Activation of toll-like receptor 2 on human dendritic cells triggers induction of IL-12, but not IL-10

Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.     

Recombinant HBsAg,an apoptotic-like lipoprotein,interferes with the LPS-induced activation of ERK-1/2 and JNK-1/2 in monocytes

Yeast expressed Hepatitis B surface antigen (rHBsAg) binds to monocytes through interaction with the LPS binding protein (LBP) and the LPS receptor CD14. Charged phospholipids of rHBsAg determine the interaction with these proteins. Although attachment of rHBsAg resembles the pro-inflammatory binding of LPS to CD14, rHBsAg does not activate monocytes and even reduces the expression of pro-inflammatory cytokines by LPS-stimulated monocytes. It is reported here that addition of rHBsAg to LPS-stimulated PBMC often results in increased secretion of IL-10, suggesting a similarity between the interaction of monocytes with apoptotic cells and rHBsAg. Using THP-1 cells, it is shown that IL-10 is not necessary to reduce TNFalpha protein levels. Addition of rHBsAg to LPS-stimulated cells reduces TNFalpha mRNA levels, but does not affect phosphorylation of p65 NF-kappaB and p38 MAP kinase. Instead, a reduced phosphorylation of ERK-1/2 and JNK-1/2 MAP kinases is observed.     

Thrombin enhancement of interleukin-1 expression in mononuclear cells: involvement of proteinase-activated receptor-1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is considered a pro-inflammatory molecule. We have previously demonstrated that differentiated monocytes express the proteolytically activated receptor for thrombin (PAR-1) and that thrombin enhances the release of interleukin (IL)-6 in human monocytes. In the present study we show that thrombin upregulates the production of both IL-1alpha and IL-1beta in phytohemagglutin (PHA)-activated human peripheral blood mononuclear cells (PBMC). Treating PHA-activated PBMC with the PAR-1 activation peptide, SFLLRN, mimics the effects of thrombin on IL-1alpha and IL-1beta production. Thus, it appears that these pro-inflammatory effects induced by thrombin may be mediated through activation of PAR-1. ELISA and RNase protection assays indicate that thrombin and SFLLRN peptide upregulates IL-1 expression at both protein and mRNA levels. Thrombin directly affects monocyte IL-1 expression, since treatment of differentiated U937 cells with thrombin and SFLLRN enhances IL-1 production. These results may help explain how thrombin can enhance IL-1 expression in normal tissue to initiate tissue repair and why thrombin and thrombin-like enzymes may contribute to inflammatory responses observed in several pathophysiological conditions.     

Adherence modifies the regulation of gene expression induced by interleukin-10

Interleukin-10 (IL-10) is a well known anti-inflammatory cytokine. However, we previously showed that it could present pro-inflammatory properties on human monocytes in the absence of adherence. In the present study, using macroarray technology, we analyzed the effects of IL-10 and adherence on the expression of 1050 genes in human monocytes cultured for 3 hours on plastic or Teflon(R) (to avoid adherence). Adherence alone induced specifically the expression of 12 genes and repressed that of 25 genes. In adherent monocytes, IL-10 induced the expression of 21 genes and repressed that of 50 genes. In non-adherent monocytes, IL-10 induced the expression of 45 genes and repressed that of 67. Only 3 common genes were induced while 35 common genes were repressed by IL-10 in the two culture conditions. Interestingly, we showed that IL-10 modulated conversely on Teflon(R) and plastic the expression of 16 genes, of which SOCS molecules, coproporphyrinogen oxidase, matrix metalloproteinases and complement receptor-1 (CD35). This study demonstrates that adherence has profound modulatory effects on the properties and the signaling induced by IL-10. The discovery that IL-10 can inhibit the production of coproporphyrinogen oxidase (an enzyme involved in the synthesis of heme) may shed some lights on the mechanisms of anaemia induced by IL-10. Furthermore, the inhibition of the expression of SOCS1 by IL-10 in the absence of adherence, may explain its priming effects on a subsequent LPS stimulation that we previously described.     

Human glioma-induced immunosuppression involves soluble factor(s) that alters monocyte cytokine profile and surface markers

Patients with gliomas exhibit deficient in vitro and in vivo T cell immune activity, and human glioblastoma culture supernatants (GCS) inhibit in vitro T lymphocyte responses. Because APC are essential for initiating and regulating T cell responses, we investigated whether GCS would affect cytokines produced by monocytes and T cells from healthy donors of PBMC. Incubation of PBMC with GCS decreased production of IL-12, IFN-gamma, and TNF-alpha, and increased production of IL-6 and IL-10. The GCS-induced changes in IL-12 and IL-10 occurred in monocytes, and involved changes in IL-12 p40 and IL-10 mRNA expression. Incubation with GCS also resulted in reduced expression of MHC class II and of CD80/86 costimulatory molecules on monocytes. The immunosuppressive effects were not the result of IL-6 or TGF-beta1 that was detected in GCS. However, it was due to a factor(s) that is resistant to pH extremes, differentially susceptible to temperature, susceptible to trypsin, and has a minimum molecular mass of 40 kDa. Our findings show that glioblastoma-generated factors that are known to suppress T cell responses alter the cytokine profiles of monocytic APC that, in turn, inhibit T cell function. This model indicates that monocytes can serve as an intermediate between tumor-generated immune-suppressive factors and the T cell responses that are suppressed in gliomas.     

Augmentation of phorbol ester-induced T cell proliferation by agents which raise intracellular cyclic adenosine monophosphate.

Although raising intracellular cyclic adenosine monophosphate (cAMP) levels is generally considered to be inhibitory on the mitogen-induced T cell proliferation, in this study we have shown that the addition of either dbcAMP (50 microM) or cholera toxin (1 ng/ml) resulted in an increase in [3H]thymidine uptake in PBMC cultures stimulated with phorbol ester, 12-tetradecanoylphorbol 13-acetate (TPA), or with a combination of TPA plus anti-CD3 mAb (mAb 235). In contrast, under similar culture conditions, the phytohemagglutinin-P (PHA-P) response was inhibited by these agents as has been reported. The augmentative effect of dbcAMP in PBMC cultures was due to an increase in IL-2 production and not to increased in IL-2R-alpha chain expression. The enhancing effect of dbcAMP and CT observed with PBMC was monocyte dependent and not seen with purified T cell preparations. The addition of monocytes reconstituted the ability of intracellular cAMP elevating agents to augment the T cell response to TPA with and without mAb to CD3. The monocytes mediate their action via soluble factor(s) with molecular weight (m.w.) of more than 10 kDa. Neither rIL-1, rIL-6, nor rTNF-alpha have any augmentative effect as contrast with the supernatant from treated monocytes. Taken together, our results indicate that cAMP can play a positive regulatory role in T cell proliferation due to factor(s) secreted by dbcAMP-treated monocytes resulting in increased IL-2 synthesis in T cells.     

Emergence of anti-inflammatory monocytes in long-term surviving hosts of IL-10-transduced liver allografts

We previously reported that transduction of IL-10 to rat liver allografts facilitates survival prolongation after orthotopic liver transplantation (OLT). In the current study, we examined Lewis hosts of IL-10-transduced allografts that had survived longer than 50 days in order to characterize peripheral blood mononuclear cells (PBMC). Phenotype analysis of the PBMC demonstrated an 18-fold increase in monocytes (CD11b/c(+)) with a massive increase in the monocyte/lymphocyte ratio. The monocytes expressed downregulated MHC class II (RT1B) but upregulated Fcgamma receptors in comparison with monocytes from the control hosts. The capacity of enriched monocytes to secrete TNF-alpha in response to LPS stimulation was downregulated in the survivors, while production of IL-10 was comparable. The monocytes from long-term survivors significantly inhibited the donor antigen stimulated secretion of IFN-gamma and IL-2. Monocytosis characterized by a shift to anti-inflammatory monocytes is associated with survival prolongation in the hosts of IL-10 transduced liver allografts.     

A novel role for accessory cells in T cell-dependent B cell differentiation

The monocyte requirement for pokeweed mitogen-induced T cell-dependent B cell activation was reexamined. We report a dichotomy in the requirement for accessory cells in B cell proliferation and differentiation. Adherent cell-depleted human peripheral blood mononuclear cells which contained only 5% monocytes generated sufficient T cell help for optimal B cell proliferation. However, the presence of 10 to 20% monocytes were required during the last 5 days of culture for stimulated B cells to become IgG-secreting cells. Similar numbers of monocytes were also needed for anti-CD3-induced B cell differentiation. Moreover, monocytes alone added to previously activated B cells could support B cell differentiation in the absence of T cells. To determine the role of cytokines in this system, we demonstrated that supernatants of adherent cell-depleted PBMC contained decreased IL-6 activity in comparison with unseparated PBMC, but not IL-1, IL-2, or BCGF. Recombinant IL-6, however, added back either alone or with other cytokines could not replace the effects of intact monocytes on B cell differentiation. Physical interaction between the accessory cells and the responder cells was also required. As a minimum, paraformaldehyde-fixed monocytes, IL-6, and IL-1 were needed to reconstitute maximal IgG secretion. These studies suggest that accessory cells capable of producing IL-1 and IL-6 can have direct effects on the terminal differentiation of stimulated B cells.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

The immunomodulatory role of syncytiotrophoblast microvesicles

Immune adaptation is a critical component of successful pregnancy. Of primary importance is the modification of cytokine production upon immune activation. With the discovery that normal pregnancy itself is a pro-inflammatory state, it was recognised that the classical Th1/Th2 cytokine paradigm, with a shift towards 'type 2' cytokine production (important for antibody production), and away from 'type 1' immunity (associated with cell mediated immunity and graft rejection), is too simplistic. It is now generally agreed that both arms of cytokine immunity are activated, but with a bias towards 'type 2' immunity. Many factors are released from the placenta that can influence the maternal cytokine balance. Here we focus on syncytiotrophoblast microvesicles (STBM) which are shed from the placenta into the maternal circulation. We show that STBM can bind to monocytes and B cells and induce cytokine release (TNFα, MIP-1α, IL-1α, IL-1β, IL-6, IL-8). Other cytokines are down-modulated, such as IP-10 which is associated with 'type 1' immunity. Therefore STBM may aid the 'type 2' skewed nature of normal pregnancy. We also observed that PBMC from third trimester normal pregnant women produce more TNFα and IL-6 in response to STBM than PBMC from non-pregnant women, confirming that maternal immune cells are primed by pregnancy, possibly through their interaction with STBM.     

Induction of IFN-alpha in peripheral blood mononuclear cells by HIV-infected monocytes. Restricted antiviral activity of the HIV-induced IFN.

PBMC cocultured with HIV-infected monocytes for 12 to 48 h released high levels of IFN activity. IFN titers were directly dependent upon time after virus infection and level of HIV replication in infected cells. IFN induction in PBMC was evident with HIV-infected monocytes and PBMC and with myeloid and lymphoblastoid cell lines with at least three different HIV strains. In HIV-infected cell line pairs in which virus infection occurs in both productive and restricted forms, IFN induction in PBMC occurred only with productive infection. IFN activity was acid stable and completely neutralized by antibodies against IFN-alpha. Induction of IFN required cell-cell contact between HIV-infected cells and PBMC, but was independent of MHC compatibility. With PBMC co-cultured with autologous HIV-infected monocytes, IFN induction was highly selective: IL-1 beta, IL-6, or TNF-alpha activity and mRNA were not detected. Cell surface determinants on HIV-infected monocytes that induced IFN in PBMC remained active after fixation in 4% paraformaldehyde. Both adherent and nonadherent PBMC produced IFN after coculture with HIV-infected monocytes. Ability to produce IFN by PBMC was not affected by depletion of T cell, NK cell, B cell, or monocyte subpopulations. The IFN activity produced by PBMC cocultured with HIV-infected cells was about 20-fold less active than equal quantities of rIFN-alpha 2b for inhibition of HIV replication in monocytes and at low concentrations enhanced virus growth. Clinical studies with HIV-infected patients and parallel findings in animal lentivirus disease suggest an adverse role for IFN in disease progression. Conditions for induction of IFN in the culture system described in this report may mimic those in the HIV-infected patient. Defining the molecular basis for IFN induction, the cells that produce IFN, and the altered biologic activity of this important cytokine may provide insight into the pathogenesis of HIV disease.     

Regulation of the expression of IL-6 in human monocytes

IL-6 is a cellular regulatory molecule with various cell-dependent functions. We have studied the control of IL-6 expression in human monocytes because they play a key role in the production of this molecule. The effects of adherence and different cytokines including CSF-1, IFN-gamma, IL-1 alpha, and granulocyte-macrophage-CSF were tested on IL-6 expression. IL-6 mRNA was usually not detected in the starting population of PBMC. Adherence induced IL-6 gene expression in monocytes in less than 2 h and subsequently IL-6 secretion. Priming of monocytes by adherence was more efficient for IL-6 overinduction by CSF-1. In contrast, high level induction of IL-6 by IFN-gamma in unfractionated PBMC did not require adherence and in situ hybridization revealed that IL-6 mRNA was present in monocytes but not in lymphocytes. A similar phenomenon was observed for IL-1 alpha and granulocyte-macrophage-CSF. Two cell lines, HL-60 and U937, in which monocytic differentiation occurs after induction by PMA, were subsequently investigated. IL-6 was not constitutively detectable in these two cell lines, whereas PMA treatment induced IL-6 expression. This effect was rapid (30 min) and transitory in HL-60, whereas IL-6 mRNA was still detected after 72 h of induction in U937. Addition of human rIL-6 on U937 and HL-60 cells inhibited their proliferation and enhanced expression of HLA class I Ag.     

Herbal melanin modulates tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF) production.

Recent studies have indicated that cytokines can enhance immunogenicity and promote tumor regression. However, the means for modulating cytokine production are not yet fully investigated. In this study we report the effects of a herbal melanin, extracted from Nigella sativa L., on the production of three cytokines [tumor necrosis factor alpha (TNF-alpha), interleukin 6 (IL-6) and vascular endothelial growth factor (VEGF)], by human monocytes, total peripheral blood mononuclear cells (PBMC) and THP-1 cell line. Cells were treated with variable concentrations of melanin and the expression of TNF-alpha, IL-6 and VEGF mRNA in cell lysates and secretion of proteins in the supernatants were detected by RT-PCR and ELISA. Melanin induced TNF-alpha, IL-6 and VEGF mRNA expression by the monocytes, PBMC and THP-1 cell line. On the protein level, melanin significantly induced TNF-alpha and IL-6 protein production and inhibited VEGF production by monocytes and PBMC. In the THP-1 cell line melanin induced production of all three cytokine proteins. These observations raise the prospects of using N. sativa L. melanin for treatment of diseases associated with imbalanced cytokine production and for enhancing cancer and other immunotherapies.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.