The adjuvants aluminum hydroxide and MF59 induce monocyte and granulocyte chemoattractants and enhance monocyte differentiation toward dendritic cells

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.     

CC-chemokine ligand 16 induces a novel maturation program in human immature monocyte-derived dendritic cells

Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host's defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses.     

Dendritic cell maturation and subsequent enhanced T-cell stimulation induced with the novel synthetic immune response modifier R-848.

Agents that enhance dendritic cell maturation can enhance T-cell activation and therefore may improve the efficiency of vaccines or improve cellular immunotherapy. Previously, we demonstrated that a novel low-molecular-weight synthetic immune response modifier, R-848, induces IL-12 and IFN-alpha secretion from monocytes and macrophages. Here we report that R-848 induces the maturation of human monocyte-derived dendritic cells. Characteristic of dendritic cell maturation, R-848 treatment induces cell surface expression of CD83 and increases cell surface expression of CD80, CD86, CD40, and HLA-DR. Additionally, R-848 induces cytokine (IL-6, IL-12, TNF-alpha, IFN-alpha) and chemokine (IL-8, MIP-1alpha, MCP-1) secretion from dendritic cells. Most significantly, R-848 enhances dendritic cell antigen presenting function, as measured by increased T-cell proliferation and T-cell cytokine secretion in both allogeneic and autologous T-cell systems. Consequently, low-molecular-weight synthetic molecules such as R-848 and its derivatives may be useful as vaccine adjuvants or as ex vivo stimulators of dendritic cells for cellular immunotherapy.     

Intracellular overexpression of HIV-1 Nef impairs differentiation and maturation of monocytic precursors towards dendritic cells

Nef functions as an immunosuppressive factor critical for HIV-1 replication, survival and development of AIDS following HIV-1 infection. What effects Nef exerts on differentiation and maturation of monocytes towards dendritic cells (DCs) remains greatly controversial. In this study, we used THP-1 (human monocytic leukemia cell line) as monocytic DC precursors to investigate how overexpression of HIV-1 Nef influences the processes of differentiation and maturation of dendritic cells. In striking contrast to negative controls, our results showed that morphological and phenotypical changes (CD11c, CD14, CD40, CD80, CD83, CD86, and HLA-DR) occurred on recombinant THP-1 expressing HIV-1 Nef (short for Nef) upon co-stimulation of GM-CSF/IL-4 or GM-CSF/IL-4/TNF-α/ionomycin. Moreover, CD4, CCR5, and CXCR4 were also down-regulated on Nef. It might be hypothesized that Nef prevents superinfection and signal transduction in HIV-1 infected monocytes. Collectively, our study demonstrates that long-lasting expression of Nef at high levels indeed retards differentiation and maturation of dendritic cells in terms of phenotype and morphology. We are hopeful that potentially, stable expression of intracellular Nef in vivo may function as a subtle mode to support long-lasting HIV-1 existence.     

Differential expression of inflammatory chemokines by Th1- and Th2-cell promoting dendritic cells: a role for different mature dendritic cell populations in attracting appropriate effector cells to peripheral sites of inflammation

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.     

Modulation of immunity against herpes simplex virus infection via mucosal genetic transfer of plasmid DNA encoding chemokines

In this study, we examined the effects of murine chemokine DNA, as genetic adjuvants given mucosally, on the systemic and distal mucosal immune responses to plasmid DNA encoding gB of herpes simplex virus (HSV) by using the mouse model. The CC chemokines macrophage inflammatory protein 1beta (MIP-1beta) and monocyte chemotactic protein 1 (MCP-1) biased the immunity to the Th2-type pattern as judged by the ratio of immunoglobulin isotypes and interleukin-4 cytokine levels produced by CD4(+) T cells. The CXC chemokine MIP-2 and the CC chemokine MIP-1alpha, however, mounted immune responses of the Th1-type pattern, and such a response rendered recipients more resistant to HSV vaginal infection. In addition, MIP-1alpha appeared to act via the upregulation of antigen-presenting cell (APC) function and the expression of costimulatory molecules (B7-1 and B7-2), whereas MIP-2 enhanced Th1-type CD4(+) T-cell-mediated adaptive immunity by increasing gamma interferon secretion from activated NK cells. Our results emphasize the value of using the mucosal route to administer DNA modulators such as chemokines that function as adjuvants by regulating the activity of innate immunity. Our findings provide new insight into the value of CXC and CC chemokines, which act on different innate cellular components as the linkage signals between innate and adaptive immunity in mucosal DNA vaccination.     

Dendritic cells exposed to MVA-based HIV-1 vaccine induce highly functional HIV-1-specific CD8(+) T cell responses in HIV-1-infected individuals

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.     

Soluble CD40 ligand induces beta-chemokine production by macrophages and resistance to HIV-1 entry

CD40 ligand (CD40L) is a cell surface molecule of CD4(+)T cells that interacts with its receptor CD40 on antigen presenting cells to mediate thymus-dependent humoral immunity and inflammatory reactions. We report here that treating monocyte-derived macrophages (MDM) with a trimeric soluble form of CD40L (CD40LT) induced them to secrete high levels of the beta-chemokines RANTES, MIP-1alpha and MIP-1beta that are ligands for CCR5 and able to inhibit HIV-1 entry. CD40LT inhibited the entry of M-tropic HIV-1 reporter viruses. Furthermore, supernatants obtained from CD40LT-stimulated macrophages protected CEMx174-CCR5 cells from infection by HIV-1(JRFL)reporter virus. The inhibitory activity appeared to be due to beta-chemokines present in the supernatant, since pretreating them with a cocktail of antibodies to RANTES, MIP-1alpha and MIP-1beta neutralized the inhibitory activity of the supernatants. In addition, treating monocytes with CD40LT caused CCR5 and CD4 to be downregulated from the cell surface. In vivo, macrophages activated through CD40 could interfere with HIV replication.     

CCL3 (MIP-1alpha) induces in vitro migration of GM-CSF-primed human neutrophils via CCR5-dependent activation of ERK 1/2

CCL3 (MIP-1alpha), a prototype of CC chemokines, is a potent chemoattractant toward human neutrophils pre-treated with GM-CSF for 15 min. GM-CSF-treated neutrophils migrate also to the selective CCR5 agonist CCL4 (MIP-1beta). CCL3- and CCL4-triggered migration of GM-CSF-primed neutrophils was inhibited by the CCR5 antagonist TAK-779. Accordingly, freshly isolated neutrophils express CCR5. Extracellular signal-regulated kinases (ERK)-1/2 and p38 mitogen-activated protein kinase (MAPK) inhibitors blocked CCL3-induced migration of GM-CSF-primed neutrophils. When the activation of ERK-1/2 and p38 MAPK by CCL3 and the classical neutrophilic chemokine CXCL8 (IL-8) were compared, both the chemokines were capable of activating p38 MAPK. On the contrary, whereas both ERK-1 and ERK-2 were activated by CXCL8, no ERK-1 band was detectable after CCL3 triggering. Finally, neutrophil pre-treatment with GM-CSF activated both ERK-1 and ERK-2. This suggests that by activating ERK-1, GM-CSF renders neutrophils rapidly responsive to CCL3 stimulation throughout CCR5 which is constitutively expressed on the cell surface.     

Regulation of inflammatory chemokine receptors on blood T cells associated to the circulating versus liver chemokines in dengue fever

Little is known about the role of chemokines/chemokines receptors on T cells in natural DENV infection. Patients from DENV-2 and -3- outbreaks were studied prospectively during the acute or convalescent phases. Expression of chemokine receptor and activation markers on lymphocyte subpopulations were determined by flow cytometry analysis, plasma chemokine ligands concentrations were measured by ELISA and quantification of CCL5/RANTES(+) cells in liver tissues from fatal dengue cases was performed by immunochemistry. In the acute DENV-infection, T-helper/T-cytotoxic type-1 cell (Th1/Tc1)-related CCR5 is significantly higher expressed on both CD4 and CD8 T cells. The Th1-related CXCR3 is up-regulated among CD4 T cells and Tc2-related CCR4 is up-regulated among CD8 T cells. In the convalescent phase, all chemokine receptor or chemokine ligand expression tends to reestablish control healthy levels. Increased CCL2/MCP-1 and CCL4/MIP-1β but decreased CCL5/RANTES levels were observed in DENV-patients during acute infection. Moreover, we showed an increased CD107a expression on CCR5 or CXCR3-expressing T cells and higher expression of CD29, CD44(HIGH) and CD127(LOW) markers on CCR4-expressing CD8 T cells in DENV-patients when compared to controls. Finally, liver from dengue fatal patients showed increased number of cells expressing CCL5/RANTES in three out of four cases compared to three death from a non-dengue patient. In conclusion, both Th1-related CCR5 and CXCR3 among CD4 T cells have a potential ability to exert cytotoxicity function. Moreover, Tc1-related CCR5 and Tc2-related CCR4 among CD8 T cells have a potential ability to exert effector function and migration based on cell markers evaluated. The CCR5 expression would be promoting an enhanced T cell recruitment into liver, a hypothesis that is corroborated by the CCL5/RANTES increase detected in hepatic tissue from dengue fatal cases. The balance between protective and pathogenic immune response mediated by chemokines during dengue fever will be discussed.     

Cytokine,chemokine, and costimulatory molecule modulation to enhance efficacy of HIV vaccines

Understanding key intervention points in developing immune responses may allow the rational inclusion of biological adjuvants into vaccines that could potentiate the immune response both quantitatively and qualitatively and enhance effective memory responses. Cytokine and chemokine combinations can potentially help target antigen to the appropriate antigen presenting cell and initiate maturation of these presenting cells, attract cells expressing different chemokine receptors, steer cellular immune responses toward Th1 and CD8 CTL, and enhance systemic and mucosal IgG and secretory IgA antibodies and determine their isotype balance. Animal protection studies suggest that synergistic combinations of cytokines and immunomodulating molecules may be required to protect from a viral challenge. For example, GM-CSF has been shown to be synergistic with IL-12 or CD40 ligand for induction of CTL and for antiviral protection, and the triple combination of GM-CSF, IL-12, and TNF alpha appears to induce the most effective protection in some mouse models. Chemokine-antigen fusions have also been shown to enhance immunogenicity of the antigen. Combinations of costimulatory molecules have been found to be synergistic when incorporated in a vaccine. Combined use of newer more potent vaccine constructs, containing codon optimized epitopes, relevant CpG motifs, cytokines, costimulatory molecules and chemokines, used in heterologous prime-boost strategies with viral vector vaccines or recombinant proteins, might afford the most potent vaccine approaches yet developed. In this review we will discuss the application and delivery of cytokines, costimulatory molecules, and chemokines toward improving current vaccine strategies.     

Allograft inflammatory factor-1 stimulates chemokine production and induces chemotaxis in human peripheral blood mononuclear cells

Allograft inflammatory factor-1 (AIF-1) is expressed by macrophages, fibroblasts, endothelial cells and smooth muscle cells in immune-inflammatory disorders such as systemic sclerosis, rheumatoid arthritis and several vasculopathies. However, its molecular function is not fully understood. In this study, we examined gene expression profiles and induction of chemokines in monocytes treated with recombinant human AIF (rhAIF-1). Using the high-density oligonucleotide microarray technique, we compared mRNA expression profiles of rhAIF-1-stimulated CD14⁺ peripheral blood mononuclear cells (CD14⁺ PBMCs) derived from healthy volunteers. We demonstrated upregulation of genes for several CC chemokines such as CCL1, CCL2, CCL3, CCL7, and CCL20. Next, using ELISAs, we confirmed that rhAIF-1 promoted the secretion of CCL3/MIP-1α and IL-6 by CD14⁺ PBMCs, whereas only small amounts of CCL1, CCL2/MCP-1, CCL7/MCP-3 and CCL20/MIP-3α were secreted. Conditioned media from rhAIF-1stimulated CD14⁺ PBMCs resulted in migration of PBMCs. These findings suggest that AIF-1, which induced chemokines and enhanced chemotaxis of monocytes, may represent a molecular target for the therapy of immune-inflammatory disorders.     

Immunization with HIV-1 Gag protein conjugated to a TLR7/8 agonist results in the generation of HIV-1 Gag-specific Th1 and CD8+ T cell responses

One strategy to induce optimal cellular and humoral immune responses following immunization is to use vaccines or adjuvants that target dendritic cells and B cells. Activation of both cell types can be achieved using specific TLR ligands or agonists directed against their cognate receptor. In this study, we compared the ability of the TLR7/8 agonist R-848, which signals only via TLR7 in mice, with CpG oligodeoxynucleotides for their capacity to induce HIV-1 Gag-specific T cell and Ab responses when used as vaccine adjuvants with HIV-1 Gag protein in mice. Injection of R-848 and CpG oligodeoxynucleotides alone enhanced the innate immune responses in vivo as demonstrated by high serum levels of inflammatory cytokines, including IL-12p70 and IFN-alpha, and increased expression of CD80, CD86, and CD40 on CD11c(+) dendritic cells. By contrast, R-848 was a relatively poor adjuvant for inducing primary Th1 or CD8(+) T cell responses when administered with HIV-1 Gag protein. However, when a TLR7/8 agonist structurally and functionally similar to R-848 was conjugated to HIV-1 Gag protein both Th1 and CD8(+) T cells responses were elicited as determined by intracellular cytokine and tetramer staining. Moreover, within the population of HIV-1 Gag-specific CD8(+) CD62(low) cells, approximately 50% of cells expressed CD127, a marker shown to correlate with the capacity to develop into long-term memory cells. Overall, these data provide evidence that TLR7/8 agonists can be effective vaccine adjuvants for eliciting strong primary immune responses with a viral protein in vivo, provided vaccine delivery is optimized.     

Natural truncation of the chemokine MIP-1 beta /CCL4 affects receptor specificity but not anti-HIV-1 activity

Activated lymphocytes synthesize and secrete substantial amounts of the beta-chemokines macrophage inflammatory protein (MIP)-1 alpha/CCL3 and MIP-1 beta/CCL4, both of which inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). The native form of MIP-1 beta secreted by activated human peripheral blood lymphocytes (MIP-1 beta(3-69)) lacks the two NH(2)-terminal amino acids of the full-length protein. This truncated form of MIP-1 beta has now been affinity-purified from the culture supernatant of such cells, and its structure has been confirmed by mass spectrometry. Functional studies of the purified protein revealed that MIP-1 beta(3-69) retains the abilities to induce down-modulation of surface expression of the chemokine receptor CCR5 and to inhibit the CCR5-mediated entry of HIV-1 in T cells. Characterization of the chemokine receptor specificity of MIP-1 beta(3-69) showed that the truncated protein not only shares the ability of intact MIP-1 beta to induce Ca(2+) signaling through CCR5, but unlike the full-length protein, it also triggers a Ca(2+) response via CCR1 and CCR2b. These results demonstrate that NH(2)-terminally truncated MIP-1 beta functions as a chemokine agonist with expanded receptor reactivity, which may represent an important mechanism for regulation of immune cell recruitment during inflammatory and antiviral responses.     

The CD8-Derived Chemokine XCL1/Lymphotactin Is a Conformation-Dependent,Broad-Spectrum Inhibitor of HIV-1

CD8+ T cells play a key role in the in vivo control of HIV-1 replication via their cytolytic activity as well as their ability to secrete non-lytic soluble suppressive factors. Although the chemokines that naturally bind CCR5 (CCL3/MIP-1α, CCL4/MIP- 1β, CCL5/RANTES) are major components of the CD8-derived anti-HIV activity, evidence indicates the existence of additional, still undefined, CD8-derived HIV-suppressive factors. Here, we report the characterization of a novel anti-HIV chemokine, XCL1/lymphotactin, a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. Experiments with stabilized variants of XCL1 demonstrated that HIV-1 inhibition requires access to the alternative, all-β conformation, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic XCL1 conformation is inactive. HIV-1 inhibition by XCL1 was shown to occur at an early stage of infection, via blockade of viral attachment and entry into host cells. Analogous to the recently described anti-HIV effect of the CXC chemokine CXCL4/PF4, XCL1-mediated inhibition is associated with direct interaction of the chemokine with the HIV-1 envelope. These results may open new perspectives for understanding the mechanisms of HIV-1 control and reveal new molecular targets for the design of effective therapeutic and preventive strategies against HIV-1.     

Diverse and potent chemokine production by lung CD11bhigh dendritic cells in homeostasis and in allergic lung inflammation

Lung CD11c(high) dendritic cells (DC) are comprised of two major phenotypically distinct populations, the CD11b(high) DC and the integrin alpha(E)beta(7)(+) DC (CD103(+) DC). To examine whether they are functionally distinguishable, global microarray studies and real-time PCR analysis were performed. Significant differences between the two major CD11c(high) DC types in chemokine mRNA expression were found. CD11b(high) DC is a major secretory cell type and highly expressed at least 16 chemokine mRNA in the homeostatic state, whereas CD103(+) DC highly expressed only 6. Intracellular chemokine staining of CD11c(high) lung cells including macrophages, and ELISA determination of sort-purified CD11c(high) cell culture supernatants, further showed that CD11b(high) DC produced the highest levels of 9 of 14 and 5 of 7 chemokines studied, respectively. Upon LPS stimulation in vitro and in vivo, CD11b(high) DC remained the highest producer of 7 of 10 of the most highly produced chemokines. Induction of airway hyperreactivity and lung inflammation increased lung CD11b(high) DC numbers markedly, and they produced comparable or higher amounts of 11 of 12 major chemokines when compared with macrophages. Although not a major producer, CD103(+) DC produced the highest amounts of the Th2-stimulating chemokines CCL17/thymus and activation-related chemokine and CCL22/monocyte-derived chemokine in both homeostasis and inflammation. Significantly, CCL22/monocyte-derived chemokine exhibited regulatory effects on CD4(+) T cell proliferation. Further functional analysis showed that both DC types induced comparable Th subset development. These studies showed that lung CD11b(high) DC is one of the most important leukocyte types in chemokine production and it is readily distinguishable from CD103(+) DC in this secretory function.     

Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation

It has previously been reported that cholera toxin (CT) is a potent mucosal adjuvant that enhances Th2 or mixed Th1/Th2 type responses to coadministered foreign Ag. Here we demonstrate that CT also promotes the generation of regulatory T (Tr) cells against bystander Ag. Parenteral immunization of mice with Ag in the presence of CT induced T cells that secreted high levels of IL-4 and IL-10 and lower levels of IL-5 and IFN-gamma. Ag-specific CD4(+) T cell lines and clones generated from these mice had cytokine profiles characteristic of Th2 or type 1 Tr cells, and these T cells suppressed IFN-gamma production by Th1 cells. Furthermore, adoptive transfer of bone marrow-derived dendritic cells (DC) incubated with Ag and CT induced T cells that secreted IL-4 and IL-10 and low concentrations of IL-5. It has previously been shown that IL-10 promotes the differentiation or expansion of type 1 Tr cells. Here we found that CT synergized with low doses of LPS to induce IL-10 production by immature DC. CT also enhanced the expression of CD80, CD86, and OX40 (CD134) on DC and induced the secretion of the chemokine, macrophage inflammatory protein-2 (MIP-2), but inhibited LPS-driven induction of CD40 and ICAM-I expression and production of the inflammatory cytokines/chemokines IL-12, TNF-alpha, MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein-1. Our findings suggest that CT induces maturation of DC, but, by inducing IL-10, inhibiting IL-12, and selectively affecting surface marker expression, suppresses the generation of Th1 cells and promotes the induction of T cells with regulatory activity.     

Chemokine production and pattern recognition receptor (PRR) expression in whole blood stimulated with pathogen-associated molecular patterns (PAMPs)

Recognition of conserved bacterial structures called pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), may lead to induction of a variety of "early immediate genes" such as chemokines. In the current study, we have in an ex vivo whole blood model studied the induction of the chemokines MIP-1alpha, MCP-1 and IL-8 by various PAMPs. The rate of appearance of Escherichia coli-Lipopolysaccharide (LPS) induced chemokines differed. The production of MIP-1alpha and IL-8 was after 1 h of stimulation significantly higher when compared to unstimulated whole blood, whereas MCP-1 was not significantly elevated until after 3 h. At peak levels the MIP-1alpha concentration induced by E. coli-LPS was 3-5-fold higher than MCP-1 and IL-8. By specific cell depletion, we demonstrated that all three chemokines were mainly produced by monocytes. However, the mRNA results showed that IL-8 was induced in both monocytes and granulocytes. The production of all three chemokines, induced by the E. coli-LPS and Neisseria meningitidis-LPS, was significantly inhibited by antibodies against CD14 and TLR4, implying these receptors to be of importance for the effects of LPS in whole blood. The chemokine production induced by lipoteichoic acid (LTA) and non-mannose-capped lipoarabinomannan (AraLAM) was, however, less efficiently blocked by antibodies against CD14 and TLR2. E. coli-LPS and LTA induced a dose-dependent increase of CD14, TLR2 and TLR4 expression on monocytes in whole blood. These data show that PAMPs may induce chemokine production in whole blood and that antibodies against PRRs inhibit the production to different extent.     

Chemokines and cytokines in patients with an occult Onchocerca volvulus infection

Repeated ivermectin treatment will clear microfilaria (Mf) of Onchocerca volvulus from skin and eyes of onchocerciasis patients while adult filaria remains alive and reproductive, and such occult O. volvulus infection may persist for years. To investigate the effect of residual adult filaria on the immune response profile, chemokines and cytokines were quantified 1) in onchocerciasis patients who developed an occult O. volvulus infection (Mf-negative) due to repeated ivermectin treatments, 2) patients who became Mf-negative without ivermectin treatments due to missing re-infection, and 3) endemic and non-endemic O. volvulus Mf-negative controls. With occult O. volvulus infection, serum levels of pro-inflammatory chemokines MCP-1/CCL2, MIP-1α/CCL3, MIP-1β/CCL4, MPIF-1/CCL23 and CXCL8/IL-8 enhanced and approached higher concentrations as determined in infection-free controls, whilst regulatory and Th2-type cytokines and chemokines MCP-4/CCL13, MIP-1δ/CCL15, TARC/CCL17 and IL-13 lessened. Levels of Eotaxin-2/CCL24, MCP-3/CCL7 and BCA-1/CXCL13 remained unchanged. At 3 days post-initial ivermectin treatment, MCP-1/CCL2, MCP-4/CCL13, MPIF-1/CCL23 and Eotaxin-2/CCL24 were strongly enhanced, suggesting that monocytes and eosinophil granulocytes have mediated Mf clearance. In summary, with occult and expiring O. volvulus infections the serum levels of inflammatory chemokines enhanced over time while regulatory and Th2-type-promoting cytokines and chemokines lessened; these changes may reflect a decreasing effector cell activation against Mf of O. volvulus, and in parallel, an enhancing inflammatory immune responsiveness.