules, including immediate early genes (IEGs), cytokines, neurotrophins, and neurotrophin receptors in the injured spinal cord. In the acute phase, expression of IEGs and cytokines mRNAs were rapidly upregulated within 1 h in nonneuronal cells in the lesioned sites and the surrounding spinal white and gray matter. Maximal expression was observed at 1 h for c-fos and TNF-alpha mRNAs, at 3 h for c-jun and IL-6 mRNAs, and at 6 h for IL-1 beta mRNA, and these signals were virtually nondetectable after 6-12 h from the onset of the injury. Some of these genes products may promote the degeneration of damaged cells and tissues, while others may be involved in the subsequent repair processes. In the subacute phase, expression of NGF, BDNF, NT-3, p75LNGFR and Trk B mRNAs began to increase in the nonneuronal cells and neuronal cells from 6 h, and peaked at 24-72 h in the area where expression of mRNAs for IEGs and cytokines overlapped. Signals for IL-6 mRNA were also observed in motoneurons at 24-72 h after the injury, with the suggestion that these molecules may be involved in promoting axonal sprouting in the injured spinal cord. Of further interest was the finding that this upregulation of IL-1 beta, BDNF, and NT-3 mRNAs in injured spinal cord was attenuated by treatment with high dose glucocorticoids, with the suggestion that the downregulation of BDNF and NT-3 might be disadvantageous to survival and axonal sprouting of spinal neurons.     

IFN-γ和TNF-α诱导的骨髓间充质干细胞的免疫抑制作用及环孢素A对其的影响

目的 观察γ干扰素(IFN-γ)和肿瘤坏死因子α(TNF-α)诱导的骨髓问充质干细胞(MSC)的免疫抑制作用及环孢素A(CsA)对其的影响.方法 取Balb/c小鼠骨髓,分离、培养、纯化及扩增MSC,分别以IFN-γ、TNF-α和IFN-γ+TNF-α(终浓度均为20 ng/ml)预处理MSC 12 h,然后与Balb/c小鼠脾细胞共培养,脾细胞以刀豆蛋白A激活72 h.混合细胞培养实验分8组进行:(1)脾细胞组;(2)MSC组,MSC+脾细胞;(3)IFN-γ处理组,IFN-γ预处理的MSC+脾细胞;(4)TNF-α处理组,TNF-α预处理的MSC+脾细胞;(5)联合处理组,IFN-γ与TNF-α联合预处理的MSC+脾细胞;(6)CsA组,CsA+脾细胞;(7)CsA联合预处理组:IFN-γ与TNF-α联合预处理的MSC+CsA+脾细胞;(8)1400W组,IFN-γ与TNF-α联合预处理的MSC+脾细胞+1400W[诱导型-氧化氮合酶(iNOS)抑制剂].各组脾细胞数量均为5000个,MSC与脾细胞按1:10混合,CsA浓度为10μg/ml.培养72 h后,以四甲基偶氮唑盐法检测脾细胞增殖情况,同时以碘化丙啶和Annexin-V凋亡双染试剂盒检测脾细胞的凋亡情况;以实时聚合酶链反应法检测经炎症因子处理的MSC的iNOS mRNA表达情况,观察CsA对MSC的iNOS mRNA表达的影响.结果 MSC组、IFN-γ处理组和TNF-α处理组的MSC对脾细胞的增殖无明显抑制作用(P>0.05),而联合处理组的脾细胞增殖明显受到抑制,CsA组的脾细胞增殖同样受到抑制,但IFN-γ与TNF-α联合预处理MSC产生的这种免疫抑制作用却可以被CsA所抑制(CsA联合预处理组,P<0.05).联合处理组、CsA组和CsA联合预处理组的脾细胞凋亡率明显高于脾细胞组、MSC组、IFN-γ处理组和TNF-α处理组(P<0.05),而联合处理组和CsA组的脾细胞凋亡率又明显高于csA联合预处理组(P<0.05).野生型MSC、分别以IFN-γ或TNF-α单独刺激的MSC均不表达iNOS mRNA,而以IFN-γ和TNF-a联合处理的MSC则高表达iNOS mRNA,但这种高表达可被CsA所抑制(P<0.05).1400W组的脾细胞增殖受到明显抑制(P<0.05).结论 IFN-γ和TNF-α联合预处理的MSC在体外可抑制脾细胞的增殖,促进脾细胞凋亡,CsA可抑制该种MSC的这一作用,其机制可能与CsA下调lFN-7和TNF-α联合预处理的MSC的iNOS表达有关.     

Differential sensitivity of renal cell carcinoma xenografts towards therapy with interferon-alpha,interferon-gamma,tumor necrosis factor and their combinations

Summary Whereas cytokine therapy has proven efficacy in the treatment of metastatic renal cell carcinoma (RCC), many questions regarding the use of these drugs remain unanswered. In the present study we evaluated the antiproliferation effects of human recombinant alphainterferon (IFN), gamma-interferon and tumor necrosis factor-alpha (TNF) on eight human RCC xenografts. In particular, the importance of the administration route, dosage and tumor load was investigated. Response to the cytokines differed widely amongst the different tumors. Of three tested routes of administration (i.v., i.p. and s.c. peritumoral), only the s.c. peritumoral route was effective against tumor growth. After 6 weeks of therapy consisting of 150 or 1,500 units IFN/g given s.c. peritumorally three times a week or 30,000 units TNF/g given five times a week, alpha-IFN treatment resulted in 2%–100% growth inhibition; gamma-IFN, in 7%–80%; and TNF, in 35%–75% as compared with the untreated control. Growth of five of eight tumor lines could be inhibited completely by combinations of IFN and TNF, whereby the tumor dimensions at the beginning of therapy were decisive for the results. In some cases IFNs had optimal doses; however, the antitumor effects of TNF were always dose-dependent. Our studies indicate that the doses at which the optimal direct effects of cytokines are measured are critically dependent on the tumor treated. Although direct effects are only one part of the mode of action of cytokines, our results indicate that dosage of cytokines may need individualisation.     

Development of a Microarray for Studying Porcine Cytokine Production in Blood Mononuclear Cells and Intestinal Biopsies

A microarray for demonstration of a limited number of porcine cytokines was initiated. Polymerase chain reaction (PCR) products were synthesized for four house‐keeping genes, cyclophilin, β‐actin, hypoxanthine phosphoribosyltransferase (HPRT) and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), and the following cytokines: interleukin (IL)‐1β, IL‐4, IL‐6, IL‐8, IL‐10, IL‐12 p35, IL‐12 p40, IL‐18, interferon (IFN)‐α, IFN‐β, IFN‐γ, tumour necrosis factor (TNF)‐α, macrophage inhibition factor (MIF) and granulocyte/macrophage colony‐stimulating factor (GM‐CSF). Cytokine production was induced by incubation of porcine peripheral blood mononuclear cells (PBMC) with Concanavalin A (ConA) or oligodeoxynucleotide (ODN) 2216. RNA was isolated after 6 or 24 h from stimulated cells or unstimulated control cells and from intestinal biopsies. Cytokine expression was analysed using a 3‐DNA Array 350^TM labelling kit from Genisphere. Data were normalized using external control genes and analysed with the genepix pro 5.0 software. All the cytokines could be induced in PBMC and expressed on the array and the cytokines IL‐6 and IFN‐α were also analysed at protein level. All but one cytokine were expressed in samples from intestinal biopsies. Densitometric analyses of PCR products of the house‐keeping genes were performed to validate the results from the microarray. Thus, this microarray will enable analyses of the cytokine profile during local and systemic infections in the pig.     

Effect of pro‐inflammatory and immunoregulatory cytokines on human tenocytes

Tendon injury induces a local inflammatory response, characterized by the induction of pro‐inflammatory cytokines. The aim of the present study was to analyze the effects of TNFα, IL‐6 and IL‐10 on key parameters of tendon homeostasis. Cultured primary human tenocytes were treated with the recombinant cytokines IL‐6, IL‐10, TNFα, or combinations of TNFα with IL‐6 and IL‐10 (10 ng/mL, 6, 24 h). Expression of type I collagen, elastin, MMP‐1, TNFα, IL‐1β, IL‐6, IL‐10, and suppressors of cytokine signaling (SOCS1, 3) was analyzed with the use of RTD‐PCR, immunocytochemistry, and Western blot analysis. In response to TNFα, tenocytes reduced their type I collagen deposition but increased their elastin gene expression and highly upregulated their expression for MMP‐1, pro‐inflammatory (TNFα, IL‐1β) and immunoregulatory (IL‐6, IL‐10) cytokines. TNFα stimulation augmented SOCS1, whereas SOCS3 expression in tenocytes was also induced by IL‐6. The treatment of tenocytes with IL‐6 and IL‐10 had no effect on cytokine expression. Neither IL‐6 nor IL‐10 modulated the observed effects of TNFα significantly. These results indicate that TNFα strongly activates the tenocytes to amplify their own TNFα expression and, subsequently, that of other regulatory cytokines and matrix degrading enzymes. However, the impact of IL‐6 and IL‐10 on tenocytes remains unclear. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1071–1077, 2010