CD38 expression and functional activities are up-regulated by IFN-gamma on human monocytes and monocytic cell lines

Human CD38, a surface molecule expressed by immature and activated T and B lymphocytes, has been characterized as a molecule transducing activation and proliferation signals, and intervening in adhesion to endothelium via its ligand CD31. CD38 is also a complex ectoenzyme featuring ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase activities, leading to the synthesis and degradation of cADPR, a Ca+-mobilizing agent. We investigated the effects of monocyte-activating stimuli (IFN-gamma, IL-2, LPS, TNF-alpha, and GM-CSF) on the expression and function of CD38, starting from the observation that human monocytes and the derived lines U937, THP-1, and Mono-Mac-6 bear the molecule on their surface. Our results indicate that IFN-gamma is a strong up-modulator of CD38, and IL-2 increases its expression only modestly. LPS, TNF-alpha, and GM-CSF had no detectable effects. Treatment with IFN-gamma produced a dose- and time-dependent up-regulation of CD38 in monocytes and monocytic lines, which was paralleled by increased ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase activities. Furthermore, CD38 ligation by specific MoAb reduced the IFN-gamma-dependent enhancement of monocyte-dynamic adhesion to endothelial monolayers. These findings identify IFN-gamma as a modulator of monocytic CD38 expression and indicate that CD38 plays a specific role in the activation and adhesion processes performed by monocytes.     

Rapid cytokine up-regulation of integrins, complement receptor 1 and HLA-DR on monocytes but not on lymphocytes.

Short-term (3 hr) incubation of whole blood with human recombinant cytokines induced rapid changes in the expression of monocyte but not of lymphocyte surface molecules. The percentage of monocytes bearing CD11b molecules was enhanced by tumour necrosis factor-beta (TNF-beta), whilst that of CD11c was increased by both TNF-alpha and TNF-beta. The mean fluorescence intensity (MFI) of monocyte CD11a was enhanced by interleukin-2 (IL-2), TNF-alpha and TNF-beta, and that of CD11b, CD11c and CD18 was increased by IL-2, IL-4, TNF-alpha and TNF-beta. The proportion of monocytes expressing HLA-DR antigens was not modified by the cytokines investigated, but its MFI was increased by IL-2, IL-4, TNF-alpha and TNF-beta. In contrast, the percentage of monocytes bearing complement receptor 1 (CD35) was enhanced by IL-2, TNF-alpha and TNF-beta but the MFI of this molecule was not modified by these cytokines. The highest up-regulation of CD18, HLA-DR and CD35 was observed with 100 U/ml of either IL-2, IL-4, TNF-alpha or TNF-beta. Decreasing the concentration of all four cytokines from 100 to 10 and 1 U/ml diminished the levels of expression of all molecules, with the exception of CD35, which reached its maximum upon incubation with 1 U/ml of TNF-alpha. IL-1 beta, IL-6 or interferon-gamma (IFN-gamma) did not modify the expression of any of the above monocyte surface determinants. Moreover, none of the lymphocyte surface molecules investigated was modified by 3-hr incubation of blood with cytokines. The demonstration that cytokines selectively and rapidly up-regulate integrins, complement receptor 1 and HLA-DR molecules, on monocytes but not on lymphocytes, suggests that similar mechanisms of mononuclear cell activation by cytokines may control the development and duration of the inflammatory process.     

IL-15-induced conversion of monocytes to mature dendritic cells.

IL-15 is produced by a wide variety of tissues in response to inflammatory stimuli. We examined the effect of IL-15 in supporting the maturation of monocytes to dendritic cells in ex vivo culture. IL-15 transformed CD14(+) monocytes to mature dendritic cells. These dendritic cells were similar to those obtained from monocyte cultures treated with a combination of the cytokines GM-CSF, IL-4 and TNF-alpha. The effects of IL-15 did not depend on endogenously produced GM-CSF. The IL-15-induced dendritic cells also expressed chemokines and stimulated strong allo-responses that were characteristic of mature dendritic cells. These data indicate that CD14(+) monocytes respond to IL-15 by undergoing morphological transformation and acquiring characteristic dendritic cell features that facilitate antigen-specific responses of T cells. Thus, the release of IL-15 by inflammatory stimuli may induce the conversion of monocytes to immuno-stimulatory dendritic cells to support primary immune responses against pathogens.     

CD16+ human monocyte-derived dendritic cells matured with different and unrelated stimuli promote similar allogeneic Th2 responses: regulation by pro- and anti-inflammatory cytokines

We previously demonstrated that tumor necrosis factor (TNF)-alpha-matured CD16- and CD16+ human monocyte-derived dendritic cells (16-mDC and 16+mDC) differentially stimulate naive CD4+ lymphocytes by inducing Th1- and Th2-like responses, respectively. Here, we further characterized the role of different DC maturation factors on Th polarization. Immature 16+mDC and 16-mDC (iDC) obtained by culture of purified monocytes with GM-CSF and IL-4 were maturated with (i) Toll-like receptor (TLR) ligands [lipopolysaccharide (LPS)], (ii) lymphocyte-derived (soluble CD40 ligand, IFN-gamma) and (iii) endogenous inflammatory stimuli [TNF-alpha, prostaglandin (PG)E2]. After activation with these stimuli, DC secrete IL-12 only in presence of LPS, and 16+mDC produced lower amounts of IL-12 and IL-10 than 16-mDC. Allogeneic CD4+CD45RO- lymphocytes co-cultured with 16+mDC secreted higher levels of IL-4 and IL-10 than those co-cultured with 16-mDC, regardless of the maturation stimuli. Results were similar when DC were activated with TLR-2 or TLR-3 ligands. The higher induction of IL-4 by 16+mDC was primarily dependent on IL-12, IL-4 and IL-10. IFN-gamma production by CD4+ T cells was similar with all the conditions except with LPS-16+mDC, which induced reduced amounts of this cytokine. Those differences were totally eliminated by neutralization of IL-12, IL-4 or IL-10. Finally, 16-mDC could reverse the Th2 phenotype of already committed lymphocytes toward a Th1 pattern in short-term cultures, whereas 16+mDC had less ability to skew this phenotype. These results indicate that 16+mDC elicit superior Th2 responses independently of the maturation factors that they received, and suggest that they could represent an important population of regulatory DC.     

Monocyte response to Th1 stimulation and effector function toward human mesangial cells are not impaired in patients with lupus nephritis

Monocytes/macrophages activated by Th1 stimulation such as interferon-gamma (IFN-gamma) and CD40 ligand (CD40L) infiltrate the kidney and play a critical role in the progression of lupus nephritis (LN). We examined the monocyte response to Th1 stimulation and their effector function toward activating renal resident cells in patients with LN. Following stimulation with IFN-gamma granulocyte macrophage-colony stimulating factor (GM-CSF)/recombinant CD40L the production of tumor necrosis factor-alpha and IL-12 p70 by PBMC was significantly higher in LN patients. In coculture experiments employing activated monocytes and human mesangial cells, there was a trend toward higher monocyte chemoattractant protein-1 production by lupus monocytes compared to normal controls. Basal expression of CD40, ICAM-1, and STAT-1 was significantly higher in monocytes from LN patients, suggesting ongoing activation. Monocyte response to IFN-gamma, as accessed by intercellular adhesion molecule-1 upregulation and phosphorylation of STAT-1, was comparable between the two groups. Thus, in contrast to earlier reports, Th1-dependent monocyte activation is not impaired. In this disease activated monocytes appear to be fully capable of inducing renal injury.     

Human Langerhans cells selectively activated via Toll-like receptor 2 agonists acquire migratory and CD4+T cell stimulatory capacity

In epidermal Langerhans cells (LCs), the expression pattern and the functions of TLRs have been poorly characterized. By using mAb, we show that LCs from human skin express TLR1, -2, -5, -6, and -9, the cognate receptors for detection of specific bacteria-derived molecules. As compared with other TLR agonists, LCs acquired a more matured phenotype when activated by specific bacterial or synthetic TLR2 agonists. In addition, monocyte-derived Langerin(+)/CD1c(+)LCs (CD1c(+)MoLCs) secreted higher amounts of IL-6 and TNF-alpha by stimulation via TLR2 than by stimulation via TLR3, -4, -5, -8, and -9. In contrast to MoLCs, dendritic cells, generated from the same donor monocytes, were activated by agonists of TLRs other than TLR2 as well. Lipopeptides triggering TLR2 induced IL-1R-associated kinase-1 phosphorylation and migration toward the chemokines CCL19 and CCL21 in epidermal LCs and CD1c(+)MoLCs. Up-regulation of CD86, CD83, and CCR7, TNF-alpha and IL-6, and NF-kappaB activation and proliferation of CD4(+)T cells could be inhibited TLR2-specific blockage using antibodies prior to TLR2 activation. Application of anti-TLR1, anti-TLR6, and anti-TLR2 indicated an exclusive role of TLR2 in IL-6 induction in human LCs. Collectively, our results show that TLR2 expressed by LCs mediates inflammatory responses to lipopeptides, which implicates a central role in sensing pathogens in human skin.     

Differential expression of Toll-like receptor 2 in human cells

Human Toll-like receptor 2 (TLR2) is a receptor for a variety of microbial products and mediates activation signals in cells of the innate immune system. We have investigated expression and regulation of the TLR2 protein in human blood cells and tissues by using two anti-TLR2 mAbs. Only myelomonocytic cell lines expressed surface TLR2. In tonsils, lymph nodes, and appendices, activated B-cells in germinal centers expressed TLR2. In human blood, CD14+ monocytes expressed the highest level of TLR2 followed by CD15+ granulocytes, and CD19+ B-cells, CD3+ T-cells, and CD56+ NK cells did not express TLR2. The level of TLR2 on monocytes was after 20 h up-regulated by LPS, GM-CSF, IL-1, and IL-10 and down-regulated by IL-4, IFN-gamma, and TNF. On purified granulocytes, LPS, GM-CSF, and TNF down-regulated, and IL-10 modestly increased TLR2 expression after 2 h. These data suggest that TLR2 protein expression in innate immune cells is differentially regulated by inflammatory mediators.     

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.     

TLR5 ligation by flagellin converts tolerogenic dendritic cells into activating antigen-presenting cells that preferentially induce T-helper 1 responses

Dendritic cells (DCs) differentiated in the presence of IL-10 preferentially induce regulatory T-cells and tolerance. Whether the tolerogenic properties displayed by these DCs (Tol-DCs) can be overcome has not been fully explored. Here we show for the first time that Tol-DCs express higher levels of TLR5 mRNA, but not TLR4 or TLR9 mRNA relative to DCs differentiated with GM-CSF and IL-4 (BM-DCs). In response to flagellin, a natural TLR-5 ligand, Tol-DCs produced IL-12 but not IL-10. Unlike Tol-DCs stimulated with LPS, which produce high levels of IL-10 and fail to generate a cognate inflammatory response in CD4⁺ T-cells, flagellin-stimulated Tol-DCs promoted the differentiation of CD4⁺ T cells with a T-helper 1 phenotype. The divergent T-cell outcomes induced by Tol-DCs in response to different TLR-ligands highlights not only their plasticity, but also points to TLR5 ligation as a potential strategy to overcome tolerance in environments that are otherwise conducive to immune unresponsiveness.     

Cytokine induction of neopterin production.

The pteridine neopterin is a marker of immunological activation and has been shown to be a useful marker of graft-versus-host disease (GVHD) in bone marrow transplant patients. High levels of both neopterin and interferon-gamma (IFN-gamma) were produced in vitro during mixed lymphocyte responses, which may be considered to be a model of the primary events leading to GVHD. Neopterin was shown to be produced by monocytes in response to stimulation with IFN-gamma, but not other cytokines. However, the interleukins IL-1 alpha, IL-1 beta, IL-2, and tumour necrosis factor (TNF) alpha and beta, but not IL-6, stimulated neopterin production by unfractionated peripheral blood mononuclear cells (PBMC), and culture supernatants from PBMC stimulated with IL-1 alpha, IL-1 beta, IL-2 and IL-6, but not TNF-alpha or TNF-beta induced neopterin production following transfer to fresh monocyte cultures. It therefore appears that cytokines may generate neopterin by induction of IFN-gamma, by synergy with low levels of induced IFN-gamma, or by non-IFN-gamma-dependent mechanisms.     

Role of the cytokine environment and cytokine receptor expression on the generation of functionally distinct dendritic cells from human monocytes

Myeloid dendritic cells (DC) and macrophages evolve from a common precursor. However, factors controlling monocyte differentiation toward DC or macrophages are poorly defined. We report that the surface density of the GM-CSF receptor (GM-CSFR) alpha subunit in human peripheral blood monocytes varies among donors. Although no correlation was found between the extent of GM-CSFR and monocyte differentiation into DC driven by GM-CSF and IL-4, GM-CSFR expression strongly influenced the generation of CD1a(+) dendritic-like cells in the absence of IL-4. CD1a(+) cells generated in the presence of GM-CSF express CD40, CD80, MHC class I and II, DC-SIGN, MR, CCR5, and partially retain CD14 expression. Interestingly, they spontaneously induce the expansion of CD4(+) and CD8(+) allogeneic T lymphocytes producing IFN-gamma, and migrate toward CCL4 and CCL19. Upon stimulation with TLR ligands, they acquire the phenotypic features of mature DC. In contrast, the allostimulatory capacity is not further increased upon LPS activation. However, by blocking LPS-induced IL-10, a higher T cell proliferative response and IL-12 production were observed. Interestingly, IL-23 secretion was not affected by endogenous IL-10. These results highlight the importance of GM-CSFR expression in monocytes for cytokine-induced DC generation and point to GM-CSF as a direct player in the generation of functionally distinct DC.     

Toll-like receptor 2 expression on human conjunctival epithelial cells: a pathway for Staphylococcus aureus involvement in chronic ocular proinflammatory responses.

BACKGROUND: Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR). OBJECTIVES: To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells. METHODS: Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry. RESULTS: Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies. CONCLUSIONS: Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.     

Gram-positive and gram-negative bacteria do not trigger monocytic cytokine production through similar intracellular pathways

Toll-like receptors (TLRs) are involved in human monocyte activation by lipopolysaccharide (LPS) and Staphylococcus aureus Cowan (SAC), suggesting that gram-positive and gram-negative bacteria may trigger similar intracellular events. Treatment with specific kinase inhibitors prior to cell stimulation dramatically decreased LPS-induced cytokine production. Blocking of the p38 pathway prior to LPS stimulation decreased interleukin-1alpha (IL-1alpha), IL-1ra, and tumor necrosis factor alpha (TNF-alpha) production, whereas blocking of the ERK1/2 pathways inhibited IL-1alpha, IL-1beta, and IL-1ra but not TNF-alpha production. When cells were stimulated by SAC, inhibition of the p38 pathway did not affect cytokine production, whereas only IL-1alpha production was decreased in the presence of ERK kinase inhibitor. We also demonstrated that although LPS and SAC have been shown to bind to CD14 before transmitting signals to TLR4 and TLR2, respectively, internalization of CD14 occurred only in monocytes triggered by LPS. Pretreatment of the cells with SB203580, U0126, or a mixture of both inhibitors did not affect internalization of CD14. Altogether, these results suggest that TLR2 signaling does not involve p38 mitogen-activated protein kinase signaling pathways, indicating that divergent pathways are triggered by gram-positive and gram-negative bacteria, thereby inducing cytokine production.     

Regulation by interleukin-3 of human monocyte pro-inflammatory mediators. Similarities with granulocyte-macrophage colony-stimulating factor.

Urokinase-type plasminogen activator (u-PA), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) activities were measured for highly purified human monocytes cultured for 18 hr with recombinant human interleukin-3 (IL-3). IL-3 alone stimulated monocyte u-PA activity, but not TNF-alpha or IL-1 activity. However, IL-3, together with interferon-gamma (IFN-gamma), stimulated the TNF-alpha, but not IL-1, activities of monocytes from several donors. In parallel cultures, granulocyte-macrophage colony-stimulating factor (GM-CSF) behaved similarly. IL-3, like GM-CSF, synergized weakly and sometimes irregularly with lipopolysaccharide (LPS) for increased TNF-alpha and IL-1 activities. Thus, IL-3 can selectively stimulate monocyte mediator production depending on the costimulus present; however, the stimulatory properties of IL-3 vary from those of IFN-gamma and IL-4. The similarities in activity between IL-3 and GM-CSF may be explained by a common or associated IL-3/GM-CSF receptor(s), as suggested by biochemical studies.     

Cultured human monocytes secrete fibronectin in response to activation by proinflammatory cytokines

We studied the effects of the cytokines IL-1alpha, IL-6, tumour necrosis factor-alpha (TNF-alpha), IL-4, IL-10, IL-13 and transforming growth factor-beta (TGF-beta) on fibronectin (FN) production by cultured-human monocytes. IL-1alpha, IL-6 and TNF-alpha all increased FN production, an indicator of monocyte activation. These cytokines increased FN production in a dose-dependent fashion, with a 4-h treatment being sufficient to measure FN production by radioimmunoassay. Conversely, IL-4, IL-10 and IL-13 strongly inhibited cytokine-induced FN production, while TGF-beta only partially inhibited FN production. The combination of suboptimal doses of cytokines (IL-1alpha + IL-6, IL-1alpha + TNF-alpha, IL-6 + TNF-alpha), which could not singly induce substantial amounts of FN, were able to induce FN production by cultured monocytes. Northern blot analysis with a cDNA specific for FN confirmed the expression of FN mRNA in cultured monocytes stimulated with a single cytokine or a combination of cytokines. Our data demonstrate that monocytes may not always require high concentrations of cytokines for activation in vitro, and that the synergistic or additive action of low levels of cytokines on monocyte activation may be sufficient to promote immune or inflammatory reactions. Our data also suggest that certain T cell cytokines may regulate monocyte activation.     

Kinetics of cytokine gene expression in human CD4+ and CD8+ T-lymphocyte subsets using quantitative real-time PCR

The time kinetics of five cytokines [interleukin-2 (IL-2), IL-5, interferon-gamma (IFN-gamma), granulocyte macrophage-colony stimulating factor (GM-CSF) and tumour necrosis factor-alpha (TNF-alpha)] and one cytotoxic effector protein (granzyme B) was analysed by real-time quantitative polymerase chain reaction (PCR) following in vitro stimulation of human CD4 and CD8 T lymphocytes. Two stimuli were used, a mitogen [phytohemagglutinin (PHA)] and a recall antigen [purified protein derivative (PPD)]. The pattern of cytokine mRNA expression was found to be dependent on the T-cell subset and stimulus used. A wide interindividual variability in the cytokine gene expression pattern was demonstrated. Two expression patterns were observed. A bell-shaped expression profile was seen for most cytokines upon PHA activation in both subsets and PPD-activated CD4 T cells, whereas a biphasic/multiphasic expression pattern was noted in CD8 T cells upon PPD stimulation. For most cytokines, the time to induction was within 30 min of activation, and maximum accumulation seemed to be obtained after 4-8 h of activation. A sustained high level could, however, be noticed for up to 24 h. Granzyme B gene expression was also induced within 30 min of activation but showed a continuous gradual increase and late maximal accumulation (48-72 h). The findings of the present study are of importance when designing studies using the cytokine gene expression profile as a marker for antigen-specific T lymphocytes. It might be recommended that cytokine gene expression (IL-2, IL-5 and IFN-gamma) should be measured after 4-8 h of specific activation but also up to 24 h of stimulation is acceptable. Granzyme B should preferentially be measured after 48-72 h of activation.     

Pivotal Advance: Th-1 cytokines inhibit, and Th-2 cytokines promote fibrocyte differentiation

CD14+ peripheral blood monocytes can differentiate into fibroblast-like cells called fibrocytes, which are associated with and are at least partially responsible for wound healing and fibrosis in multiple organ systems. Signals regulating fibrocyte differentiation are poorly understood. In this study, we find that when added to human PBMCs cultured in serum-free medium, the profibrotic cytokines IL-4 and IL-13 promote fibrocyte differentiation without inducing fibrocyte or fibrocyte precursor proliferation. We also find that the potent, antifibrotic cytokines IFN-gamma and IL-12 inhibit fibrocyte differentiation. In our culture system, IL-1beta, IL-3, IL-6, IL-7, IL-16, GM-CSF, M-CSF, fetal liver tyrosine kinase 3, insulin growth factor 1, vascular endothelial growth factor, and TNF-alpha had no significant effect on fibrocyte differentiation. IL-4, IL-13, and IFN-gamma act directly on monocytes to regulate fibrocyte differentiation, and IL-12 acts indirectly, possibly through CD16-positive NK cells. We previously identified the plasma protein serum amyloid P (SAP) as a potent inhibitor of fibrocyte differentiation. When added together, the fibrocyte-inhibitory activity of SAP dominates the profibrocyte activities of IL-4 and IL-13. The profibrocyte activities of IL-4 and IL-13 and the fibrocyte-inhibitory activities of IFN-gamma and IL-12 counteract each other in a concentration-dependent manner. These results indicate that the complex mix of cytokines and plasma proteins present in inflammatory lesions, wounds, and fibrosis will influence fibrocyte differentiation.     

Toll-like receptor 2 and 4 induced interleukin-19 dampens immune reactions and associates inversely with spondyloarthritis disease activity

Spondyloarthritis (SpA) is a group of immune mediated inflammatory diseases affecting joints, gut, skin and entheses. The inflammatory process involves activation of Toll-like receptor (TLR)-2 and TLR-4 and production of cytokines and chemokines such as monocyte chemoattractant protein 1 (CCL2/MCP-1). This proinflammatory chemokine recruits monocytes to sites of inflammation and is central in the development of several immune-mediated inflammatory diseases. Interleukin (IL)-19 is a member of the IL-10 family of cytokines. IL-19-deficient mice are more susceptible to innate-mediated colitis and develop more severe inflammation in response to injury. In this work, we studied inducers of IL-19 production and effect of IL-19 on the production of CCL2/MCP-1 and proinflammatory cytokines in peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and in PBMCs and synovial fluid mononuclear cells (SFMCs) from SpA patients. Further, we measured IL-19 in plasma from HCs and in plasma and synovial fluid from SpA patients. Constitutive IL-19 expression was present in both PBMCs and SFMCs and the secretion of IL-19 was increased by TLR-2 and TLR-4 ligands. Neutralizing IL-19 in HC PBMCs and SpA SFMCs resulted in increased production of CCL-2/MCP-1. IL-19 concentrations were decreased in synovial fluid compared with plasma and associated inversely with disease activity in SpA. SpA SFMCs produced less IL-19 in response to LPS compared with HC PBMCs. These findings indicate that IL-19 production is diminished in SpA. Taken together, impaired IL-19 control of the innate immune system might be involved in the pathogenesis of SpA.     

Effects of granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-2, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and IL-6 on the production of immunoreactive IL-1 and TNF-alpha by human monocytes.

The effects of GM-CSF, IL-2, IFN-gamma, TNF-alpha and IL-6 on the production of IL-1 (both secreted and cell associated) and TNF-alpha by peripheral blood monocytes were studied. Monocytes were cultured for 20 h in suspension and in serum-free conditions which minimized background stimulation of monokine production. GM-CSF, IL-2 and TNF-alpha directly induced the production of cell-associated IL-1 but little or no IL-1 or TNF-alpha secretion. Combination of GM-CSF with IFN-gamma, IL-2 or TNF-alpha synergistically enhanced IL-1 secretion and had an additive effect on cell-associated IL-1 production. Combination of IL-2 with IFN-gamma or TNF-alpha also synergistically enhanced IL-1 secretion but the effect on cell-associated IL-1 production was less than additive. GM-CSF synergistically enhanced TNF-alpha secretion induced by IFN-gamma but not by lipopolysaccharide. GM-CSF did not enhance TNF-alpha secretion induced by IL-2 or TNF-alpha. In contrast, IL-2 synergistically enhanced TNF-alpha secretion induced by IFN-gamma. These results are discussed in relation to cytokine involvement in rheumatoid arthritis.     

Differential constitutive and cytokine-modulated expression of human Toll-like receptors in primary neutrophils, monocytes, and macrophages

Human Toll-like receptors (TLRs) comprise a family of proteins that recognizes pathogen-associated molecular patterns (PAMPs) and initiates host innate immune responses. Neutrophils, monocytes, and macrophages are critical cellular components of the human innate immune system. Proinflammatory cytokines, such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), and interferon-gamma (IFN-gamma), have been shown to up-regulate microbicidal activity in these effector cells of innate immunity. Currently, the cellular and molecular mechanisms responsible for these effects are not completely understood. We hypothesized that these cytokines may up-regulate TLR expression as a mechanism to facilitate microbial recognition and augment the innate immune response. Using quantitative realtime rt-PCR technology, we examined constitutive expression of TLR2, TLR4, TLR5, and TLR9 mRNA and the effects of G-CSF, GM-CSF, M-CSF, and IFN-gamma on TLR mRNA expression in purified populations of normal human neutrophils, monocytes, and monocyte-derived macrophages. Relative constitutive expression of TLR2, TLR4, and TLR9 was similar in neutrophils and monocytes. Constitutive expression of TLR5 was less in neutrophils compared to monocytes. Constitutive expression of TLR4 was greater and that of TLR9 lower in monocyte-derived macrophages compared to monocytes. Of the cytokines examined, IFN-gamma and GM-CSF caused the greatest effects on TLR expression. IFN- gamma up-regulated TLR2 and TLR4 in neutrophils and monocytes. GM-CSF up-regulated expression of TLR2 and TLR4 in neutrophils and TLR2 in monocytes. TLR5 was down-regulated by inflammatory cytokines in monocytes. These results suggest a potential role for IFN- gamma and/or GM-CSF as therapeutic immunomodulators of the host defense to infection.