Neutrophils produce biologically active macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19.

Macrophage inflammatory protein-3alpha (MIP-3alpha)/CCL20 and MIP-3beta/CCL19 are members of the CC chemokine subfamily which exert their effects through specific receptors, CCR6 and CCR7, respectively. Previously, we have reported that human neutrophils have the capacity to produce a number of chemokines, including IL-8/CXCL8, GROalpha/CXCL1, IP-10/CXCL10, and MIG/CXCL9. Herein, we show that neutrophils also have the ability to express and release MIP-3alpha/CCL20 and MIP-3beta/CCL19 when cultured with either LPS or TNF-alpha. We also report that MIP-3alpha/CCL20 and MIP-3beta/CCL19 production by LPS-stimulated neutrophils is negatively modulated by IL-10. Remarkably, we found that supernatants harvested from stimulated neutrophils not only induced chemotaxis of both immature and mature dendritic cells (DC), but also triggered rapid integrin-dependent adhesion of CCR6- and CCR7-expressing lymphocytes to purified VCAM-1 and ICAM-1, respectively. Importantly, both chemotaxis and rapid integrin-dependent adhesion were dramatically suppressed by anti-MIP-3alpha/CCL20 and anti-MIP-3beta/CCL19 neutralizing antibodies, indicating that MIP-3alpha/CCL20 and MIP-3beta/CCL19 present in the supernatants were both biologically active. As these chemokines are primarily chemotactic for DC and specific lymphocyte subsets, the ability of neutrophils to produce MIP-3alpha/CCL20 and MIP-3beta/CCL19 might be significant in orchestrating the recruitment of these cell types to the inflamed sites and therefore in contributing to the regulation of the immune response.     

The role of cytokines in the immune response to influenza A virus infection

Influenza A virus is one of the most important causes of respiratory tract diseases. It replicates in epithelial cells and leukocytes resulting in the production of immune mediators--cytokines, substances with various biological effects. Cytokines, as a part of innate immunity, favor the development of antiviral and TH 1-type immune responses. Cytokines also affect the adaptive immune response and disease manifestation. In the organism, the virus infection results in the production of chemotactic [a regulated upon activation, normal T cell-expressed and -secreted cytokine (RANTES), monocyte chemoattractant proteins (MCP) MCP-1, MCP-3, macrophage inflammatory protein 1 alpha (MIP- 1 alpha), interferon gamma-induced protein 10 (IP-10), and interleukin 8 (IL-8)], pro-inflammatory [IL- 1beta, IL-6, IL-18, and tumor necrosis factor alpha(TNF-alpha)] and antiviral [interferon (IFN) alpha/beta] cytokines. Whilst knowledge of the mechanisms underlying host and tissue specificity has advanced significantly, we still know relatively little about the function of cytokines released from different cells following influenza infection. In this review we deal with the role and mode of possible impact of cytokines on the disease pathogenesis and host immune response.     

Production of Interferon-β by Murine T-Cell Lines Induced by 10-Carboxymethyl-9-Acridanone

Besides the established T-cell property of producing gamma interferon (IFN-gamma), murine T cells additionally possess the ability to produce IFN-alpha and IFN-beta when appropriate inducers such as 10-carboxymethyl-9-acridanone (CMA) or Newcastle disease virus (NDV) are used. Interleukin 2 (IL-2)-dependent murine T-cell lines, but not purified resting splenic T cells, responded to CMA and NDV with production of IFN-alpha, beta. The IFN production by these T cells was not restricted to a special subset, since T cells expressing the Lyt 1+2- and the Lyt 1-2+ phenotype responded to these inducers with IFN production. After prolonged passaging of the T-cell lines in IL-2-containing medium, the ability to respond to CMA with production of antiviral activity was sustained longer than the ability for concanavalin A-induced IFN-gamma production. Whereas the NDV-induced T-cell supernates contained both IFN-alpha and IFN-beta, the induction with CMA resulted exclusively in the synthesis of IFN-beta by the T-cell lines.     

Regulation by endogenous interferon of virus-induced cytokine gene expression in mouse macrophages.

In macrophages from inbred mice the magnitude of the interferon (IFN) response to Newcastle disease virus (NDV) infection is under genetic control of the locus If-1, with C57BL/6 carrying the 'high-producer' allele If-1h whereas BALB/c have the 'low-producer' allele If-1l. The IFN produced consists of 90% IFN-beta and there are 10-fold differences between macrophages from If-1h and If-1l mice. Recently, we observed that interleukin-6 (IL-6) is coinduced by NDV in macrophages and seems to be under the same genetic control. Noninduced macrophages have been shown to secrete low amounts of antiviral activity endogenously when cultured in the presence of the macrophage-colony-stimulating factor (M-CSF). Here, we report that the amount of this endogenous IFN varies between macrophages from different mouse strains. Macrophages from BALB/c were found to secrete 5-10 times more endogenous IFN compared to C57BL/6. The antiviral activity could be identified as IFN-beta. Interestingly, we observed that endogenous IFN specifically down-regulates NDV-induced IFN and IL-6 production. Preculture of BALB/c macrophages in M-CSF plus anti-IFN-beta to neutralize the biological effects of the endogenous IFN provoked a 30- to 50-fold increase in NDV-induced cytokine production, resulting in a nearly complete abrogation of the genetically determined difference since the same treatment only caused a 6-fold increase in C57BL/6 macrophages following NDV infection. This increase in cytokine gene expression was specific for NDV and marked by a strong additional activation of IFN-alpha genes. Addition of mouse recombinant IFN-alpha 4 to anti-IFN-beta-treated macrophages for 18 h prior to NDV infection down-regulated again IFN gene expression and reestablished the genetic differences between macrophages from If-1h and If-1l mice.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

Infection of human primary hepatocytes with dengue virus serotype 2

While the impact of the dengue viruses on liver function is prominent as shown by hepatomegaly, liver enzyme abnormality, occasional fulminant hepatic failure and histological changes including hepatocellular necrosis, significant debate exists as to the possible involvement of the predominant cell type in the liver, hepatocytes, in the disease process. To address this issue purified human primary hepatocytes were exposed to dengue virus serotype 2 and the production of de novo viral progeny was established by standard plaque assay, RT-PCR and immunocytochemistry. To investigate the response of the primary hepatocytes to infection, the expression of a panel of 9 cytokine genes (IFN-beta, TRAIL, MCP-1, IL-6, IL-1beta, IL-8, MIP-1alpha, MIP-1beta, and RANTES) was semi-quantitatively investigated by RT-PCR and up-regulation of TRAIL, MIP-1alpha, IFN-beta, MIP-1beta, IL-8, and RANTES was observed in response to infection. The induction of IL-8 in response to infection was accompanied by the secretion of IL-8 as verified by ELISA assay. The ability of hepatocytes to be infected with dengue virus serotype 2 in vitro support evidence implicating human hepatocytes as a target cell in cases of dengue virus infection, and provide the first experimental evidence to support the large number of clinical studies that implicate the liver as a critical target organ in severe cases of dengue infection.     

Viruses and the type I interferon antiviral system: induction and evasion

The type I interferon (IFN) system responds to viral infection and induces an "antiviral state" in cells, providing an important first line of defense against virus infection. Interaction of type I IFNs (IFN alpha and IFN beta) with their receptor induces hundreds of cellular genes. Of the proteins induced by IFN, the antiviral function of only a few is known, and their mechanisms of action are only partly understood. Additionally, although viral-encoded mechanisms that counteract specific components of the type I IFN response have been known for some time, it has recently become clear that many (if not most) viruses encode some form of IFN-antagonist. Understanding the interplay between viral-encoded IFN antagonists and the interferon response will be essential if the therapeutic potential of IFNs is to be fully exploited.     

Monocyte-derived human macrophages and peripheral blood mononuclear cells infected with ebola virus secrete MIP-1alpha and TNF-alpha and inhibit poly-IC-induced IFN-alpha in vitro

Ebola virus infection of humans is associated with high levels of circulating inflammatory chemokines and cytokines. We demonstrate that direct infection of human PBMC results in the induction of MCP-1, MIP-1alpha, RANTES, and TNF-alpha as early as 24 h p.i. in response to live virus. Monocyte-derived macrophages infected with live Ebola-virus secreted MIP-1alpha and TNF-alpha specifically while RANTES and MCP-1 were secreted by with both live or inactivated virus stimulation and do not require viral replication. Type I interferons (IFN-alpha and -beta), IL-1beta and IL-10, were not induced by Ebola virus. Furthermore, live virus infection of both PBMCs and monocytes-derived macrophages inhibited IFN-alpha induced by double-stranded RNA in vitro. These data provide the first direct evidence of a role for macrophages in the pathogenesis to Ebola virus and suggest that Ebola virus can inhibit cellular antiviral mechanisms mediated by type I interferons.     

Sex hormone modulation of interferon (IFN) alpha/beta and gamma production by mouse spleen cell subsets following picornavirus infection

Replication of the diabetogenic variant of encephalomyocarditis virus (EMCV-D) in spleen cells and its association with subpopulations of spleen cells (L3T4+, Lyt-2+, Mac 1+, 33D1+ and AGM1+ cells) from both sexes of ICR Swiss mice were examined. Virus replication was limited to less than 0.5 log in suspensions of whole spleen cells, nonadherent cells or a B cell subfraction from both sexes of ICR Swiss mice following infection with EMCV-D at an MOI of 10; no virus replication was seen in adherent spleen cells from either sex. After 1 hour adsorption of EMCV-D onto spleen cells at a multiplicity of infection (MOI) of either 10 or 0.1, virus-associated cells were isolated using a monoclonal murine anti-EMCV-D and anti-mouse IgG conjugated to magnetic beads. Using an MOI of 0.1, less than 1% of spleen cells bound virus particles after 1 hour adsorption at 4 degrees C. Among the virus-positive cells, relatively higher percentages of adherent cell populations (Mac 1+ and 33D1+ cells) of both sexes bound virus particles within the first hour post-infection (PI) than did the other spleen cell subpopulations. Interferon (IFN) alpha/beta production was detected as early as 4 hours PI in female spleen cell cultures infected with EMCV-D at an MOI of 0.1 while no IFN alpha/beta activity was found in comparably infected male spleen cell cultures. Inhibiting IFN alpha/beta activity in the virus-infected spleen cell cultures during the first 20 hours of infection using polyclonal rabbit anti-mouse IFN alpha/beta serum eliminated production of IFN gamma as well as IFN alpha/beta. Spleen cell cultures depleted of adherent cells were unable to produce IFN alpha/beta or IFN gamma in the first 24 hours PI. The capacity to produce IFN gamma at 12 hours after virus infection of spleen cells from both sexes of mice was restored to adherent cell-depleted cultures by addition of mouse IFN alpha/beta at the time of infection. These results suggest that IFN alpha/beta and adherent cells play critical roles in the early production of IFN gamma (less than 16 hours PI) characteristic of the infected spleen cell cultures of females. Production of IFN alpha/beta and IFN gamma by spleen cells from both sexes of ICR Swiss mice was enhanced by administrating estrone to donor mice during the week before harvesting spleen cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calling in the troops: regulation of inflammatory cell trafficking through innate cytokine/chemokine networks

The recruitment of immune effector cells to localized sites of infection is crucial for the effective delivery of innate immune mechanisms. Under the conditions of infections with murine cytomegalovirus (MCMV), a herpesvirus with pathogenic potential, early immune functions are essential in the control of virus replication and virus-induced pathology. Our studies have demonstrated that the chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) is critical for natural killer (NK) cell inflammation and delivery of interferon (IFN)-gamma to mediate downstream protective responses against MCMV infection in liver. Moreover, IFN-alpha/beta-dependent mechanisms promote MIP-1alpha production and subsequently the accumulation of NK cells in liver. Taken together, the studies highlighted in this review define a unique in vivo pathway mediated by innate cytokines in regulating chemokine responses that are essential in the promotion of NK cell inflammation for localized antiviral defense. In addition, the downstream consequences of these events in enhancing endogenous adaptive immune responses will also be discussed. Overall, the innate cytokine/chemokine networks that are described emphasize the emerging importance of chemokine functions for protective immune responses during infection with viruses.     

Single point mutations in the helicase domain of the NS3 protein enhance dengue virus replicative capacity in human monocyte‐derived dendritic cells and circumvent the type I interferon response

Dengue is the most prevalent arboviral disease worldwide. The outcome of the infection is determined by the interplay of viral and host factors. In the present study, we evaluated the cellular response of human monocyte‐derived DCs (mdDCs) infected with recombinant dengue virus type 1 (DV1) strains carrying a single point mutation in the NS3_hel protein (L435S or L480S). Both mutated viruses infect and replicate more efficiently and produce more viral progeny in infected mdDCs compared with the parental, non‐mutated virus (vBACDV1). Additionally, global gene expression analysis using cDNA microarrays revealed that the mutated DVs induce the up‐regulation of the interferon (IFN) signalling and pattern recognition receptor (PRR) canonical pathways in mdDCs. Pronounced production of type I IFN were detected specifically in mdDCs infected with DV1‐NS3_hel‐mutated virus compared with mdDCs infected with the parental virus. In addition, we showed that the type I IFN produced by mdDCs is able to reduce DV1 infection rates, suggesting that cytokine function is effective but not sufficient to mediate viral clearance of DV1‐NS3_hel‐mutated strains. Our results demonstrate that single point mutations in subdomain 2 have important implications for adenosine triphosphatase (ATPase) activity of DV1‐NS3_hel. Although a direct functional connection between the increased ATPase activity and viral replication still requires further studies, these mutations speed up viral RNA replication and are sufficient to enhance viral replicative capacity in human primary cell infection and circumvent type I IFN activity. This information may have particular relevance for attenuated vaccine protocols designed for DV.     

Intermittent interferonemia and interferon responses in multiple sclerosis.

In view of the immunoregulatory and antiviral properties of the interferons (IFNs), the production of and response to these cytokines in vivo and in vitro were assessed in 42 patients with multiple sclerosis (MS), a disease with features of autoimmunity and a viral infection. Serum IFN, determined by bioassay of antiviral activity at 10 intervals over 18 months, was detectable at levels ranging from 16 to 250 IU/ml, at least once and up to five times in 37 of the 42 patients. Of 420 samples tested, 88 (21%) were positive. None of the 71 serum samples from 37 healthy subjects contained detectable IFN activity. Neutralization of antiviral activity by antibodies showed that the serum IFN type was IFN-alpha in 82 samples, IFN-gamma only in 2, and both IFN-alpha and IFN-gamma were present in 4. At the initial time point the activity of 2'-5' oligoadenylate synthetase (OAS), an IFN-induced enzyme, was elevated in peripheral blood leucocytes (PBL) from 13 patients, but not in 7 patients seropositive for IFN, indicating that in some patients there was a failure of PBL to respond to endogenous IFN. In most patients the capacity of PBL in vitro to produce IFNs-alpha/beta or -gamma after induction by virus or mitogens, respectively, was likewise reduced. These various abnormalities in IFN responses could not be correlated with clinical assessments of disease activity but may reflect subclinical attacks. The abnormalities described, in particular the intermittent interferonemia in MS, are more striking than in other diseases previously reported, indicating an unusual component to the stimulus for IFN production (viral or other) or the response to it. The effects of endogenous IFN production may have implications for the scheduling of therapy with IFN in MS.     

Blocking of interferon synthesis in murine macrophages by pretreatment with interferon

The influence of pretreatment with interferon (IFN) on subsequent IFN synthesis was investigated in macrophage cultures of DBA/2 and C57BL/6 mice. The doses of IFN alpha/beta for pretreatment ranged from 10,000 U/ml to 100 U/ml and the incubation time was between 18 and 2 h. No blocking effect was observed for chemical induction with poly I:poly C or CMA. However, for viral infection with NDV, blocking was observed. This inhibition of IFN synthesis was dependent on the dose and time of IFN pretreatment and of the titer of the inducing virus. Similarly in mouse fibroblast cultures no blocking activity was observed for induction with poly I:poly C/DEAE-dextran. Again, with NDV as inducer, pretreatment with IFN resulted in inhibition of interferon synthesis. Thus, our data show that blocking occurs only with a viral inducer and suggest that it is caused by an antiviral effect.     

Studies of chemotactic, chemotactic movement-inhibiting and random movement-inhibiting effects of interleukin-1 alpha and beta, tumour necrosis factors alpha and beta and interferon gamma on human neutrophils in assays using 'sparse-pore' polycarbonate (Nuclepore) membranes in the Boyden chamber

Interleukin-1 alpha and beta (IL-1 alpha and beta), tumour necrosis factors alpha and beta (TNF alpha and beta) and interferon gamma (IFN gamma) were tested for their chemotactic effects, their effects on chemotactic movement towards N-formyl-methionyl-leucyl-phenylalanine (FMLP) and their effects on random locomotion of human peripheral blood neutrophils through polycarbonate membranes in Boyden-type chambers. Both IL-1 alpha and beta, but no other cytokine tested were chemotactic for neutrophils using 'sparse-pore' polycarbonate membrane. Both TNFs, but no other cytokine, inhibited neutrophil chemotactic movement towards FMLP using the same membrane. No cytokine influenced random migration of neutrophils through polycarbonate membrane of standard pore density. These results suggest that IL-1 may have a role as a chemotactic mediator of inflammation, but that TNFs may inhibit chemotactic migration of neutrophils into inflammatory lesions.     

Receptors and ligands involved in viral induction of type I interferon production by plasmacytoid dendritic cells

Virus infection is sensed by the innate immune system which then rapidly initiates biosynthesis of type I interferon (IFN). The IFN signaling systems produce a broadly effective innate antiviral response by creating an antiviral state in both an autocrine and paracrine manner in cells and by activating innate and adaptive immunity. Plasmacytoid dendritic cells (pDCs) have the unique ability to produce very high levels of type I IFN following viral infection in vivo. Most recent research has focused on oligonucleotide-mediated induction of type I IFN production, implicating viral genome and replication intermediates as the stimulus for this response. However there are additional viral ligands which can potentially induce type I IFN production in pDCs, such as envelope glycoproteins, viral glycolipids, tegument, capsid or nuclear proteins. This area of viral immunology, which has been neglected in the literature, will be discussed here.     

Gene expression contributing to recruitment of circulating cells in response to vesicular stomatitis virus infection of the CNS

During acute Vesicular Stomatitis Virus (VSV) infection of the mouse central nervous system, neutrophils, natural killer (NK) cells, macrophages, and CD4+ and CD8+ T cells are recruited from the circulation in response to chemokines and cytokines. This study elucidated the production of these factors and infiltration of these peripheral cells. Chemokines that were observed included CCL1, CXCL10 (IP-10), CCL5 (RANTES), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), CXCL1 (MIP-2), CCL2 (MCP-1), and CCL11 (eotaxin). Cytokines produced in response to the infection include IL-1 and interferon-gamma, but not type I interferons. Neutrophils are the first recruited cell type, appearing as early as 24 h after intranasal application of the virus. NK cells follow, but T cells are not detected until 6 days postinfection.     

Microbicidal protein psoriasin is a multifunctional modulator of neutrophil activation

As effector cells in host defence, neutrophils actively destroy invading microorganisms via a potent antimicrobial arsenal composed of oxidants and antimicrobial peptides. Psoriasin, an Escherichia coli-cidal antimicrobial protein, has been found to be overexpressed in psoriasis, a skin disease characterized by infiltration of neutrophils. In addition to its microbicidal activities and chemotaxis of neutrophils reported previously, we hypothesized that psoriasin might regulate other neutrophil functions such as cytokine and chemokine production, reactive oxygen species generation, and release of antimicrobial peptides. In the current study, we demonstrate that psoriasin activates neutrophils to produce a range of cytokines and chemokines including interleukin-6 (IL-6), IL-8/CXCL8, tumour necrosis factor-alpha, macrophage inflammatory protein-1alpha (MIP-1alpha)/CCL3, MIP-1beta/CCL4 and MIP-3alpha/CCL20. Furthermore, psoriasin induces phosphorylation of mitogen-activated protein kinase p38 and extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK), both of which are required for the production of cytokines and chemokines as evidenced by the inhibitory effects of p38 and ERK inhibitors on psoriasin-mediated neutrophil activation. Moreover, psoriasin stimulates the generation of reactive oxygen species from neutrophils, most likely via nicotinamide adenine dinucleotide phosphate oxidase activation. Finally, we demonstrate that psoriasin enhances messenger RNA expression of alpha-defensins, termed human neutrophil peptides (HNP) 1 to 3, and induces their extracellular release. Besides its antimicrobial properties, therefore, psoriasin may contribute to innate immunity through enhancing neutrophil host defence functions at sites of inflammation or infection.