Severe acute respiratory syndrome (SARS) is an acute infectious disease of the respiratory system. Although a novel coronavirus has been identified as the causative agent of SARS, the pathogenic mechanisms of SARS are not understood. In this study, sera were collected from healthy donors, patients with SARS, patients with severe SARS, and patients with SARS in convalescence. The serum concentrations of interleukin-1 (IL-1), IL-4, IL-6, IL-8, IL-10, tumor growth factor beta (TGF-beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were measured by enzyme-linked immunosorbent assays (ELISA). The concentrations of IL-1 and TNF-alpha were not significantly different in different groups. The IL-6 concentration was increased in SARS patients and was significantly elevated in severe SARS patients, but the IL-6 concentrations were similar in convalescent patients and control subjects, suggesting that there was a positive relationship between the serum IL-6 concentration and SARS severity. The concentrations of IL-8 and TGF-beta were decreased in SARS patients and significantly reduced in severe SARS patients, but they were comparable in convalescent SARS patients and control subjects, suggesting that there was a negative relationship between the IL-8 and TGF-beta concentrations and SARS severity. The concentrations of IFN-gamma, IL-4, and IL-10 showed significant changes only in convalescent SARS patients. The IFN-gamma and IL-4 levels were decreased, while the levels of IL-10 were increased, and the differences between convalescent SARS patients and other patient groups were statistically significant. These results suggest that there are different immunoregulatory events during and after SARS and may contribute to our understanding of the pathogenesis of this syndrome.

Type I interferon (IFN-alpha/beta) comprises a family of immunomodulatory cytokines that are critical for controlling viral infections. In cell culture, many RNA viruses trigger IFN responses through the binding of RNA recognition molecules (RIG-I, MDA5, and TLR-3) and induction of interferon regulatory factor IRF-3-dependent gene transcription. Recent studies with West Nile virus (WNV) have shown that type I IFN is essential for restricting infection and that a deficiency of IRF-3 results in enhanced lethality. However, IRF-3 was not required for optimal systemic IFN production in vivo or in vitro in macrophages. To begin to define the transcriptional factors that regulate type I IFN after WNV infection, we evaluated IFN induction and virus control in IRF-7(-/-) mice. Compared to congenic wild-type mice, IRF-7(-/-) mice showed increased lethality after WNV infection and developed early and elevated WNV burdens in both peripheral and central nervous system tissues. As a correlate, a deficiency of IRF-7 blunted the systemic type I IFN response in mice. Consistent with this, IFN-alpha gene expression and protein production were reduced and viral titers were increased in IRF-7(-/-) primary macrophages, fibroblasts, dendritic cells, and cortical neurons. In contrast, in these cells the IFN-beta response remained largely intact. Our data suggest that the early protective IFN-alpha response against WNV occurs through an IRF-7-dependent transcriptional signal.

Pip is a lymphoid-restricted IRF transcription factor that is recruited to composite elements within immunoglobulin light-chain gene enhancers through a specific interaction with the Ets factor PU.1. We have examined the transcriptional regulatory properties of Pip as well as the requirements for its interaction with PU.1 and DNA to form a ternary complex. We demonstrate that Pip is a dichotomous regulator; it specifically stimulates transcription in conjunction with PU.1, but represses alpha/beta-interferon-inducible transcription in the absence of PU.1. Thus, during B-cell activation and differentiation, Pip may function both as an activator to promote B cell-specific gene expression and as a repressor to inhibit the antiproliferative effects of alpha/beta-interferons. Mutational analysis of Pip reveals a carboxy-terminal segment that is important for autoinhibition of DNA binding and ternary complex formation. A domain of Pip containing this segment confers autoinhibition and PU.1-dependent binding activity to the DNA-binding domain of the related IRF family member, p48. On the basis of these and other data we propose a model for PU.1/Pip ternary complex formation.

Fractalkine (now also called CX3CL1) is a unique chemokine that functions not only as a chemoattractant but also as an adhesion molecule and is expressed on endothelial cells activated by proinflammatory cytokines, such as interferon-gamma and tumor necrosis factor-alpha. The fractalkine receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes, including natural killer (NK) cells and cytotoxic T lymphocytes, which contain high levels of intracellular perforin and granzyme B, and on macrophages. Soluble fractalkine causes migration of NK cells, cytotoxic T lymphocytes, and macrophages, whereas the membrane-bound form captures and enhances the subsequent migration of these cells in response to secondary stimulation with other chemokines. Furthermore, stimulation through membrane-bound fractalkine activates NK cells, leading to increased cytotoxicity and interferon-gamma production. Recently, accumulating evidence has shown that fractalkine is involved in the pathogenesis of various clinical disease states or processes, such as atherosclerosis, glomerulonephritis, cardiac allograft rejection, and rheumatoid arthritis. In addition, polymorphisms in CX3CR1, which reduce its binding activity to fractalkine, have been reported to increase the risk of HIV disease and to reduce the risk of coronary artery disease. This review will examine new concepts underlying fractalkine-mediated leukocyte migration and tissue damage, focusing primarily on the pathophysiological roles of fractalkine in various clinical conditions, especially in atherosclerosis and vascular injury.

Interferon (IFN)-alpha/beta-mediated negative regulation of interleukin 12 (IL-12) and IFN-gamma proteins is reported here. Both IFN-alpha and IFN-beta inhibited fixed Staphylococcus aureus Cowan strain induction of IL-12 and IFN-gamma production by mouse splenic leukocytes in culture. Extended studies with IFN-alpha demonstrated that inhibition was at the level of biologically active IL-12 p70. Effects were selective, as induction of tumor necrosis factor was unaffected and induction of IL-6 was enhanced. Neutralization of IFN-alpha/beta expressed endogenously during infections with murine cytomegalovirus (MCMV) enhanced early IL-12 and IFN-gamma protein production. Furthermore, during infections of mice with lymphocytic choriomeningitis virus (LCMV), this treatment revealed a previously undetected early IL-12 and IFN-gamma protein expression, and mice deficient in IFN-alpha/beta receptor function, but not control mice, also expressed endogenous LCMV-induced IL-12. The effects of IFN-alpha/beta neutralization on production of IL-12 and IFN-gamma during the viral infections were detected in both serum samples and medium conditioned with splenic leukocytes isolated from infected animals. In vitro studies demonstrated that splenic leukocytes isolated from LCMV-infected mice were primed to produce IL-12 in response to stimulation with Staphylococcus aureus Cowan strain, but that this responsiveness was sensitive to added IFN-alpha. Moreover, endogenous IFN-alpha/beta induced by LCMV inhibited in vivo lipopolysaccharide stimulation of IL-12 production. These results demonstrate a new pathway for regulating cytokine responses, and suggest a mechanism for inhibition of IL-12-dependent immune responses during viral infections.

Cytochromes P450 (P450s) are down-regulated in hepatocytes in response to inflammation and infection. This effect has been extensively studied in animal models, but significantly less is known about responses in humans and even less about responses in the absence of inducing agents. This article focuses on the effects of bacterial lipopolysaccaride (LPS), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF), interferon gamma (IFN), transforming growth factor-beta (TGF) and interleukin-1 beta (IL-1) on expression of CYP2B6 and the CYP2C mRNAs in human hepatocytes. These effects were compared with responses of the better studied and more abundant CYP3A4. CYP3A4 and CYP2C8 were down-regulated by all cytokine treatments. CYP2C18, which is expressed at very low levels in liver, was unaffected by cytokine treatments. The other CYP2Cs and CYP2B6 showed cytokine-specific effects. CYP2C9 and CYP2C19 showed almost identical response patterns, being down-regulated by IL-6 and TGF but not significantly affected by LPS, TNF, IFN, or IL-1. CYP2B6 mRNA responded only to IL-6 and IFN. IL-6 down-regulated the mRNAs of all P450s studied. Western blot analysis of P450 protein expression supported the mRNA data to a large extent, although some inconsistencies were observed. Our results show that human CYP2C8, 2C9, 2C18, 2C19, 2B6, and 3A4 responses to inflammation are independently regulated and indicate that this fine control may have a critical effect on human drug responses in disease states.

Hantavirus pulmonary syndrome (HPS) is characterized by the rapid onset of pulmonary edema and a high case-fatality rate. Hantavirus antigens have been demonstrated in pulmonary capillary endothelial cells, but the mechanisms causing capillary leakage remain unclear. Immunohistochemical staining was used to enumerate cytokine-producing cells (monokines: interleukin [IL]-1alpha, IL-1beta, IL-6, and tumor necrosis factor [TNF]-alpha; lymphokines: interferon-gamma, IL-2, IL-4, and TNF-beta) in tissues obtained at autopsy from subjects with HPS. High numbers of cytokine-producing cells were seen in the lung and spleen tissues of HPS patients, but only low numbers in the livers and kidneys. A modest increase in the numbers of cytokine-producing cells was detected in the lungs of patients who died with non-HPS acute respiratory distress syndrome (ARDS), and very few (or no) cytokine-producing cells were detected in the lungs of patients who died of causes other than ARDS. These results suggest that local cytokine production may play an important role in the pathogenesis of HPS.

Fifty-nine patients with prior hepatitis B virus infection underwent orthotopic liver transplantation. During the first 2 mo, mortality was not significantly different in the hepatitis B virus-infected group (25.5%) vs. a hepatitis B virus-immune control group (21%). Beyond 2 mo, the mortality, rate of graft loss, need for retransplantation and incidence of abnormal liver function were significantly higher in the hepatitis B virus-infected group. Treatment of the hepatitis B virus infection was attempted with passive immunization, combined active and passive immunization, alpha-interferon or nothing. The clinical outcome was not significantly influenced by any of these therapies. However, of the patients who lived more than 60 days, 6 of 22 treated with active plus passive immunization were cleared of HBsAg, something achieved once in 16 patients treated with alpha-interferon, never in 3 patients with passive immunization only and once in 4 patients with no therapy. In patients with recurrent hepatitis B virus infection, the pace of hepatitis development in the graft appeared to be accelerated, and this was particularly striking in patients who underwent multiple retransplantations at progressively shorter intervals. None of the patients who became HBsAg-negative had HBeAg preoperatively.

Reactive oxygen species (ROS) generated during inflammation are believed to play critical roles in various ocular diseases. However, the underlying mechanisms remain poorly understood. We investigated if pro-inflammatory cytokines, tumor necrosis factor (TNF)-alpha, interleukin-1 beta (IL-1 beta), and interferon-gamma (IFN-gamma), induce ROS in human retinal pigment epithelial (RPE) cells. TNF-alpha, IL-1 beta and IFN-gamma increased both intracellular and extracellular ROS production in a time- and dose-dependent manner. Thenoyltrifluoroacetone (TTFA), an inhibitor of mitochondrial respiratory chain, blocked TNF-alpha- and IFN-gamma-, but not IL-1 beta-induced ROS, whereas other two mitochondrial respiratory chain inhibitors, rotenone and antimycin A, had no effect. NADPH oxidase inhibitor (diphenylene iodinium) abolished the ROS production induced by IL-1 beta or IFN-gamma, but not by TNF-alpha, whereas 6-aminonicotinamide (6AN), an inhibitor of the hexose monophosphate shunt (HMS), had no significant effects on the ROS induced by all three cytokines. ROS scavengers, pyrrolidinedithiocarbamate (PDTC) and N-acetyl-cysteine (NAC), reduced the levels of ROS induced by TNF-alpha, IL-1 beta and IFN-gamma (P<0.05). Collectively, these results demonstrate that TNF-alpha, IL-1 beta and IFN-gamma increase mitochondrial- and NADPH oxidase-generated ROS in human RPE cells.

Leishmania major promastigotes were found to avoid activation of mouse bone marrow-derived macrophages (BMM0) in vitro for production of cytokines that are typically induced during infection with other intracellular pathogens. Coexposure of BMM0 to the parasite and other microbial stimuli resulted in complete inhibition of interleukin (IL) 12 (p40) mRNA induction and IL-12 release. In contrast, mRNA and protein levels for IL-1(alpha), IL-1(beta), tumor necrosis factor (TNF) alpha, and inducible NO synthase (iNOS) were only partially reduced, and signals for IL-10 and monocyte chemoattractant protein (MCP-1/JE) were enhanced. The parasite could provide a detectable trigger for TNF-alpha and iNOS in BMM0 primed with interferon (IFN) gamma, but still failed to induce IL-12. Thus IL-12 induction is selectively impaired after infection, whereas activation pathways for other monokine responses remain relatively intact. Selective and complete inhibition of IL-12(p40) induction was observed using BMM0 from either genetically susceptible or resistant mouse strains, as well as IL-10 knockout mice, and was obtained using promastigotes from cutaneous, visceral, and lipophosphoglycan-deficient strains of Leishmania. The impaired production of the major physiological inducer of IFN-gamma is suggested to underlie the relatively prolonged interval of parasite intracellular survival and replication that is typically associate with leishmanial infections, including those producing self-limiting disease.

Nuclear protein IRF-1 (interferon regulatory factor 1) was earlier shown to bind to cis-acting regulatory elements present on interferon (IFN)-alpha/beta genes and some IFN-inducible genes. Here we show that in both human FS-4 and murine L929 cells, steady-state levels of IRF-1 mRNA were increased by treatment with tumor necrosis factor (TNF), interleukin 1 (IL-1), poly(I).poly(C), or IFN-beta. IRF-1 mRNA induction was also demonstrated in cells treated with calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, but not with epidermal growth factor, dibutyryl-cAMP, or the adenylate cyclase activator forskolin. To determine whether stimulation of IRF-1 mRNA levels correlates with IFN-beta induction, we compared IRF-1 and IFN-beta mRNA levels in cells exposed to various stimuli. In L929 cells, treatment with poly(I).poly(C) under conditions that failed to induce significant levels of IFN-beta mRNA led to a very low induction of IRF-1 mRNA, but "priming" cells with IFN prior to the addition of poly(I).poly(C) greatly increased both IRF-1 and IFN-beta mRNAs. In FS-4 cells an increase in IFN-beta mRNA (examined by the polymerase chain reaction) was seen after treatment with TNF, IL-1, A23187, or poly(I).poly(C), but not with IFN-beta, epidermal growth factor, dibutyryl-cAMP, or forskolin. Thus, all treatments that increased steady-state levels of IFN-beta mRNA also enhanced IRF-1 mRNA levels. However, treatment with IFN-beta, which caused a marked stimulation in IRF-1 mRNA, failed to produce a detectable increase in IFN-beta mRNA. It appears that IRF-1 may be necessary but not sufficient for IFN-beta induction. The ability of TNF and IL-1 to increase both IRF-1 and IFN-beta mRNAs may be responsible for some similarities in the actions of TNF, IL-1, and the IFNs.

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-alpha/beta exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-beta, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-beta activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.

Cytoplasmic viral RNAs with 5' triphosphates (5'ppp) are detected by the RNA helicase RIG-I, initiating downstream signaling and alpha/beta interferon (IFN-alpha/beta) expression that establish an antiviral state. We demonstrate here that the hepatitis C virus (HCV) 3' untranslated region (UTR) RNA has greater activity as an immune stimulator than several flavivirus UTR RNAs. We confirmed that the HCV 3'-UTR poly(U/UC) region is the determinant for robust activation of RIG-I-mediated innate immune signaling and that its antisense sequence, poly(AG/A), is an equivalent RIG-I activator. The poly(U/UC) region of the fulminant HCV JFH-1 strain was a relatively weak activator, while the antisense JFH-1 strain poly(AG/A) RNA was very potent. Poly(U/UC) activity does not require primary nucleotide sequence adjacency to the 5'ppp, suggesting that RIG-I recognizes two independent RNA domains. Whereas poly(U) 50-nt or poly(A) 50-nt sequences were minimally active, inserting a single C or G nucleotide, respectively, into these RNAs increased IFN-beta expression. Poly(U/UC) RNAs transcribed in vitro using modified uridine 2' fluoro or pseudouridine ribonucleotides lacked signaling activity while functioning as competitive inhibitors of RIG-I binding and IFN-beta expression. Nucleotide base and ribose modifications that convert activator RNAs into competitive inhibitors of RIG-I signaling may be useful as modulators of RIG-I-mediated innate immune responses and as tools to dissect the RNA binding and conformational events associated with signaling.

The advent of specific antiviral therapy for chronic hepatitis C has increased the importance of establishing the correct etiology of chronic hepatitis in patients, especially because interferon alfa (IFN-alpha) has been reported to exacerbate autoimmune hepatitis (AIH), whereas corticosteroids increase viral replication in chronic hepatitis C. In our medical center, we have treated many patients with apparent chronic hepatitis C and serological or clinical evidence of autoimmunity. Our aim was to estimate the prevalence of this association and to learn whether demographic or clinical features distinguished between patients with or without autoimmune markers. We performed a retrospective review of the records of 244 unselected patients seen at the Clinics and Hospital of the University of Massachusetts between May 1991 and November 1993, who had elevated serum aminotransferases. One hundred seventeen patients had chronic hepatitis C defined by elevations of serum alanine transaminase (ALT) for at least 6 months, positive serum antibodies to hepatitis C virus (HCV; second-generation enzyme immunoassay [EIA2] or recombinant immunoblot assay [RIBA]), and absence of hepatitis B surface antigen in the serum. Records were reviewed for results of autoimmune markers in sera, including anti-nuclear antibodies (ANAs), anti-smooth muscle antibodies (SMAs), rheumatoid factor (RF), antimitochondrial antibodies (AMAs), anti-liver and kidney microsomal (LKM) antibodies, and cryoglobulins. We found a high prevalence of positivity, particularly for anti-SMAs (66%) and RF (76%) in both men and women. Forty of 41 patients tested negative for anti-LKM antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

BACKGROUND

Neuromyelitis optica (NMO) is assumed to be immunologically distinct from multiple sclerosis (MS). Adequate studies about cytokines and chemokines in NMO have been lacking.

OBJECTIVE

To investigate the contribution of cytokines/chemokines in the pathogenesis of NMO.

METHODS

We measured 27 cytokines/chemokines and Th17 cell-associated cytokines in the cerebrospinal fluid (CSF) of 31 NMO, 29 MS and 18 other non-inflammatory neurological disorders patients. The serum levels of some cytokines/ chemokines were also measured. The correlations between clinical characteristics/laboratory findings and levels of cytokines/chemokines in NMO were examined.

RESULTS

The CSF levels of interleukin (IL)-1 receptor antagonist, IL-6, IL-8, IL-13 and granulocyte colony-stimulating factor were significantly increased in NMO, while IL-9, fibroblast growth factor-basic, granulocyte macrophage colony-stimulating factor, macrophage inflammatory protein-1-beta and tumor necrosis factor-alpha were increased in MS. IL-10 and interferon-gamma-inducible protein-10 were elevated in NMO and MS. In serum analyses, only the IL-6 level showed significant elevation in NMO. The CSF IL-6 level had a significant correlation with the CSF glial fibrillary acidic protein level and CSF cells, and a weak correlation with anti-aquaporin-4 antibody titers.

CONCLUSIONS

Different immunological status and pathophysiologies exist between NMO and MS, and IL-6 may play important roles in the pathogenesis of NMO.

Severe acute respiratory syndrome coronavirus (SARS-CoV) caused an acute human respiratory illness with high morbidity and mortality in 2002-2003. Several studies have demonstrated the role of neutralizing antibodies induced by the spike (S) glycoprotein in protecting susceptible hosts from lethal infection. However, the anti-SARS-CoV antibody response is short-lived in patients who have recovered from SARS, making it critical to develop additional vaccine strategies. SARS-CoV-specific memory CD8 T cells persisted for up to 6 years after SARS-CoV infection, a time at which memory B cells and antivirus antibodies were undetectable in individuals who had recovered from SARS. In this study, we assessed the ability of virus-specific memory CD8 T cells to mediate protection against infection in the absence of SARS-CoV-specific memory CD4 T or B cells. We demonstrate that memory CD8 T cells specific for a single immunodominant epitope (S436 or S525) substantially protected 8- to 10-month-old mice from lethal SARS-CoV infection. Intravenous immunization with peptide-loaded dendritic cells (DCs) followed by intranasal boosting with recombinant vaccinia virus (rVV) encoding S436 or S525 resulted in accumulation of virus-specific memory CD8 T cells in bronchoalveolar lavage fluid (BAL), lungs, and spleen. Upon challenge with a lethal dose of SARS-CoV, virus-specific memory CD8 T cells efficiently produced multiple effector cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], and interleukin 2 [IL-2]) and cytolytic molecules (granzyme B) and reduced lung viral loads. Overall, our results show that SARS-CoV-specific memory CD8 T cells protect susceptible hosts from lethal SARS-CoV infection, but they also suggest that SARS-CoV-specific CD4 T cell and antibody responses are necessary for complete protection.

OBJECTIVE

Virus-specific CD8 T cells are required for pathogen clearance following primary SARS-CoV infection. However, the role of SARS-CoV-specific memory CD8 T cells in mediating protection after SARS-CoV challenge has not been previously investigated. In this study, using a prime-boost immunization approach, we showed that virus-specific CD8 T cells protect susceptible 8- to 10-month-old mice from lethal SARS-CoV challenge. Thus, future vaccines against emerging coronaviruses should emphasize the generation of a memory CD8 T cell response for optimal protection.

BACKGROUND

Despite growing recognition of an etiologic role for inflammation in lung carcinogenesis, few prospective epidemiologic studies have comprehensively investigated the association of circulating inflammation markers with lung cancer.

METHODS

We conducted a nested case-control study (n = 526 lung cancer patients and n = 592 control subjects) within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial. Control subjects were matched to lung cancer case patients on age, sex, follow-up time (median = 2.9 years), randomization year, and smoking (pack-years and time since quitting). Serum levels of 77 inflammation markers were measured using a Luminex bead-based assay. Conditional logistic regression and weighted Cox models were used to estimate odds ratios (ORs) and cumulative risks, respectively.

RESULTS

Of 68 evaluable markers, 11 were statistically significantly associated with lung cancer risk (P trend across marker categories < .05), including acute-phase proteins (C-reactive protein [CRP], serum amyloid A [SAA]), proinflammatory cytokines (soluble tumor necrosis factor receptor 2 [sTNFRII]), anti-inflammatory cytokines (interleukin 1 receptor antagonist [IL-1RA]), lymphoid differentiation cytokines (interleukin 7 [IL-7]), growth factors (transforming growth factor alpha [TGF-A]), and chemokines (epithelial neutrophil-activating peptide 78 [ENA 78/CXCL5], monokine induced by gamma interferon [MIG/CXCL9], B cell-attracting chemokine 1 [BCA-1/CXCL13], thymus activation regulated chemokine [TARC/CCL17], macrophage-derived chemokine [MDC/CCL22]). Elevated marker levels were associated with increased lung cancer risk, with odds ratios comparing the highest vs the lowest group ranging from 1.47 (IL-7) to 2.27 (CRP). For IL-1RA, elevated levels were associated with decreased lung cancer risk (OR = 0.71; 95% confidence interval = 0.51 to 1.00). Associations did not differ by smoking, lung cancer histology, or latency. A cross-validated inflammation score using four independent markers (CRP, BCA-1/CXCL13, MDC/CCL22, and IL-1RA) provided good separation in 10-year lung cancer cumulative risks among former smokers (quartile [Q] 1 = 1.1% vs Q4 = 3.1%) and current smokers (Q1 = 2.3% vs Q4 = 7.9%) even after adjustment for smoking.

CONCLUSIONS

Some circulating inflammation marker levels are associated with prospective lung cancer risk.

We investigated the influence of interferons alpha, beta, and gamma (IFN-alpha, -beta, and -gamma) on the production of basic fibroblast growth factor (bFGF) by human renal carcinoma cells. The human renal carcinoma cell metastatic line SN12PM6 was established in culture from a lung metastasis and SN12PM6-resistant cells were selected in vitro for resistance to the antiproliferative effects of IFN-alpha or IFN-beta. IFN-alpha and IFN-beta, but not IFN-gamma, down-regulated the expression of bFGF at the mRNA and protein levels by a mechanism independent of their antiproliferative effects. Down-regulation of bFGF required a long exposure >> 4 days) of cells to low concentrations >> 10 units/ml) of IFN-alpha or IFN-beta. The withdrawal of IFN-alpha or IFN-beta from the medium permitted SN12PM6-resistant cells to resume production of bFGF. The incubation of human bladder, prostate, colon, and breast carcinoma cells with noncytostatic concentrations of IFN-alpha or IFN-beta also produced down-regulation of bFGF production.

PML has been identified through its fusion to the RAR alpha gene in acute promyelocytic leukemia (APL). The PML protein is specifically associated to nuclear bodies (NBs) whose alterations in APL were proposed to contribute to leukemogenesis. The role of this nuclear domain (which also harbors the Sp100 autoantigen and the NDP52 protein) is unknown. Here, we show that the PML protein, like Sp100 and NDP52, is induced by interferons (IFNs alpha, beta and gamma) in a large variety of human cells. Interestingly, the NBs that contain the three IFN-induced proteins appear to be associated to speckles labelled by the IFN-mediator Mx1. These observations link NBs to IFN response pathways, which may contribute to the elucidation of the biological role of these structures. In APL cells, IFNs induced both PML and PML/RAR alpha expression, resulting in an increased sequestration of PML and RXRs in the microspeckles induced by the fusion protein. As PML has growth suppressing properties, it may mediate some of the antiproliferative effects of IFN. In APL, inactivation of PML may result in disruption of growth control.

Interferons IFN-alpha/beta and IFN-gamma act through independent cell-surface receptors, inducing gene expression through tyrosine phosphorylation of cytoplasmic transcription factors . IFN-alpha stimulates phosphorylation and nuclear localization of the 84/91K and 113K subunits of latent ISGF3 (interferon-stimulated gene factor 3), which combine with the 48K DNA-binding subunit to bind regulatory elements of IFN-alpha-responsive genes. IFN-gamma activates p91 alone, inducing IFN-gamma-responsive genes through a distinct DNA element. Genetic complementation studies implicated the tyrosine kinase Tyk2 in IFN-alpha signalling and, more recently, the related Jak2 kinase in IFN-gamma signalling. We now present biochemical evidence for Jak-family kinase involvement in IFN signal transduction. Jak1 was activated in response to IFN-alpha and IFN-gamma; Jak2 responded exclusively to IFN-gamma. Overexpression of either Jak1 or Jak2 stimulated p91 DNA-binding activity and p91-dependent transcription. Overexpression also activated endogenous Jak kinases, suggesting that interactions between Jak kinases are required during interferon signalling.

The role of different T cell subsets in antiviral host defense was investigated by treating thymectomized C57BL/6 and CBA/J mice with monoclonal rat anti-Lyt-2 or anti-L3/T4 IgG 2b antibodies 14 and 10 days before infection. This treatment depleted the respective T cell subsets to undetectable levels in peripheral blood when assayed by immunofluorescence. In mice treated with anti-Lyt-2, induction of cytotoxic T cells was reduced to less than 1 to 2% after intravenous infection with Armstrong strain of lymphocytic choriomeningitis virus (LCMV). In addition, no primary swelling of the footpad could be detected following local inoculation of the virus. In animals treated with anti-L3/T4, antiviral cytotoxic T lymphocyte responses were reduced by a factor of 10. These L3/T4+ cell-depleted mice showed delayed footpad swelling after local injection of LCMV Armstrong. After intracerebral infection with LCMV, anti-Lyt-2-treated mice were resistant and those injected with anti-L3/T4 were totally susceptible to LCMV Armstrong-triggered immunopathologic disease. Virus could be detected in the blood of antibody-treated mice 7 days after inoculation; however, no virus could be measured in the blood of surviving anti-Lyt-2-treated animals 15 days after intracerebral infection. Serum titers of interferon-alpha,beta induced by viral infection remained unaffected by depletion of T cell subsets. Anti-L3/T4 antibody-treated C57BL/6 mice failed to generate IgG antibodies against the New Jersey strain of vesicular stomatitis virus, whereas Lyt-2+ cell-depleted mice had normal antivesicular stomatitis virus (New Jersey strain) IgG antibody titers.

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

Suppressor of cytokine signaling (SOCS) proteins constitute a class of negative regulators for Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. These intracellular proteins are induced by cytokine signaling, but they can also be induced by stimulation of Toll-like receptors (TLR). It has even been suggested that SOCS proteins are important negative regulators of TLR signaling. Here we have elucidated the nature of the regulatory role of SOCS in TLR signaling. Induction of SOCS-3 and cytokine-inducible Src homology 2-containing protein (CIS) by TLR stimulation was strictly dependent on MyD88 but showed differing needs in case of SOCS-1. However, induction of SOCS proteins by TLR ligands was independent of type I interferon. In macrophages overexpressing SOCS, we were not able to observe an inhibitory effect of SOCS-1, SOCS-2, SOCS-3, or CIS on prototypical TLR target genes such as tumor necrosis factor-alpha. However, we found that TLR-2, TLR-3, TLR-4, and TLR-9 stimulation induced interferon-beta (IFN-beta), which is able to exert auto- and paracrine signaling, leading to the activation of secondary genes like IP-10. SOCS-1 and, to a lesser extent, SOCS-3 and CIS were able to inhibit this indirect signaling pathway following TLR stimulation, whereas neither MAP kinase nor NF kappa B signaling were affected. However, STAT-1 tyrosine phosphorylation following TLR triggering was severely impaired by SOCS-1 overexpression. Thus, our data suggest that SOCS proteins induced by TLR stimulation limit the extent of TLR signaling by inhibiting type I IFN signaling but not the main NF kappa B pathway.

BACKGROUND

Increasing evidence demonstrates that atherosclerosis is an immunologically mediated disease in which the secretion of atherogenic and atheroprotective cytokines, by infiltrating blood mononuclear cells, plays an important role. It is not known whether long-term exercise alters this atherogenic and atheroprotective activity directly.

OBJECTIVE

To determine the effect of long-term exercise on the atherogenic activity of blood mononuclear cells in persons at risk of developing ischemic heart disease.

METHODS

Before-after trial using a 6-month individualized, supervised exercise program, with an enrollment period from December 1996 to October 1997.

METHODS

Hospital-based community wellness center.

METHODS

Of 110 persons who responded to a public request for volunteers, 52 met the inclusion criteria (risk ratio for myocardial infarction>> or =1.7 based on serum complement and/or C-reactive protein levels, and normal exercise treadmill test results). Forty-three of the 52 enrollees (25 women [mean age, 49.7 years] and 18 men [mean age, 48.1 years]) completed the study; 9 withdrew for personal reasons. Additional risk factors for ischemic heart disease included hypercholesterolemia (65.1 %), a family history of coronary heart disease (62.8%), inactivity (60.5%), hypertension (32.6%), obesity (25.6%), smoking (11.6%), and diabetes mellitus (4.7%).

METHODS

Blood levels were compared at baseline and after the exercise program had been completed for the following: spontaneous and phytohemagglutinin-induced production of interleukin 1 alpha, tumor necrosis factor alpha, and interferon gamma (atherogenic cytokines), and interleukin 4, interleukin 10, and transforming growth factor beta 1 (atheroprotective cytokines) by blood mononuclear cells; lymphocyte phenotypes and mitogenic responses to phytohemagglutinin; and serum C-reactive protein levels.

RESULTS

Subjects exercised for a mean of 2.5 (range, 0.3-7.4) hours per week. Mononuclear cell production of atherogenic cytokines fell by 58.3 % (P<.001) following the exercise program, where as the production of atheroprotective cytokines rose by 35.9% (P<.001). Changes in transforming growth factor beta 1 and in phytohemagglutinin-induced atherogenic cytokine production after the exercise program were proportionate to the time subjects spent performing repetitive lower-body motion exercises (P<.02), indicating a dose-response relationship. After the exercise program, changes in cellular function were reflected systemically by a 35% decrease in serum levels of C-reactive protein (P=.12).

CONCLUSIONS

Our data suggest that long-term exercise decreases the atherogenic activity of blood mononuclear cells in persons at risk of developing ischemic heart disease. This may be a mechanism whereby physical activity protects against ischemic heart disease.