Interleukin-10 Directly Inhibits the Interleukin-6 Production in T-Cells

IL-6 is a potent regulator of T-cell activation, proliferation and differentiation. Since IL-10 inhibits cytokine production by T cells, the effect of IL-10 on IL-6 production by T cells was investigated. IL-6 production by purified monocytes or T cells was detected from cell-free culture supernatants by ELISA after stimulation of the cells with LPS or an anti-CD3 monoclonal antibody for 3 days. Although the main source of IL-6 are LPS activated monocytes (29.6 × lOng/ml), T cells secreted sufficiently high levels of IL-6 (790 × 200pg/ml) to stimulate the high affinity IL-6 receptor. IL-10 decreased anti-CD3 induced IL-6 mRNA expression by up to 80%. In addition, IL-10 significantly inhibited IL-6 release from T-cells. Highly purified, anti-CD3 activated T-cells secreted 600 × 150pg/ml IL-6 compared to 21 × 2pg/ml IL-6 following addition of IL-10 (10ng/ml; P <0.001). FACS analysis revealed a monocyte contamination of the T-cell preparations of less than 0.5%. In addition, no IL-1 production was detectable. Thus, in our experiments the effect of TL-10 on IL-6 production was independent of the presence of monocytes. Finally, inhibition of IL-6 production was not reversed by IL-2 (100U/ml). In conclusion, IL-10 suppressed the synthesis of IL-6 by T-cells via a monocyte-and IL-2-independeni mechanism. These results may help to understand the complex regulation of T-cell mediated cytokine production by IL-10.     

Human Brain Endothelial Cells and Astrocytes Produce IL-1β but not IL-10

The ability of human brain endothelial cells to produce mRNA for interleukin-10, and release IL-10 in culture supernatants after in vitro stimulation with LPS, TNF-α and γ-IFN was assessed and compared to that of astrocytes, peripheral blood mononuclear cells and human umbilical vein endothelial cells. IL-1β and β₂-microglobulin release were also analysed. IL-10 and TNF-α mRNA presence was investigated in normal brain as well as in three plaques from two multiple sclerosis patients. While increased IL-1β and β₂-microglobulin release in the supernatants of stimulated cells could be detected in all the studied cell lineages, IL-10 mRNA and protein release was only seen in LPS-stimulated PBMNCs. Similarly, mRNA for IL-10 was not detected in CNS tissues, while TNF-α was present in all plaques. The lack of production of significant amounts of IL-10 by astrocytes and human brain endothelial cells suggests that these cells may not be the primary source of in vivo IL-10-mediated down-regulation of immune reactions within the central nervous system.     

Contribution of Intestinal Epithelial Cells to Innate Immunity of the Human Gut – Studies on Polarized Monolayers of Colon Carcinoma Cells

The aim was to establish an in vitro model for studies of innate defence mechanisms of human intestinal epithelium. Ultrastructural characterization and determination of mRNA expression levels for apical glycocalyx and mucous components showed that polarized, tight monolayers of the colon carcinoma cell lines T84 and Caco2 acquire the features of mature- and immature columnar epithelial cells, respectively. Polarized monolayers were challenged with non-pathogenic Gram+ and Gram− bacteria from the apical side and the proinflammatory cytokines interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) from the basolateral side. Immune responses were estimated as changes in mRNA expression levels for the mucous component mucin-2 (MUC2), the glycocalyx components carcinoembryonic antigen (CEA), CEA-related cell adhesion molecule-1 (CEACAM1), CEACAM6, CEACAM7 and MUC3, the antimicrobial factors human β-defensin-1 (hBD1), hBD2, hBD3 and lysozyme, the chemokine IL-8 and the cytokines IL-6 and TNF-α. Tight monolayer cells were generally unresponsive to bacterial challenge, but increased their hBD2 levels when challenged with Bacillus megaterium. T84 cells also increased their TNF-α levels upon bacterial challenge. Tight monolayer cells responded to cytokine challenge suggesting awareness of basolateral attack. TNF-α induced significantly increased levels of IL-8 and TNF-α itself in both cell lines suggesting recruitment and activation of immune cells in the underlying mucosa in vivo . Cytokine challenge also increased levels of CEACAM1, which includes two functionally different forms, CEACAM1-L and CEACAM1-S. In T84 cells, IFN-γ was selective for CEACAM1-L while TNF-α upregulated both forms. Increased CEACAM1 expression may influence epithelial function and communication between epithelial cells and intraepithelial lymphocytes.     

Interleukin-6 and interleukin-6 soluble receptor regulate proliferation of normal, human papillomavirus-immortalized, and carcinoma-derived cervical cells in vitro.

A variety of sexually transmitted diseases frequently accompany infection with human papillomavirus and stimulate inflammation of the cervical mucosa. Inflammation and cell injury cause release of proinflammatory cytokines, which in turn might regulate growth of human papillomavirus-infected cells. This study compared the interaction of the proinflammatory cytokine, interleukin-6 (IL-6), and its soluble receptor with normal ecto- and endocervical cells, human papillomavirus-immortalized ectocervical cells, and squamous carcinoma-derived cell lines. Proliferation of normal cervical cells was enhanced by IL-6 but inhibited by its soluble receptor. However, both IL-6 and its soluble receptor significantly stimulated growth of the three immortal and four cervical carcinoma-derived cell lines analyzed. Stimulation by IL-6 was dose dependent and was blocked by an antibody that neutralized IL-6 activity. IL-6-mediated proliferation was accompanied by increased expression of RNAs encoding transforming growth factor-alpha and amphiregulin, two epidermal growth factor receptor ligands. Furthermore, growth stimulation by IL-6 was significantly inhibited by antibodies that either blocked signal transduction by the epidermal growth factor receptor or that neutralized transforming growth factor-alpha or amphiregulin activity. Thus, IL-6 stimulates proliferation of human papillomavirus-immortalized cervical cells via an epidermal growth factor receptor-dependent pathway involving autocrine stimulation by transforming growth factor-alpha and amphiregulin.     

Human B cells and macrophages cooperate in T-cell-independent type 2 response

We have analysed separately the role of B-cell receptor (BCR) stimulation and the soluble second signal in the T-cell-independent type 2 (TI-2) B-cell response. We were able to show that human B cells and macrophages (Mφ) could function together in TI-type microbial response. Interestingly, BCR cross-linking of peripheral blood (PB) B cells enhanced IgG production induced by Mφ-derived growth factors whereas interleukin (IL)-12 + IL-18 had milder effect on IgG production. We demonstrated that B-cell-derived soluble mediators primed lipopolysaccharide (LPS)-stimulated Mφ for tumour necrosis factor-α (TNF-α) and IL-6 production significantly better than IFN-γ, confirming the role of B cells in the activation of Mφ. We could show that human PB B cells were active cytokine producers and could be induced to produce interferon (IFN)-γ mRNA in the presence of known Mφ cytokines, like IL-12 and IL-18. BCR stimulation also stabilized and enhanced the IFN-γ mRNA production induced by IL-12 and IL-18. In addition, our novel finding was that a known Mφ cytokine, IL-10, induced the expression of IFN-γ mRNA from human B-cell line (HF28R0) cells. In summary, we propose a model for the active role of B cells in the induction of the inflammatory response during TI antigen challenge in close collaboration with Mφ.     

A mechanism underlying the sexually dimorphic ACTH response to lipopolysaccharide in rats: sex steroid modulation of cytokine binding sites in the hypothalamus

It is well established that the hypothalamic-pituitary-adrenal responses to immune stressors are sexually dimorphic in rodents (females > males), but the underlying mechanism is still unclear. To investigate the mechanism, in this study we examined whether the sex steroid environment affects the following variables in male and female rats: (1) plasma levels of ACTH, interleukin (IL)-1β, IL-6 and tumour necrosis factor-α (TNF-α) after systemic lipopolysaccharide (LPS) administration; (2) static concentrations of corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) in the mediobasal hypothalamus (MBH) and those of ACTH in the anterior pituitary (AP); and (3) the binding characteristics of IL-1β, IL-6 and TNF-α in the MBH and AP. LPS-induced ACTH release was significantly higher in female than in male rats, and this sexual difference was abolished by performing gonadectomy in both sexes. Administration of physiological doses of testosterone and oestradiol to gonadectomized males and females, respectively, restored the altered ACTH responses to normal. Changes in the sex steroid milieu did not affect plasma cytokine responses to LPS, tissue contents of CRH, AVP and ACTH, or the IL-6 binding characteristics in the MBH and AP. However, the number of IL-1β and TNF-α binding sites, but not their binding affinities, in the MBH showed significant changes according to altered sex hormone milieu, in the same direction as the LPS-induced ACTH response. These results suggest that the hypothalamic sensitivity to peripheral IL-1β and TNF-α may be an important mechanism underlying the sexually dimorphic ACTH response to LPS in rats.     

Epstein-Barr virus-immortalized B cells produce IL-6 as an autocrine growth factor.

The continuous proliferation of Epstein-Barr virus (EBV)-immortalized B cells is enhanced by autocrine as well as paracrine growth factors. In the present study, the possibility that EBV-immortalized B cells might produce interleukin-6 (IL-6) proteins in an autocrine manner was examined. It was found that culture supernatants from EBV-transformed B cells, but not from Burkitt's lymphoma lines, augmented the proliferation of an IL-6-dependent murine hybridoma clone, MH60.BSF2. This growth-promoting activity for hybridoma cells found in culture supernatants of EBV-transformed B cells was specifically neutralized by rabbit anti-recombinant (r) IL-6 antibody. The IL-6 activity in culture supernatants of EBV-transformed B cells, though much less than that of lipopolysaccharide (LPS)-stimulated monocytes, was increased by the addition of phorbol myristate acetate. Western blot experiments using rabbit anti-rIL-6 antiserum demonstrated that supernatants from cultured EBV-transformed B cells contained the distinct forms of IL-6, with a peak of 23,000 MW. When examined by in situ hybridization analysis, it was found that IL-6 mRNA were expressed on EBV-transformed B cells. It was noted that a fraction, but not all, of these cells expressed IL-6 mRNA strongly, implying their cell cycle-dependent expression. In addition, it was shown that rIL-6 promoted the growth of EBV-transformed B cells at low cell densities. The results suggest that IL-6 serves as an autocrine growth factor in EBV-transformed B cells.     

TNF-α Dominates Cytokine mRNA Expression in Lymphoid Tissues of Rats Developing Collagen- and Oil-Induced Arthritis

Experimental arthritis can be induced in the DA rat strain with rat type II collagen (RCII) administered in Freund's incomplete adjuvant oil (FIA) or with only FLA. If ovalbumin (Ova), is added to these arthritogens the development of arthritis is blocked. To investigate the mechanisms responsible for induction of arthritis, as well as inhibition of arthritis, a kinetic study of the local cytokine expression in lymph nodes has been performed after immunization with the above mentioned agents. By using in situ hybridization techniques, mRNA expression of TNF-α, IL-2, IFN-γ and IL-4 was determined. The results show a rapid and pronounced accumulation of TNF-α mRNA expression, in RCII/FIA and FIA immunized rats. This pronounced expression of TNF-α mRNA was not recorded in the Ova/FIA immunized animals, which instead were the only animals in which the IL-4 gene was expressed. The expression of IFN-γ mRNA was limited in RCII/FIA- and FIA-immunized rats, whereas IL-2 mRNA expression was detected only after RCII/FIA injection. Lymph node cells from RCII-immunized animals generated a high amount of TNF-α mRNA after restimulation with RCII, whereas restimulation with the mitogen Con A generated a cytokine mRNA response dominated by IL-2 and IFN-γ.
These and other results indicate that a strong local expression of TNF-α, induced by arthritogenic stimuli, may be important for the induction of arthritis. Moreover, the elicitation of an immune reaction against Ova, may inhibit arthritis development by contributing to a shift in the initial arthritogenic cytokine response.     

Interleukin-10 Response Abnormalities in Systemic Lupus Erythematosus

It has been previously reported that the production of interleukin-6 (IL-6) is often enhanced in systemic lupus erythematosus (SLE). The authors examined the secretion of IL-6, tumour necrosis factor-α (TNF-α), granulocyte–macrophage colony-stimulating factor, IL-1α and IL-4 by B cells and monocytes from lupus patients and compared this to the production in normal controls and in rheumatoid arthritis patients. IL-6 production was increased an average of 3.4-fold compared to that in normal subjects and 8.4-fold compared to rheumatoid arthritis patients. In SLE, a strongly positive correlation was found between the levels of IL-6 and TNF-α ( R  = 0.8987, P  = 0.002). Since production of both IL-6 and TNF-α is regulated by IL-10, the enhancement of the production of these cytokines could reflect a defect in either IL-10 production or responsiveness. However, spontaneous production of IL-10 was enhanced in cultures of B cells and monocytes from lupus patients, compared to normal controls, the levels being increased 3.1- to 6-fold for monocytes and B cells, respectively. The finding of increased secretion of these cytokines implies an abnormality in IL-10-mediated suppression in SLE. To assess this possibility, the authors examined recombinant human IL-10-mediated suppression of IL-6 production by monocytes and B cells from lupus patients, compared to normal controls, and found that whereas IL-10 caused a concentration-dependent suppression of IL-6 production in normal B cells and monocytes, this suppression was deficient in B cells and monocytes from lupus patients. In SLE, it therefore appears that there may be an intrinsic defect in IL-10-induced suppression of cytokine synthesis. This could explain the increased levels of IL-10 and IL-6 found in this condition, and may also be responsible for the characteristic polyclonal B-cell activation that is seen.     

Possible contribution of aminopeptidase N (APN/CD13) to invasive potential enhanced by interleukin-6 and soluble interleukin-6 receptor in human osteosarcoma cell lines

This study aimed at clarifying the role of Aminopeptidase N (APN), a Zn²⁺-dependent ectopeptidase localized on the cell surface of human osteosarcoma cell lines treated with proinflammatory cytokines. We investigated the proinflammatory cytokines interleukin-1 beta (IL-1β), IL-6 and tumor necrosis factor alpha (TNF-α) as well as the anti-inflammatory cytokine transforming growth factor beta (TGF-β) for their influence on APN regulation. Soluble IL-6 receptor (sIL-6R) was always used together with IL-6 to achieve a stable effect. In addition, the invasive potential of the osteosarcoma cell lines MG63 and HOS was examined. Competitive RT-PCR and Ala-pNA activity assays revealed that IL-6 and sIL-6R significantly increased the mRNA expression and activity of APN in both osteosarcoma cell lines. Although IL-1β significantly stimulated APN mRNA expression in both cell lines, it influenced the enzyme activity only in MG63. TNF-α and TGF-β, however, had an effect neither on mRNA expression nor on the enzyme activity of APN in both cell lines. In the Matrigel invasion assay, IL-6 and sIL-6R significantly up-regulated the transmigration of these cell lines, whereas other cytokines did not. The up-regulated invasion was inhibited by bestatin, a specific inhibitor of APN. Cellular migration correlated highly with APN activity (r = 0.79, P < 0.002). These findings suggest that APN contributes to the invasive potential of human osteosarcomas enhanced by IL-6 and SIL-6R. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of Tumour Necrosis Factor-α on the Expression of Fc IgG and IgA Receptors, and Other Markers by Cultured Human Blood Monocytes and U937 Cells

The expression of Fc receptors for IgG (FcγR) and IgA (FcαR) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor-α (TNF-α) and other cytokines, was investigated by flow cytomelry. TNF-α, as well as interferon-γ (IFN-γ) or interleukin-6 (IL-6) had a significant up-regulating effect on U937 expression of FcγRI/CD64. Furthermore, the action of TNF-α was augmented by IL-6, and more evidently by IFN-γ. IFN-α alone had only a marginal effect, but was able to increase the TNF-α-driven FcγRI expression. In contrast to U937 cells, TNF-α did not enhance significantly FcγRI expression on human monocytes. Interestingly, on both U937 cells and monocytes. FcαR was augmented markedly by TNF-α. Furthermore, TNF-α induced the expression of HLA-DR and HLA-DP antigens on monocytes and U937 cells. The expression of FcγRII/CD32, FcγRIII/CD16. CD14, complement receptor type 1 (CR1/CD35). CR4 (CD11c/CD18), and MHC class-I antigens, was not influenced significantly by TNF-α. The results of this study show that TNF-α may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell-surface antigens.     

Constitutive and Inducible Cytokine mRNA Expression in the Human Mast Cell Line HMC-1

The discovery that mast cells are a potential source of cytokines has suggested new ways in which mast cells can act in immunological and inflammatory responses. In this study we have used the HMC-1 cell line as a model for human mast cells to study the constitutive and inducible mRNA expression of interleukins, colony-stimulating factors, interferons, tumour necrosis factors α and β, tumour growth factor β and platelet-derived growth factor A and B. We found that HMC-1 cells constitutively expressed mRNA for TNF-α and TGF-β, and a low level of M-CSF. After treatment with the phorbol ester TPA or the calcium ionophore ionomycin expression of several cytokines, i. e. IL-1β, IL-3, IL-6, GM-CSF, TNF-β and PDGF-A, could be detected. Both TPA and ionomycin induced the same set of cytokines, but the effect of TPA was more prominent. The relative induction was calculated to be 70X for IL-1β and IL-3, 30X for GM-CSF and PDGF-A and 3 - 10X for IL-6, M-CSF and TNF-β. This study shows that human mast cells have the capacity to express not only cytokines mediating an immune response but also cytokines affecting other cell types, e. g. fibroblasts and endothelial cells, involved in later steps of the inflammatory response.     

Modulation of Glucocorticosteroid Binding in Human Lymphoid, Monocytoid and Hepatoma Cell Lines by Inflammatory Cytokines Interleukin (IL)-lβ, IL-6 and Tumour Necrosis Factor (TNF)-α

In order to elucidate the role of the inflammatory cytokines in regulating glucocorticosteroid binding (GCSB) and glucocorticosteroid receptor (GR) level we incubated a B-cell line (CESS), a promonocytic cell line (U937) and a hepatoma cell Iine(HepG2)in the presence of varying concentrations of IL-1β, IL-6 and TNF-α(for 24 h. Glucocorticosteroid binding was determined by the method of whole cell uptake', and the cellular appearance of the glucocorticosteroid receptor was detected by immunocytochemistry, A rise in the glucocorticosteroid binding was induced by all three cytokines. The increase in level of glucocorticosteroid receptors in the cells shown by immunocytochemistry was much more pronounced. However, antagonistic effects were demonstrated by both methods between IL-6 and TNF-α. and between IL-1β and TNF-α when they were applied simultaneously, in U937. Present data suggest that local imbalance in the ratio of these three cytokines in different pathological cases might influence the glucocorticosteroid sensitivity of the lymphocytes, monocytes and hepatocytes as target cells.     

Interleukin-6 and its receptor during homeostasis,inflammation, and tumor growth

Summary This review focuses on describing the specific role of interleukin-6 within the network of inflammatory mediators in man. Sites of interleukin-6 synthesis, regulation of its expression, and the biological functions of this molecule are here outlined. The potential role of interleukin-6 as a diagnostic monitor is discussed. Particular attention is paid to experimental evidence that interleukin-6 and its receptor may be involved in the pathogenesis of autocrine tumor growth. A recently proposed therapeutical use of cytotoxic interleukin-6 fusion proteins in order to selectively destroy certain interleukin-6 receptor bearing tumor cells is discussed in the light of the finding, that not only hepatocytes, but also normal peripheral blood monocytes express the interleukin-6 receptor.Abbreviations ACTH Adenocorticotropic hormone - BCDF B-cell differentiation factor - BSF-2 B-cell stimulatory factor 2 - bp base pairs - CAT chloramphenicol acetyltransferase - cDNA complementary deoxyribonucleic acid - cAMP cyclic adenosinmonophosphate - CHX cycloheximid - CRP C-reactive protein - DNA deoxyribonucleic acid - EBV Epstein Barr Virus - FACS fluorescence activated cell sorter - GMCSF granulocyte-monocyte colony stimulating factor - HSF hepatocyte stimulating factor - HGF hybridoma growth factor - IFN 2 interferon beta 2 - IL-1 interleukin-1 - IL-3 interleukin-3 - IL-6 interleukin-6 - IL6R interleukin-6 receptor - kDa kilo-dalton - LPS lipopolysaccharide/endotoxin - M-CSF macrophage colony stimulating factor - mRNA messenger ribonucleic acid - PDGF platelet-derived growth factor - RNA ribonucleic acid - SAC Staphylococchus aureus Cowan I - TNF tumor necrosis factor/cachectin Dedicated to Hedwig     

HIV-1 Inhibits Leishmania-lnduced Cell Proliferation but not Production of Interleukin-6 and Tumour Necrosis Factor Alpha

The immune response of normal human peripheral blood mononuclear cells (PBMC) after stimulation with human immunodeficiency virus-1 (HIV-1) antigens plus Letshmania donovani promastigotes in vitro was investigated. HIV-I-antigen stimulation of PBMC did not induce the intracellular accumulation of interleukin-6 (IL-6),. tumour necrosis factor-alpha (TNF-α), or Interferon-gamma (IFN-γ). However, cells stimulated with L. donovani antigens exhibited the production of IL-6 and TNF-α, but not IFN-γ. Furthermore, co-stimulation of PBMC with HIV-1 antigen plus L. donovani resulted in the intracellular accumulation of IL-6 and TNF-α comparable to that of cells that were activated with L. donovani antigen alone. Heat-inactivated HIV-1 antigen did not appear to induce or suppress cytokine production by PBMC. However, the same HIV antigens did suppress L. donovani-induced proliferation as well as PPD-induced proliferation in a dose-dependent fashion. Elevated levels of serum cytokines have been demonstrated in patients with HIV infection indicating their role in the pathogenesis of HIV-associated immunosuppression. The results may partially support the idea that the abnormally increased cytokine levels in the sera of HIV-infected subjects is due to the various opportunistic pathogens that these patients contract, rather than a response to HIV antigens. As cytokines have been shown to up-regulatc HIV replication, the data suggest a role for opportunistic infections in cytokine-induced transactivation of HIV-1 and disease progression.     

Downregulation of CXCR6 and CXCR3 in lymphocytes from birch-allergic patients

Preferential expression of chemokine receptors on Th1 or Th2 T-helper cells has mostly been studied in cell lines generated in vitro or in animal models; however, results are less well characterized in humans. We determined T-cell responses through chemokine receptor expression on lymphocytes, and cytokine secretion in plasma from birch-allergic and healthy subjects. The expression of CCR2, CCR3, CCR4, CCR5, CCR7, CXCR3, CXCR4, CXCR6, IL-12 and IL-18R receptors was studied on CD4⁺ and CD8⁺ cells from birch-allergic ( n  = 14) and healthy ( n  = 14) subjects by flow cytometry. The concentration of IL-4, IL-5, IL-10, IL-12, IFN-γ and TNF-α cytokines was measured in plasma from the same individuals using a cytometric bead array human cytokines kit. The similar expression of CCR4 in T cells from atopic and healthy individuals argues against the use of the receptor as an in vivo marker of Th2 immune responses. Reduced percentages of CD4⁺ cells expressing IL-18R, CXCR6 and CXCR3 were found in the same group of samples. TNF-α, IFN-γ, IL-10, IL-5, IL-4 and IL-12 cytokines were elevated in samples from allergic individuals. Reduced expression of Th1-associated chemokine receptors together with higher levels of Th1, Th2 and anti-inflammatory cytokines in samples from allergic patients indicate that immune responses in peripheral blood in atopic diseases are complex and cannot be simplified to the Th1/Th2 paradigm. Not only the clinical picture of atopic diseases but also the clinical state at different time points of the disease might influence the results of studies including immunological markers associated with Th1- or Th2-type immune responses.     

Interleukin-6 production by endothelial cells via stimulation of protease-activated receptors is amplified by endotoxin and tumor necrosis factor-alpha.

Human endothelial cells respond to extracellular proteases, endotoxin (lipopolysaccharide, LPS), and inflammatory cytokines. Endothelial cells express several protease-activated receptors (PAR), including the thrombin-activated receptors PAR-1 and PAR-3 and a thrombin-independent, protease-activated receptor, PAR-2. To examine the potential cooperation between PAR and inflammatory stimuli, we investigated the effects of the PAR-1 agonist peptide Ser-Phe-Leu-Leu-Arg-Asn (SFLLRN) and PAR-2 agonist peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) on endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured in vitro with SFLLRN or SLIGKV in the presence and absence of LPS or tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) levels in the culture supernatants were assayed. Both SFLLRN and SLIGKV induced detectable levels of IL-6 production in a dose-dependent fashion, with the PAR-1 receptor agonist being more potent. In the presence of all stimulatory concentrations of LPS or TNF-alpha tested, both peptides were found to further enhance IL-6 production. The effects of SFLLRN and SLIGKV were specific, as related peptides with identical amino acid compositions, but lacking in consensus sequences, were biologically inactive either alone or in the presence of LPS. Both the direct and the amplifying effects of PAR agonist peptides on IL-6 production were pertussis toxin sensitive and caused an increase in the intracellular levels of calcium, implicating G-proteins and calcium mobilization in these pathways. Furthermore, the amplifying effect of LPS or TNF-alpha on PAR-mediated cytokine production was associated with corresponding increases in nuclear NF-kappaB proteins. The results demonstrate significant potentiation of PAR-induced signaling by LPS and TNF-alpha and indicate the potential cooperation of proteases and inflammatory stimuli in amplifying vascular inflammation.