Effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus

We studied a well-selected population of patients with active rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE) without immunosuppressive therapy. Control and patient peripheral blood mononuclear cells (PBMC) were incubated with IL-1beta, IL-10, TGF-beta or LPS for 20 h and the in vitro basal and stimulated secretions of IL-6, TNF-alpha, IL-1beta and IL-1ra were measured by ELISA. We found that in the SLE patients the basal secretion of IL-6 was significantly lower and that of IL-1ra significantly higher than in control subjects, while in the RA group the basal IL-1ra secretion was higher than in healthy subjects. SLE and RA PBMC responded to LPS and IL-1beta reaching higher cytokine secretion values than controls. The in vitro response of SLE and RA PBMC to TGFbeta was normal, while that to IL-10 was defective: IL-10 was able to stimulate the production of IL-6 and IL-1ra in PBMC from normal subjects, but it was unable to enhance IL-6 secretion in RA cells and it was also completely ineffective in inducing IL-1ra secretion in both SLE and RA PBMC. Our work add new data useful for the evaluation of IL-10 and IL-1ra as therapeutic agents in rheumatic diseases.     

Levels of MCP-1 and GM-CSF mRNA Correlated with Inflammatory Cytokines mRNA Levels in Experimental Autoimmune Myocarditis in Rats

Infiltration of monocytes and T cells is known to be an essential trigger for the progression of experimental autoimmune myocarditis (EAM) in rats. Monocyte chemotactic protein-1 (MCP-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were shown to mediate the migration of monocytes and T cells into inflammatory sites and to proliferate monocytes. Thus, we evaluated levels of MCP-1 and GM-CSF mRNA in the myocardium of EAM in rats using a real time quantitative PCR method. We also examined the correlation of MCP-1 or GM-CSF mRNA levels with those of inflammatory cytokines such as tumor necrosis factor- &#102 (TNF- &#102 ), interleukin-1 &#103 (IL-1 &#103 ) and interleukin-6 (IL-6) in the same lesion. Levels of MCP-1, GM-CSF, TNF- &#102, IL-1 &#103 and IL-6 mRNA increased with the progression of myocarditis which was accompanied by the accumulation of ED-1 positive cells. The MCP-1 and GM-CSF mRNA levels were positively correlated with TNF- &#102, IL-1 &#103 and IL-6 mRNA levels in the same lesion of EAM. We also demonstrated that serum MCP-1 concentrations were increased during the active stage of EAM, and were correlated with MCP-1 mRNA levels in the myocardium of each rat. These findings suggest that elevated MCP-1 and GM-CSF may associate with the migration and proliferation of monocytes/macrophages in EAM. Thus, MCP-1 and GM-CSF may play an important role in the progression of EAM.     

Decreased IL-4 Producing CD4 + T Cells in Patients with Active Systemic Lupus Erythematosus-relation to IL-12R Expression

The balance of interferon- &#110 (IFN- &#110 ) and/or interleukin-4 (IL-4) producing T cells and interleukin-12 receptor (IL-12R) expression on T cells were evaluated in patients with active systemic lupus erythematosus (SLE). Assessment of intracellular IFN- &#110 and/or IL-4 were conducted with cytoplasmic staining. IL-12R presenting T cells were also assessed by flowcytometry without in vitro stimulation. In SLE, the number of IFN- &#110 producing CD4 + T cells was increased, and the absolute number of IL-4 producing CD4 + T cells was significantly decreased. Although the ratio of IL-12R presenting CD4 + T cells was significantly greater, the absolute number did not increase. The ratio of IFN- &#110 /IL-4-producing CD4 + T cells correlated with the SLE disease activity index (SLEDAI) and was significantly higher among patients with lupus nephritis. Therefore, the imbalance of IFN- &#110 /IL-4 producing CD4 + T cells was due to the decrease in IL-4 producing CD4 + T cells and may play an important pathogenic role in active SLE.     

Inflammation and Colorectal Cancer: Does Aspirin Affect the Interaction between Cancer and Immune Cells?

The effect of aspirin on colon-cancer-cell-induced cytokine secretion by peripheral blood mononuclear cells (PBMC) was examined. Aspirin was added to human colon cancer cells (HT-29 and RKO) or to PBMC incubated separately or jointly. The secretion of IFNγ, IL-6, and IL-10 induced by HT-29 cells was decreased, that of IL-1β was slightly increased, whereas IL-1ra production was not affected. With RKO cells, aspirin reduced IL-6, IL-1ra, and IL-10 synthesis and enhanced IFNγ secretion, while IL-1β remained unchanged. Conditioned media from colon cancer cells incubated without or with aspirin stimulated cytokine productions by PBMC similarly, suggesting that aspirin acts on the cell-to-cell interaction between cancer cells and PBMC. The results indicate that aspirin alter the balance between pro- and anti-inflammatory cytokines generated by interaction between colon cancer and immune cells disclosing an additional role of the drug in affecting inflammation-induced colon cancer.     

Peripheral blood mononuclear cells from patients with rheumatoid arthritis spontaneously secrete vascular endothelial growth factor (VEGF): specific up-regulation by tumour necrosis factor-alpha (TNF-alpha) in synovial fluid.

This study was designed to investigate VEGF production from peripheral blood mononuclear cells (PBMC) from patients with rheumatoid arthritis (RA) compared with healthy controls and to identify the predominant cellular source in PBMC isolated from RA patients. The regulation of PBMC VEGF production by cytokines and synovial fluid (SF) was studied. PBMC were isolated from RA patients and healthy controls and stimulated with lipopolysaccharide (LPS), IL-1beta, IL-4, IL-6, IL-8, IL-10, TNF-alpha and transforming growth factor-beta (TGF-beta) isoforms for varying time points up to 72 h at 37 degrees C/5% CO2. The effect of SF on VEGF secretion by PBMC was also studied. Supernatant VEGF levels were measured using a flt-1 receptor capture ELISA. RA patients had significantly higher spontaneous production of VEGF compared with controls, and monocytes were identified as the predominant cellular source. RA PBMC VEGF production was up-regulated by TGF-beta isoforms and TNF-alpha and down-regulated by IL-4 and IL-10, with no effect observed with IL-1beta, IL-6 and IL-8. Antibody blocking experiments confirmed that TNF-alpha and not TGF-beta isoforms in SF increased VEGF secretion by RA PBMC. These results emphasize the importance of monocytes as a source of VEGF in the pathophysiology of RA. Several cytokines known to be present in SF can modulate the level of VEGF secretion, but the predominant effect of SF in VEGF up-regulation is shown to be dependent on TNF-alpha.     

SLE患者外周血单个核细胞分泌白细胞介素6和IgG的研究

我们研究了21例SLE患者和正常人外周血单个核细胞(PBMC)体外培养自发和丝裂原刺激后细胞增殖、IL—6产生、IgG分泌的情况。发现SLE 患者PBMC经PHA和PWM刺激后,细胞增殖的指数降低;用IL—6细胞依赖株(MH_(60)·BSF_2)检测培养上清液中的IL—6,发现活动期SLE患者PBMC自发和PHA刺激后产生的IL—6明显高于缓解期SLE患者(P<0.001)和正常人(P<0.001);活动期和缓解期SLE患者PBMC体外自发分泌IgG量增高,分别为正常人自发分泌量的5.68倍和4.48倍;我们首次用统计学方法分析了SLE患者细胞培养上清液中的IL—6水平与分泌的IgG量的关系,发现两者有显著的相关性(r=0.7046,P<0.001)。     

Treatment of Induced Murine SLE with a Peptide Based on the CDR3 of an Anti-DNA Antibody Reverses the Pattern of Pathogenic Cytokines

A peptide based on the complementarity determining region (CDR) 3 of a pathogenic anti-DNA monoclonal antibody that bears the 16/6 idiotype (Id) was shown previously to be a dominant T-cell epitope in experimental SLE, and to be capable of inhibiting SLE-associated responses. When injected, concomitant with active immunization with the pathogenic human anti-DNA, 16/6 Id + mAb, pCDR3 inhibited the proliferation of LN-derived T cells stimulated in vitro with the 16/6 Id mAb. The inhibition of the specific proliferative responses could be reversed by the addition of exogenous IL-2 to the cultures. Analysis of secreted cytokine profile in supernatants of these cultures demonstrated that pCDR3 treatment reduced significantly the levels of both IL-2 and IFN- &#110 that were elevated further in cells of the 16/6 Id-immunized mice. The CDR3-based peptide was shown here to immunomodulate in vivo experimental SLE, induced by the human anti-DNA 16/6 Id + antibody. The beneficial effects of pCDR3 on the clinical manifestations of SLE were associated with downregulation of the Th1-type (IL-2, IFN- &#110 ) and proinflammatory (TNF- &#102 ) cytokines, whereas the immunosuppressive cytokine TGF- &#103 was up regulated.     

Production of IL-1beta, IL-1 receptor antagonist and IL-10 by mononuclear cells from patients with SLE.

Depositions of immune-complexes are responsible for many of the pathological features of systemic lupus erythematosus (SLE). For example, immune-complex-induced tissue damage in glomerulonephritis has been shown to be mediated, at least in part, by interleukin (IL)-1. Inappropriate production or function of IL-1 may therefore contribute to disease manifestations in SLE. We investigated lipopolysaccharide (LPS)- and adherent IgG-stimulated release of IL-1beta, IL-1 receptor antagonist (IL-1ra) and IL-10, a potent modulator of IL-1, by blood mononuclear cells from patients with SLE. Mediator production was measured as ng cytokines/10(6) monocytes and compared with clinical parameters. Release of IL-1beta was only detectable in LPS-stimulated cultures and substantially reduced in patients with both active and inactive disease (P < 0.001). LPS-stimulated IL-1ra release was normal and the IL-1ra/IL-1beta ratio was therefore increased (P < 0.05) and correlated inversely to prednisolone dosage (P = 0.009). IgG-stimulated release of IL-1ra was reduced in patients with active disease compared to those with inactive disease and controls (P = 0.002). IL-10 release was similar in patients and controls. We conclude that monocytes from patients with active SLE are deficient in Fc gamma-R-mediated production of IL-1ra, whereas LPS-stimulated IL-1beta release by SLE monocytes is reduced regardless of disease activity. The former may contribute to immune-complex-mediated tissue damage in SLE.     

Paradoxical priming effects of IL-10 on cytokine production.

IL-10 is a well-known immunosuppressive and/or anti-inflammatory cytokine. However, we report in vitro experimental studies in which IL-10 primed leukocytes and led to an enhanced production of tumor necrosis factor (TNF) upon further stimulation by lipopolysaccharide (LPS). Monocytes and peripheral blood mononuclear cells (PBMC) prepared from whole blood maintained for 20 h at 37 degrees C in the presence of recombinant human IL-10 had an enhanced capacity to produce TNF in response to LPS. In addition to TNF, LPS-induced IL-6 and spontaneous IL-1ra production were also enhanced. When isolated PBMC were first cultured for 20 h in the presence of IL-10 on Teflon to prevent adherence, washed to remove IL-10 and then further cultured in plastic dishes for an additional 20 h in the presence of LPS or IL-1beta, an enhanced release of TNF was observed. This was not the case when PBMC were pre-cultured in plastic multidishes in the presence of IL-10. TNF mRNA expression induced by LPS was decreased when the pre-treatment of PBMC with IL-10 was performed on plastic, whereas this was not the case when cells were pre-cultured with IL-10 on Teflon. Furthermore, NFkappaB translocation following LPS activation was higher after IL-10 pre-treatment on Teflon than on plastic. Interestingly, an enhanced frequency of CD16 and CD68(+) cells among the CD14(+) cells was observed in the presence of IL-10, independently of the pre-culture conditions of the PBMC. Altogether, these results indicate that the IL-10-induced up-regulation of cytokine production depends on the prevention of monocyte adherence by red cells in the whole blood assays or by cultures of PBMC on Teflon. In contrast, the adherence parameter has no effect on the IL-10-induced modulation of some monocyte surface markers.     

Spontaneous and LPS-induced secretion of cytokines by villous chorion tissue

Secretion of IL-1α, IL-1α, IL-6, IL-10, and TNF-α cytokines by villous chorion cultures (7–14 weeks) during normal pregnancy and in spontaneous abortions was studied. Secretion of IL-1α and IL-6 increased 4.5 and 7.3 times in miscarriages, while secretion of IL-1α, IL-10, and TNF-α decreased. LPS stimulated the production of IL-1α and IL-6 in samples obtained during surgical abortion. LPS stimulated IL-1α and TNF-α secretion in miscarriages, while the level of IL-6 production decreased significantly. It is hypothesized that increased production of IL-1α and IL-6 and attenuation of the antiinflammatory effect of IL-10 play an important role in the pathogenesis of miscarriages at early stages of gestation. The results suggest that cytokine regulation of the fetus rejection is different at early and late stages of gestation. __________ Translated from Byulleten” Eksperimental’noi Biologii i Meditsiny, Vol. 141, No. 6, pp. 668–671, June, 2006     

Effects of Electromagnetic Fields on Proteoglycan Metabolism of Bovine Articular Cartilage Explants

Electromagnetic field (EMF) exposure has been proposed for the treatment of osteoarthritis. In this study, we investigated the effects of EMF (75 Hz, 2,3 mT) on proteoglycan (PG) metabolism of bovine articular cartilage explants cultured in vitro, both under basal conditions and in the presence of interleukin-1 &#103 (IL-1 &#103 ) in the culture medium. Proteoglycan synthesis and the residual PG tissue content resulted significantly higher in EMF-exposed explants than in controls, whereas no effect was observed on PG release and nitric oxide (NO) production. IL-1 &#103 induced both a reduction in PG synthesis and an increase in PG release, related to a strong stimulation of NO production, which resulted in a net loss of tissue PG content. In IL-1 &#103 -treated explants, EMF increased PG synthesis, whereas in spite of a slight stimulation of NO production EMF did not modify PG release. This resulted in the residual PG tissue content being maintained at the control level. In both experimental conditions, the effects of EMF were associated with an increase in lactate production. The results of our study show that EMFs are able to promote anabolic activities and PG synthesis in bovine articular cartilage explants. This effect also is maintained in the presence of IL-1 &#103 , thus counteracting the catabolic activity of the cytokine. Altogether, these data suggest that EMF exposure exerts a chondroprotective effect on articular cartilage in vitro.     

Th1 (IL-2, interferon-gamma (IFN-γ)) and Th2 (IL-10, IL-4) cytokine production by peripheral blood mononuclear cells (PBMC) from patients with systemic lupus erythematosus (SLE)

We investigated the production of IL-2, IFN-γ, IL-10 and IL-4 by PBMC from 24 patients with SLE and 10 healthy individuals. Basal and mitogen-stimulated (lipopolysaccharide and phytohaemagglutinin (LPS + PHA)) cytokine production was determined in a whole blood assay (WBA). Supernatants were collected and assayed with specific ELISAs. Although the IL-2 and IFN-γ contents did not differ significantly between patients and controls under both conditions, statistically significant correlations were found between each cytokine and disease activity (SLAM index) after stimulation (respectively, r= 0.501, P = 0.01 and r = 0.631, P = 0.001). PBMC IL-10 production was significantly higher for patients than controls (P = 0.05), but no correlation between IL-10 levels and the SLAM index was obtained. IL-4 production was not statistically different between SLE patients and controls. For stimulated WBAs, the IL-10/IL-2 and IL-10/IFN-γ ratios were significantly correlated with disease severity (P = 0.02; P = 0.001, respectively). Overall, our data suggest that SLE is characterized by an elevated production of IL-10, reflecting the basal state of activation of the immune system. During exacerbation of SLE, IL-2 and IFN-γ are synthesized in larger amounts and may cause the tissue damage observed.     

Th1 and Th2 Cytokine Modulation by IL-10/IL-12 Imbalance in Autoimmune Haemolytic Anaemia (AIHA)

Recent studies about autoimmune diseases in animal models and in humans focused their attention on lymphocyte activation and in vitro cytokine production. The respective contribution of the Th1 and Th2 cytokines to the pathogenesis of autoimmune diseases is still a matter of debate. In this study the role of IL-2, IL-4, IFN- &#110, IL-10 and IL-12 cytokines were investigated by examining their spontaneous and mitogen-induced (OKT3 and PHA or LPS) synthesis and T-cells proliferative response by peripheral blood mononuclear cells to determine their role in the pathogenesis of AIHA. Thirteen patients affected by AIHA, idiopathic or associated with other diseases, and 13 healthy subjects, randomly selected from a group of blood donors, were investigated. This study indicated that AIHA is characterised by increased basal synthesis of IL-4 and decreased levels of IFN- &#110 compared with healthy controls ( p <0,01). These results suggest that there is a basal decrease of Th1 cytokine and an increase of the Th2 ones. Enhanced IL-2 levels in AIHA patients are likely due to the necessity of a T-cell proliferation stimulus rather than produced as Th1 prevalent stimulation. Furthermore, it has been observed a significant increase in IL-12 production in LPS stimulated cultures from healthy controls, but not in AIHA patients, that shows IL-10 increased levels, which could cause a secondary decrease in IFN- &#110 production and a stimulation of Th2 differentiation. These observations indicate that decreased production of Th1-type cytokines and prevalent Th2 ones leading to autoantibodies production in AIHA may be secondary to the imbalance between IL-10 and IL-12. These results strongly suggest that manipulation of the cytokine network, i.e. IL-10/IL-12 balance, maintained by cells of the innate immune system, can have a strong effect on the incidence of AIHA and their modulation might be useful for a therapeutic control of the disorder.     

Effects of whole-body heat acclimation on cell injury and cytokine responses in peripheral blood mononuclear cells

To test the hypothesis that whole-body heat acclimation (HA) would increase peripheral blood mononuclear cells’ (PBMC) tolerance to heat shock (HS) and/or alter the release of cytokines (IL-1β, IL-6, IL-10 and TNF-α) to bacterial lipopolysaccharide (LPS), we heat acclimated nine subjects by exercising them for 100 min in a hot environment for 10 days. The subjects’ PBMC were separated and cultured on days 1 and 10 of HA pre- and post-exercise. Pre-exercise PBMC were allocated to three treatments: control (PRE, 37°C), HS (42.5°C for 2 h), or LPS (1 ng mL⁻¹ for 24 h). Post-exercise samples were incubated at 37°C. PBMC lactate dehydrogenase release increased (p < 0.05) after HS but it was not different (p > 0.05) between days 1 and 10 (0.100 ± 0.012 and 0.102 ± 0.16 abs., respectively). LPS treatment induced an increased (p < 0.05) release of cytokines but HA did not alter this response (p > 0.05). Pre-exercise intracellular heat shock protein 72 (Hsp72) was higher (p < 0.05) on day 10 compared to day 1 of HA (13 ± 5 and 8 ± 5 ng mL⁻¹, respectively). HS treatment caused a greater increase (p < 0.05) in Hsp72 than the exercise sessions on HA days 1 and 10. In addition, after HA, the Hsp72 response to HS was reduced (day 1, 129 ± 46; day 10, 80 ± 32 ng mL⁻¹, p < 0.05). In conclusion, HA increases PBMC Hsp72 but it does not reduce cellular damage to HS or alter cytokine response to LPS. We speculate that the stress applied during HA is not sufficient to modify the PBMC response.     

In vitro and in vivo reactivity to fungal cell wall agents in sarcoidosis

Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n=22) and controls (n=20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate β-glucan (S-glucan, P-glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 were measured. The severity of sarcoidosis was determined using a chest X-ray-based score. Serum cytokines (IL-2R, IL-6, IL-10 and IL-12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters. N-acetylhexosaminidase (NAHA) on the filters was measured as a marker of fungal cell biomass. The induced secretion of cytokines was higher from peripheral blood mononuclear cells (PBMC) from subjects with sarcoidosis. P-glucan was more potent than S-glucan inducing a secretion. Chitin had a small effect. Among subjects with sarcoidosis there was a significant relation between the spontaneous PBMC production of IL-6, IL-10 and IL-12 and the NAHA levels at home. The P-glucan induced secretion of IL-12 was related to the duration of symptoms at the time of diagnosis. Their X-ray scores were related to an increased secretion of cytokines after stimulation with LPS or P-glucan. Subjects with sarcoidosis have a higher reactivity to FCWA in vitro and to home exposure. The influence of FCWA on inflammatory cells and their interference with the inflammatory defense mechanisms in terms of cytokine secretion could be important factors for the development of sarcoidosis.     

Influence of Low Molecular Weight Heparin (Certoparin) and Unfractionated Heparin on the Release of Cytokines from Human Leukocytes

We analyzed the influence of heparins (unfractionated heparin, UFH and low molecular weight heparin certoparin) on the generation of IL-1ra, IL-6, IL-10, and IL-12p40 and from leukocyte fractions in vitro. Polymorphonuclear neutrophil leukocytes (PMN) and peripheral blood mononuclear cells (PBMC) from 16 different healthy donors were isolated and adjusted to 1 × 10⁶ cells/ml supplemented RPMI 1640. Leukocyte fractions were differentially stimulated (PMN with 1 g and 5 g LPS, PBMC with 10 ng TSST-1 or 2 g ConA) in the presence or absence of heparins (1 U/ml, 2 U/ml, and 4 U/ml) for 24 h at 37°C. Cytokine release was analyzed by ELISA. Certoparin but not UFH led to a dose-dependent increase in IL-6 from non-stimulated PBMC. In contrast, the release of IL-1ra, IL-10, and IL-12p40 was not modulated by heparins in a dose-dependent fashion. Increases in these cytokines occurred only as single incidents at intermediate heparin levels. An influence of the heparins on the apoptosis of PMN (measured as DNA-fragmentation in non-stimulated or LPS-stimulated cell-fractions) was not observed.     

In vitro effect of statins on cytokine production and mitogen response of human peripheral blood mononuclear cells

The in vitro effect of the hydrophilic statin - pravastatin - and three hydrophobic statins - atorvastatin, lovastatin and simvastatin - on the production of IL-1beta, IL-1ra, IL-2, IL-6 and IFN-gamma by human peripheral blood mononuclear cells (PBMC) and their response to mitogens was examined. Lovastatin and simvastatin increased the production of IL-1beta in a dose dependent manner and reduced secretion of IL-1ra at high concentration. These two statins exerted a dose dependent inhibitory effect on IL-2 production and reduced the secretion of IFNgamma at high dose. Atorvastatin did not affect IL-1beta, but suppressed IL-1ra, IL-2 and IFNgamma production. Atorvastatin, lovastatin and simvastatin caused a dose dependent inhibition of mitogen-induced proliferation of PBMC. IL-6 production was not affected by any one of the statins. While pravastatin caused a slight reduction in the proliferative response of PBMC to PHA, it did not affect either their response to Con A and PWM, or the secretion of cytokines tested.