Interleukin-1 receptor antagonist blocks chemokine production in the mixed lymphocyte reaction

The mixed lymphocyte reaction (MLR) has previously been used to elucidate pathways of cytokine activation and T-lymphocyte proliferation and is regarded as a model that simulates responses in allograft rejection. Studies have indicated that interleukin-1 (IL-1), a potent inflammatory cytokine, may have an important activating role in the MLR response. The discovery of a naturally occurring IL-1 receptor antagonist protein (IRAP) has renewed interest in control of IL-1--dependent responses both in vitro and in vivo. MLR cultures were used to study the role of IL-1 and IRAP in the regulation of subsequent cytokines during a T-lymphocyte-mediated alloantigen response. The temporal expression of IL-1 and IRAP during 5-day one-way MLR assays suggested antagonistic production of the two cytokines. IL-1 was produced early in the response, peaking at 4 hours through day 2, subsequently declining to near-background levels on day 5 of culture. In contrast, production of IRAP was delayed until day 2, steadily increased on days 3 and 4, and peaked on day 5 of culture, which correlated with the declining levels of IL-1. The addition of graded doses of IRAP (25 to 1,000 ng/mL) to MLR cultures decreased IL-1 production but had no effect on T-lymphocyte proliferative response. In addition, IRAP had little effect on the production of either IL-2 or tumor necrosis factor. The addition of 25 ng/mL of IRAP to MLR assays showed significantly decreased levels of two potent chemotactic cytokines, IL-8 and macrophage inflammatory protein-1 alpha (MIP-1 alpha), at peak chemokine production on day 5 of culture. The levels of IL-8 and MIP-1 alpha could be restored by the addition of IL-1 to the IRAP-treated cultures. IL-8 and MIP-1 alpha represent the two different families of chemotactic cytokines, C-X-C (IL-8) and C-C (MIP-1 alpha), and potentially play important roles in the recruitment of leukocytes to a site of immune allogeneic response. These studies indicate that regulation of IL-1 by IRAP does not significantly reduce T-lymphocyte activation but can regulate the production of chemokines involved in leukocyte recruitment.     

Regulation of activin A and inhibin B secretion by inflammatory mediators in adult rat Sertoli cell cultures

The regulation of Sertoli cell activin A and inhibin B secretion during inflammation was investigated in vitro. Adult rat Sertoli cells were incubated with the inflammatory mediators, lipopolysaccharide (LPS), interleukin-1beta (IL-1beta), IL-6 and the IL-1 receptor antagonist (IL-1ra) over 48 h in culture. Activin A, inhibin B and IL-1alpha were measured in the culture medium by specific two-site ELISAs. Both IL-1beta- and LPS-stimulated activin A and inhibited inhibin B secretion. LPS also stimulated the production of IL-1alpha in the cultures. In contrast to IL-1beta, IL-6 had no effect on activin A, although it did have a significant inhibitory effect on inhibin B secretion. Ovine follicle-stimulating hormone (FSH) and the cAMP analogue dibutyryl cAMP opposed the actions of IL-1 and LPS by suppressing activin A and IL-1alpha secretion and by stimulating inhibin B. Blocking IL-1 activity in the cultures by addition of an excess of IL-1ra completely prevented the response of activin A to exogenous IL-1beta, and reduced the response to LPS by 50%. In the presence of IL-1ra, basal secretion of inhibin B was increased, but IL-1ra was unable to reverse the suppression of inhibin B by LPS. These data indicate the importance of both IL-1 isoforms in regulating secretion of activin A and inhibin B by mature Sertoli cells during inflammation. The data also establish that inflammation exerts its effects on activin A and inhibin B secretion via other pathways in addition to those mediated by IL-1, and that hormonal stimulation by FSH and cAMP moderates the Sertoli cell response to inflammation. Interference with the complex interactions between these cytokines and hormones may contribute to the disruption of reproductive function that can accompany infection and illness in men.     

The effects of thalidomide on chemotactic migration of multiple myeloma cell lines

We examined the effect of thalidomide and dexamethasone on the migration of multiple myeloma (MM) cell lines, U266, RPMI8226, and NCI-H929, using chemotaxis chamber plates. U266 underwent chemotactic migration in response to stromal-cell derived factor-1 alpha (SDF-1alpha), and other cell lines underwent random migration in response to SDF-1alpha or monocyte chemotactic protein-1 alpha. Following preincubation with 1 mug/ml thalidomide, the cell lines showed reduced migratory capacity in response to SDF-1alpha. Concerning the corresponding receptors, CXC chemokine receptor 4 was detected only on the surface of U266, by flow cytometry, whereas chemokine (C-C motif) receptor 2 was not detected on all three cell lines. Moreover, decreased migration by thalidomide was not accompanied by altered expression of the corresponding receptors of each cell line. This is the first report to show the effects of thalidomide on the migration of MM cell lines. The results suggest that the inhibition of chemotactic migration might be one of the mechanisms of the success of thalidomide in controlling MM.     

Interleukin-8 (IL-8): the major neutrophil chemotactic factor in the lung

A number of novel chemotactic cytokines are becoming increasingly recognized as important participants in the elicitation of specific inflammatory cells from the peripheral blood to sites of inflammation. Recent observations have now demonstrated that certain chemotactic cytokines possess specificity for the selected movement of individual immune/inflammatory cell populations. One of the more studied chemotactic cytokines is a neutrophil chemotactic factor identified as interleukin-8 (IL-8). This polypeptide mediator is produced in abundance by mononuclear phagocytic cells, as well as a number of non-inflammatory cells. This latter list includes both fibroblasts and epithelial cells. Moreover, the synthesis of IL-8 by fibroblasts and epithelial cells involves stimulus specificity, as the production of this mediator by non-inflammatory cells is dependent upon an initial host response. In the context of the lung, the alveolar macrophage appears to play a central role by generating factors, such as interleukin-1 and tumor-necrosis factor, which are potent stimuli for the induction of IL-8 by the lung fibroblasts and type II epithelial cells. The cascade-like interaction may lead to the rapid production of significant quantities of IL-8 by the lung and may selectively recruit neutrophils to the pulmonary interstitium and/or airspace. This sequence of events, which leads to cytokine networking in the lung, may be an important phenomenon for the generation of a major chemotaxin important to a variety of lung diseases.     

Functionally distinct dendritic cell (DC) populations induced by physiologic stimuli: prostaglandin E(2) regulates the migratory capacity of specific DC subsets

Migration of antigen (Ag)-loaded dendritic cells (DCs) from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. We evaluated monocyte-derived DCs (MoDCs) and peripheral blood DCs (PBDCs) to respond to proinflammatory mediators, CD40L, and intact bacteria. All classes of stimuli induced DC phenotypic maturation. However, for MoDCs, only prostaglandin E(2) (PGE(2))-containing stimuli induced migratory-type DCs. Thus, immature MoDCs that encountered proinflammatory cytokines or CD40L or intact bacteria in the presence of PGE(2) acquired migratory capacity but secreted low levels of cytokines. Conversely, MoDCs that encountered pathogens or CD40L alone become nonmigratory cytokine-secreting cells (proinflammatory type). Interestingly, both migratory- and proinflammatory-type DCs expressed equivalent levels of chemokine receptors, suggesting that the role of PGE(2) was to switch on migratory function. We demonstrate that PGE(2) induces migration via the E-prostanoid 2/E-prostanoid 4 (EP(2)/EP(4)) receptors and the cAMP pathway. Finally, migratory-type MoDCs stimulated T-cell proliferation and predominantly IL-2 secretion, whereas proinflammatory-type MoDCs induced IFN-gamma production. In contrast, CD1b/c(+) PBDC rapidly acquired migratory capacity irrespective of the class of stimulus encountered and secreted low levels of cytokines. This suggests that not all mature stages of DCs are destined to migrate to lymphoid organs and that the sequence in which stimuli are encountered significantly affects which functions are expressed. Thus, certain immature DC subsets recruited from the resting precursor pool may have multiple functional fates that play distinct roles during the induction and effector phases of the immune response. These findings have important implications for the clinical utility of DCs in immunotherapy.     

Mast cell migratory response to interleukin-8 is mediated through interaction with chemokine receptor CXCR2/Interleukin-8RB

To explore the role of chemokines in mast cell chemotaxis and accumulation at sites of inflammation, we first investigated the response of human mast cells to 18 different chemokines by induction of intracellular calcium mobilization in the human mast cell line, HMC-1. Only a subgroup of CXC chemokines defined by the conserved sequence motif glutamic acid-leucine-arginine (ELR) tripeptide motif, which included interleukin-8 (IL-8), growth-regulated oncogene alpha (GROalpha), neutrophil-activating peptide-2 (NAP-2), and epithelial cell-derived neutrophil activating peptide-78 (ENA-78), induced calcium flux in the cells. These observations suggested that the receptor CXCR2 (IL-8RB) should be expressed on the surface of these cells. Using the RNAse protection assay, CXCR2 mRNA, but not CXCR1 (IL-8RA) mRNA expression was detected in HMC-1 cells. Flow cytometry analysis documented the surface expression of CXCR2. A binding analysis performed with 125I-IL-8 determined that there were approximately 3,600 high affinity IL-8 binding sites per HMC-1 cell, with a calculated kd of 1.2 to 2 nmol/L. The activity of this receptor was further explored using IL-8, which was found to induce dose-dependent chemotactic and haptotactic responses in both HMC-1 cells and in vitro cultured human cord blood-derived mast cells. These results show the expression of functional CXCR2 receptors on the surface of human mast cells, which may play an important role in mast cell recruitment during the genesis of an inflammatory response.     

RANTES- and interleukin-8-induced responses in normal human eosinophils: effects of priming with interleukin-5

We report that responses of normal human eosinophils toward the chemokines RANTES and interleukin-8 (IL-8) are modulated and upregulated by priming with IL-5. In a modified Boyden chamber assay, we studied migratory responses toward the members of the chemokine family RANTES, monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) (C-C subfamily), and IL-8, platelet factor-4 (PF-4), and neutrophil-activating peptide-2 (NAP-2) (C-x-C subfamily). These chemokines were also studied in terms of actin polymerization and ([Ca2+]i)-mobilizing properties, intracellular signals that are thought to play a role during migratory responses. We found that eosinophils showed significant migratory responses toward RANTES and IL-8 at concentrations of 10(-9) to 10(-7) mol/L only after priming with IL-5 (10 pmol/L). At these concentrations, PF-4, NAP-2, MCP-1, and MIP-1 alpha induced no significant migratory responses after priming. Unprimed eosinophils only showed a significant migratory response toward RANTES (10(-6) mol/L). Changes in [Ca2+]i were found after addition of RANTES, MIP-1 alpha, and NAP-2 (10 nmol/L) to unprimed eosinophils. RANTES (10(-9) to 10(-7) mol/L) significantly induced actin polymerization both in primed and unprimed eosinophils, whereas IL-8 (10(-9) to 10(-8) mol/L) and MIP-1 alpha (10(-8) mol/L) only induced actin polymerization after priming with IL-5. NAP-2, PF-4, and MCP-1 did not affect actin polymerization. These findings are further evidence for the hypothesis that cytokines like IL-5 and locally secreted chemokines like RANTES and IL-8 are both at the basis of specific eosinophil influx into the allergic inflammatory locus.     

Interleukin-1 is a potent growth factor for immature rat sertoli cells

Testes from rats of different maturational ages were explored for presence of paracrine sertoli cell growth factors. Pubertal and adult testes contained a 17 kDa protein, with potent stimulatory effect on immature Sertoli cell multiplication in vitro. The bioactivity of this protein was mimicked by rat interleukin-1 (IL-1) and neutralized by IL-1 receptor antagonist. A receptor-mediated action was further supported by the demonstration of IL-1 receptor type I mRNA and protein expression in the cultured sertoli cells and in intact immature rat testes. IL-1alpha showed higher efficacy in stimulating proliferation than IL-1beta and follicle-stimulating hormone (FSH), and displayed synergistic action in combination with FSH. As IL-1alpha is constitutively produced by the rat testis and IL-1beta readily inducible by proinflammatory stimuli, our results suggest that IL-1 may serve as a growth factor for Sertoli cells under physiological and pathophysiological conditions.     

Stem cell factor and interleukin-7 synergize to enhance early myelopoiesis in vitro

Interleukin-7 (IL-7) has been shown to be a critical factor in murine lymphoid development. It stimulates pre-B cells to divide in the absence of stroma cells and it is an important growth regulator of immature and mature T cells. IL-7 has been shown to synergize with stem cell factor (SCF) to provide a potent growth stimulus for pre-B cells. However, the combined effects of IL-7 and SCF on murine primitive hematopoietic cells in vitro have not been established. In the present study, the effects of recombinant rat (rr) SCF and recombinant human (rh) IL-7 on primitive murine bone marrow progenitors (Lin-Sca1+) were investigated in single-cell cloning experiments. rhIL-7 alone had no proliferative effect on Lin-Sca1+ cells, but in a dose-dependent manner directly enhanced rrSCF-induced colony formation, with an average increase in colony numbers of 2.7-fold. Interestingly, the cells formed in response to SCF and IL-7 were predominantly mature granulocytes. Thus, SCF and IL-7 synergize to stimulate early myelopoiesis in vitro.     

The c-kit receptor ligand functions as a mast cell chemoattractant.

Mast cells accumulate at sites of neovascularization, solid tumors, and many immune reactions. Such accumulation requires directed migration of mature mast cells or their precursors. The nature of the chemoattractants that regulate mast cell motility and the identity of the receptors that mediate the chemotactic response are poorly understood. We have tested the ability of stem cell factor (SCF), a mast cell growth factor, to stimulate mast cell migration. Our results show that SCF is a potent mast cell attractant that stimulates directional motility of both mucosal and connective tissue-type mast cells. The activity is potentiated by costimulation with interleukin-3 (IL-3), another mast cell chemoattractant. SCF, a known ligand for the c-kit tyrosine kinase receptor, was unable to stimulate motility in W42 mutant mast cells, which have a defective c-kit tyrosine kinase. However, W42 mast cells were still able to migrate in response to IL-3. These results show that SCF is a chemotactic factor as well as a growth factor and that the c-kit receptor can transduce signals leading to both cell proliferation and increased directional cell motility.     

P2Y receptor signaling regulates phenotype and IFN-alpha secretion of human plasmacytoid dendritic cells

Plasmacytoid dendritic cells (PDCs) play powerful regulatory roles in innate and adaptive immune responses and are a major source of type I interferon (IFN) following viral infection. During inflammation and mechanical stress, cells release nucleotides into the extracellular space where they act as signaling molecules via G protein-coupled P2Y receptors. We have previously reported on the regulation of myeloid dendritic cell (DC) function by nucleotides. Here, we report that human PDCs express several subtypes of P2Y receptors and mobilize intracellular calcium in response to nucleotide exposure. As a functional consequence, PDCs acquire a mature phenotype that is further enhanced in the context of CD40 ligation. Strikingly, nucleotides strongly inhibit IFN-alpha secretion induced by influenza virus or CpG-A. This effect is most pronounced for the uridine nucleotides UDP and UTP and the sugar nucleotide UDP-glucose, ligands of P2Y(6), P2Y(4), and P2Y(14), respectively. Nucleotide-induced inhibition of IFN-alpha production is blocked by suramin, a P2Y receptor antagonist. Pharmacological data point toward a role of protein kinase C in the negative regulation of type I IFN. Manipulating PDC function with P2Y receptor agonists may offer novel therapeutic strategies for autoimmune diseases or cancer.     

IL-10 regulates plasmacytoid dendritic cell response to CpG-containing immunostimulatory sequences

Immunostimulatory sequences (ISS) are short oligonucleotides containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides that stimulate innate immune responses through Toll-like receptor-9 on B cells and plasmacytoid dendritic cell (PDC) precursors. The anti-inflammatory cytokine interleukin (IL)-10 is predicted to be a potent inhibitor of many of the activities described for ISS, and this may impact the use of ISS in disease states characterized by elevated IL-10. As the activities of ISS on PDCs are central to many clinical applications of ISS, we have studied the effects of IL-10 on PDC stimulation by 3 distinct classes of ISS. IL-10 inhibited cytokine production and survival of ISS-activated PDCs; however, IL-12 induction was much more sensitive to inhibition than interferon (IFN)-alpha induction. Within the PDC population are cells that respond to ISS by producing either IL-12 or IFN-alpha but not both cytokines. IL-12-producing PDCs require costimulation through CD40 and appear more mature than IFN-alpha-producing PDCs. The 3 distinct classes of ISS differed with respect to induction of PDC maturation and T-cell priming capacity. IL-10 regulated PDC activation but did not inhibit the subsequent T-cell-priming ability of PDCs already activated by ISS.     

Mouse CD11c(+) B220(+) Gr1(+) plasmacytoid dendritic cells develop independently of the T-cell lineage

The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8alpha(+) and CD8alpha(-) DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c(+) B220(+) DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNalpha)-producing human plasmacytoid DCs (PDCs). We show here that CD11c(+) B220(+) mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8alpha and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DC precursor.     

Immature mouse dendritic cells enter inflamed tissue, a process that requires E- and P-selectin, but not P-selectin glycoprotein ligand 1.

Inflammatory processes are associated with the rapid migration of dendritic cells (DCs) to regional lymph nodes and depletion of these potent antigen-presenting cells (APCs) from the inflamed tissue. This study examined whether sites of cutaneous inflammation can be repopulated with DCs from a pool of immature DCs circulating in the blood. In adoptive transfer experiments with ex vivo-generated radioactively labeled primary bone marrow-derived DCs injected into mice challenged by an allergic contact dermatitis reaction, immature DCs were actively recruited from the blood to sites of cutaneous inflammation, whereas mature DCs were not. Immature, but not mature, DCs were able to adhere specifically to immobilized recombinant E- and P-selectin under static as well as under flow conditions. P-selectin-dependent adhesion of immature DCs correlates with their higher level of expression of the carbohydrate epitope cutaneous lymphocyte-associated antigen (CLA) and is blocked by a novel inhibitory antibody against mouse P-selectin glycoprotein ligand 1 (PSGL-1). Surprisingly, however, emigration of immature DCs into inflamed skin is retained in the presence of this anti-PSGL-1 antibody and is also normal when immature DCs are generated from fucosyltransferase (Fuc-T) Fuc-TVII-deficient mice. By contrast, emigration of wild-type immature DCs is reduced by adhesion-blocking anti-E- and P-selectin antibodies, and immature DCs generated ex vivo from Fuc-TVII/Fuc-TIV double-deficient mice emigrate poorly. Thus, fucosylated ligands of the endothelial selectins, determined in part by Fuc-TIV, and independent of PSGL-1, are required for extravasation of DCs into sites of cutaneous inflammation.     

Plasmacytoid dendritic cells, antigen, and CpG-C license human B cells for plasma cell differentiation and immunoglobulin production in the absence of T-cell help

It has been reported that interferon alpha (IFN-alpha) enhances humoral immunity and that dendritic cells of the myeloid lineage promote B-cell differentiation. Here we studied whether the plasmacytoid dendritic cell (PDC), a subset of dendritic cells specialized for the production of IFN-alpha, is involved in regulating B-cell differentiation and immunoglobulin production. The recently identified class of CpG oligonucleotides (CpG-C) was used to activate both B cells and PDCs via Toll-like receptor 9 (TLR9). The presence of PDCs synergistically enhanced CD86 expression, cytokine production (interleukin 6 [IL-6], tumor necrosis factor alpha, and IL-10) and plasma cell differentiation of isolated human peripheral blood B cells stimulated through CpG-C and B-cell antigen receptor (BCR) ligation. This stimulation protocol was sufficient to drive purified naive B cells into IgM-producing plasma cells and to trigger IgG synthesis in memory B cells. PDCs contributed to B-cell activation via IFN-alpha secretion. Up-regulation of TLR9 on B cells was not involved. These results demonstrate that CpG-stimulated PDCs induce plasma cell differentiation in naive and memory B cells in the absence of T-cell help, providing an explanation for the excellent activity of CpG oligonucleotides as a humoral vaccine adjuvant.     

Adenosine affects expression of membrane molecules,cytokine and chemokine release,and the T-cell stimulatory capacity of human dendritic cells

Dendritic cells (DCs) express functional purinergic type 1 receptors, but the effects of adenosine in these antigen-presenting cells have been only marginally investigated. Here, we further characterized the biologic activity of adenosine in immature DCs (iDCs) and lipopolysaccharide (LPS)-matured DCs (mDCs). Chronic stimulation with adenosine enhanced the macropinocytotic activity and the membrane expression of CD80, CD86, major histocompatibility complex (MHC) class I, and HLA-DR molecules on iDCs. Adenosine also increased LPS-induced CD54, CD80, MHC class I, and HLA-DR molecule expression in mDCs. In addition, adenosine dose-dependently inhibited tumor necrosis factor alpha and interleukin-12 (IL-12) release, whereas it enhanced the secretion of IL-10 from mDCs. The use of selective receptor agonists revealed that the modulation of the cytokine and cell-surface marker profile was due to activation of A(2) adenosine receptor. Functionally, adenosine reduced the allostimulatory capacity of iDCs, but not of mDCs. More important, DCs matured in the presence of adenosine had a reduced capacity to induce T helper 1 (Th1) polarization of naive CD4(+) T lymphocytes. Finally, adenosine augmented the release of the chemokine CCL17 and inhibited CXCL10 production by mDCs. In aggregate, the results provide initial evidence that adenosine diminishes the capacity of DCs to initiate and amplify Th1 immune responses.