Serum cytokine/chemokine profiles in patients with dengue fever (DF) and dengue hemorrhagic fever (FHD) by using protein array

BackgroundDENV infection can induce different clinical manifestations varying from mild forms to dengue fever (DF) or the severe hemorrhagic fever (DHF). Several factors are involved in the progression from DF to DHF. No marker is available to predict this progression. Such biomarker could allow a suitable medical care at the beginning of the infection, improving patient prognosis.ObjectivesThe aim of this study was to compare the serum expression levels of acute phase proteins in a well-established cohort of dengue fever (DF) and dengue hemorrhagic fever (DHF) patients, in order to individuate a prognostic marker of diseases severity.Study designThe serum levels of 36 cytokines, chemokines and acute phase proteins were determined in DF and DHF patients and compared to healthy volunteers using a multiplex protein array and near-infrared (NIR) fluorescence detection. Serum levels of IL-1ra, IL-23, MIF, sCD40 ligand, IP-10 and GRO-α were also determined by ELISA.ResultsAt the early stages of infection, GRO-α and IP-10 expression levels were different in DF compared to DHF patients. Besides, GRO-α was positively correlated with platelet counts and IP-10 was negatively correlated with total protein levels.ConclusionsThese findings suggest that high levels of GRO-α during acute DENV infection may be associated with a good prognosis, while high levels of IP-10 may be a warning sign of infection severity.     

Antibodies play a greater role than immune cells in heterologous protection against secondary dengue virus infection in a mouse model

The four serotypes of dengue virus (DENV1–4) are causative agents of dengue fever and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Previous DENV infection is a risk factor for DHF/DSS during subsequent infection by a different serotype. Nonetheless, most primary and secondary DENV infections are asymptomatic. To investigate the possible mechanisms of immune protection in vivo, 129/Pas mice lacking IFN-α/β and -γ receptors (AG129) were used to model secondary infection using both DENV1–DENV2 and DENV2–DENV4 sequences. At intervals between sequential infections of 4 to 52 weeks, protection against secondary heterologous DENV infection was observed. Passive transfer of DENV-immune serum was protective against replication of heterologous challenge virus in all tissues tested, whereas adoptive transfer of DENV-immune cells significantly protected mice from replication of the challenge virus only when a lower inoculum was administered. These findings are relevant for understanding both natural and vaccine-induced immunity to DENV.     

Galectin-9 plasma levels reflect adverse hematological and immunological features in acute dengue virus infection

BackgroundDengue virus (DENV) infection remains a major public health burden worldwide. Soluble mediators may play a critical role in the pathogenesis of acute DENV infection. Galectin-9 (Gal-9) is a soluble β-galactoside-binding lectin, with multiple immunoregulatory and inflammatory properties.ObjectiveTo investigate plasma Gal-9 levels as a biomarker for DENV infection.Study designWe enrolled 65 DENV infected patients during the 2010 epidemic in the Philippines and measured their plasma Gal-9 and cytokine/chemokine levels, DENV genotypes, and copy number during the critical and recovery phases of illness.ResultsDuring the critical phase, Gal-9 levels were significantly higher in DENV infected patients compared to healthy or those with non-dengue febrile illness. The highest Gal-9 levels were observed in dengue hemorrhagic fever (DHF) patients (DHF: 2464 pg/ml; dengue fever patients (DF): 1407 pg/ml; non-dengue febrile illness: 616 pg/ml; healthy: 196 pg/ml). In the recovery phase, Gal-9 levels significantly declined from peak levels in DF and DHF patients. Gal-9 levels tracked viral load, and were associated with multiple cytokines and chemokines (IL-1α, IL-8, IP-10, and VEGF), including monocyte frequencies and hematologic variables of coagulation. Further discriminant analyses showed that eotaxin, Gal-9, IFN-α2, and MCP-1 could detect 92% of DHF and 79.3% of DF, specifically (P < 0.01).ConclusionGal-9 appears to track DENV inflammatory responses, and therefore, it could serve as an important novel biomarker of acute DENV infection and disease severity.     

High Producing Tumor Necrosis Factor Alpha Gene Alleles in Protection against Severe Manifestations of Dengue

Dengue virus (DENV) infection usually presents with mild self-limiting dengue fever (DF). Few however, would present with the more severe form of the disease, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In the present study, the association between IL-12B, IL-10 and TNF-α gene polymorphisms and dengue severity was investigated. Methods: A case-control study was performed on a total of 120 unrelated controls, 86 DF patients and 196 DHF/DSS patients. The polymorphisms in IL-12B, IL-10 and TNF-α genes were genotyped using PCR-RFLP and PCR-sequencing methods. Results: A protective association of TNF-α -308A allele and -308GA genotype against DHF/DSS was observed, while TNF-α -238A allele and -238GA genotype were associated with DHF/DSS. A combination of TNF-α -308GA+AA genotype and IL-10 non-GCC haplotypes, IL-12B pro homozygotes (pro1/pro1, pro2/pro2) and IL-12B 3''UTR AC were significantly correlated with protective effects against DHF/DSS. An association between the cytokine gene polymorphisms and protection against the clinical features of severe dengue including thrombocytopenia and increased liver enzymes was observed in this study. Conclusion: The overall findings of the study support the correlation of high-producer TNF-α genotypes combined with low-producer IL-10 haplotypes and IL-12B genotypes in reduced risk of DHF/DSS.     

The pathology of dengue hemorrhagic fever

An estimated 2.5 billion people are at risk of dengue infection, and of the 100 million cases of dengue fever per year, up to 500,000 develop dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), the life-threatening forms of the infection. The large majority of DHF/DSS occurs as the result of a secondary infection with a different serotype of the virus. While not completely understood, there is evidence that the target cells include dendritic reticulum cells, monocytes, lymphocytes, hepatocytes, and vascular endothelial cells. Viral replication appears to occur in dendritic cells, monocytes, and possibly circulating lymphoid cells, and damage to these and other target cells occurs through immune-mediated mechanisms related to cross-reacting antibodies and cytokines released by dendritic cells, monocytes, and vascular endothelium. There is evidence of a concomitant cellular activation as well as immune suppression during the infection. The activation of memory T cells results in cascades of inflammatory cytokines, including tumor necrosis factor-alpha, interleukins (IL-2, IL-6, and IL-8), and other chemical mediators that increase vascular endothelial permeability or trigger death of target cells through apoptosis. Pathological studies in humans are uncommon, and a suitable animal model of DHF/DSS does not exist. The current treatment of DHF/DSS is symptomatic, and prevention is through vector control. As such, there is a great impetus for the development of vaccines and novel therapeutic molecules to impede viral replication in infected cells or counteract the effects of specific inflammatory mediators on target cells. The role of genetics in relation to resistance to DHF/DSS also requires clarification.     

Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT^?/? mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.     

Elevated serum PAR-1 levels as an emerging biomarker of inflammation to predict the dengue infection severity

The present study was designed to check the serum levels of protease-activated receptor (PAR-1) in patients during different phases of dengue severity. Moreover, a correlation between serum PAR-1 levels and hematological parameters, inflammatory cytokine levels, and liver functional changes was also determined. Based on the World Health Organization criteria, the study population was divided into: nonsevere dengue fever (DF; n = 30), severe dengue hemorrhagic fever (DHF; n = 19), and severe dengue shock syndrome (DSS; n = 11). The platelet count (PLT) and hematocrit (HCT) were analyzed using an automated hematology analyzer and liver function enzymes aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphate (ALP), bilirubin were checked by auto-analyzer using diagnostic kits. Moreover, the levels of inflammatory mediators C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-17 (IL-17), and PAR-1 were determined using respective ELISA kits. The HCT levels were elevated and platelet count decreased significantly during dengue complications (DHF and DSS) compared to the DF patients, while the levels of liver functional biomarkers AST, ALT, ALP, and bilirubin remained elevated in DHF and DSS groups than in the corresponding DF group. Similarly, the inflammatory cytokine levels of CRP, TNF-α, IL-6, and IL-17 in DHF and DSS subjects were markedly increased when observed against DF subjects. Notably, the PAR-1 levels were significantly elevated in DHF and DSS groups than in the DF group and positively correlated with changes in HCT levels, inflammatory biomarkers, and liver enzymes. Our findings conclude that PAR-1 levels persistently increased with the severity of the dengue infection and are strongly associated with various clinical manifestations. Thus, PAR-1 levels can be used as a diagnostic marker for assessing dengue severity.     

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Severe acute respiratory syndrome (SARS) is a recently emerged infectious disease caused by a novel coronavirus, but its immunopathological mechanisms have not yet been fully elucidated. We investigated changes in plasma T helper (Th) cell cytokines, inflammatory cytokines and chemokines in 20 patients diagnosed with SARS. Cytokine profile of SARS patients showed marked elevation of Th1 cytokine interferon (IFN)-gamma, inflammatory cytokines interleukin (IL)-1, IL-6 and IL-12 for at least 2 weeks after disease onset, but there was no significant elevation of inflammatory cytokine tumour necrosis factor (TNF)-alpha, anti-inflammatory cytokine IL-10, Th1 cytokine IL-2 and Th2 cytokine IL-4. The chemokine profile demonstrated significant elevation of neutrophil chemokine IL-8, monocyte chemoattractant protein-1 (MCP-1), and Th1 chemokine IFN-gamma-inducible protein-10 (IP-10). Corticosteroid reduced significantly IL-8, MCP-1 and IP-10 concentrations from 5 to 8 days after treatment (all P < 0.001). Together, the elevation of Th1 cytokine IFN-gamma, inflammatory cytokines IL-1, IL-6 and IL-12 and chemokines IL-8, MCP-1 and IP-10 confirmed the activation of Th1 cell-mediated immunity and hyperinnate inflammatory response in SARS through the accumulation of monocytes/macrophages and neutrophils.     

Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection

Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.     

Viral replication and paracrine effects result in distinct, functional responses of dendritic cells following infection with dengue 2 virus

Dengue virus (DENV), a re-emerging arbovirus, readily infects dendritic cells (DC) in culture and in vivo. However, there have been contradictory reports regarding the effect of DENV infection on DC activation and maturation. DC undergo a series of functional changes following exposure to infectious agents, including cytokine production and costimulatory and MHC molecule induction, culminating in stimulation of adaptive immune responses. Immunological memory to primary DENV infection critically influences disease severity during subsequent infections with heterologous serotypes. To explore these phenomena, we examined DENV infection-dependent and -independent effects on DC secretory, phenotypic, and allostimulatory functions. DENV infection of DC resulted in the secretion of a broad array of cytokines and chemokines. Type I IFN produced by DC inhibited propagation of infection and induced the chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10). Based on intracellular cytokine staining, infected DC produced less IP-10 but more TNF-alpha than uninfected bystander cells in the same culture. DENV exposure activated surface molecule expression on infected and bystander cells; infected DC had enhanced programmed death ligand 2 (PD-L2) and MHC II expression but reduced levels of PD-L1, CD80, CD86, and MHC I relative to bystander DC. Dengue-infected DC cultures stimulated resting allogeneic CD4 T cell proliferation, although an increasing multiplicity of infection was associated with decreasing stimulatory capacity of DC. These data demonstrate that functional maturation of DC in response to dengue infection is modified by the presence of virus through IFN-dependent and -independent mechanisms with consequences for the development of adaptive immunity.     

Seminal plasma differentially regulates inflammatory cytokine gene expression in human cervical and vaginal epithelial cells

Exposure to semen elicits an inflammatory response in the female reproductive tract of rodents and other animals. The nature and regulation of any similar response in humans is poorly understood. This study investigated seminal plasma induction of inflammatory cytokine and chemokine gene regulation in human cervical and vaginal epithelial cells in vitro. Affymetrix microarray gene profiling revealed that inflammatory cytokine genes were prevalent among 317 known genes differentially expressed in immortalized ectocervical epithelial (Ect1) cells after incubation with pooled human seminal plasma. A dose- and time-dependent induction by seminal plasma of IL8, IL6, CSF2 and CCL2 mRNA expression in Ect1 cells was verified by quantitative RT-PCR. This was accompanied by increases in Ect1 secretion of immunoactive gene products IL-8, IL-6, GM-CSF and MCP-1. Similar cytokine responses were elicited in primary ectocervical epithelial cells. Endocervical epithelial (End1) and vaginal epithelial (Vk2) cells were less responsive to seminal fluid, with induction of IL-8 and MCP-1, but not GM-CSF or IL-6. In a panel of 10 seminal plasma samples, considerable variation in inflammatory cytokine-inducing activity was evident. These experiments show that seminal plasma can elicit expression of a range of inflammatory cytokines and chemokines in reproductive tract epithelia, and implicate the ectocervix as the primary site of responsiveness, with gene-specific differences in the kinetics and site-restrictedness of the response. Seminal factor regulation of inflammatory cytokines in the cervical epithelium is implicated in controlling the immune response to seminal antigens, and defence against infectious agents introduced at intercourse.     

Induction of cytokine granulocyte-macrophage colony-stimulating factor and chemokine macrophage inflammatory protein 2 mRNAs in macrophages by Legionella pneumophila or Salmonella typhimurium attachment requires different ligand-receptor systems.

The attachment of bacteria to macrophages is mediated by different ligands and receptors and induces various intracellular molecular responses. In the present study, induction of cytokines and chemokines, especially granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 2 (MIP-2), was examined, following bacterial attachment, with regard to the ligand-receptor systems involved. Attachment of Legionella pneumophila or Salmonella typhimurium to cultured mouse peritoneal macrophages increased the steady-state levels of cellular mRNAs for the cytokines interleukin 1beta (IL-1beta), IL-6, and GM-CSF as well as the chemokines MIP-1beta, MIP-2, and KC. However, when macrophages were treated with alpha-methyl-D-mannoside (alphaMM), a competitor of glycopeptide ligands, induction of cytokine mRNAs was inhibited, but the levels of chemokine mRNAs were not. Pretreatment of the bacteria with fresh mouse serum enhanced the level of GM-CSF mRNA but not the level of MIP-2 mRNA. In addition, serum treatment reduced the inhibitory effect of alphaMM on GM-CSF mRNA. These results indicate that bacterial attachment increases the steady-state levels of the cytokine and chemokine mRNAs tested by at least two distinct receptor-ligand systems, namely, one linked to cytokine induction and involving mannose or other sugar residues and the other linked to chemokine induction and relatively alphaMM insensitive. Furthermore, opsonization with serum engages other pathways in the cytokine response which are relatively independent of the alphaMM-sensitive system. Regarding bacterial surface ligands involved in cytokine mRNA induction, evidence is presented that the flagellum may be important in stimulating cytokine GM-CSF message but not chemokine MIP-2 message. Analysis of cytokine GM-CSF and chemokine MIP-2 signaling pathways with protein kinase inhibitors revealed the involvement of calmodulin and myosin light-chain kinase in GM-CSF but not MIP-2 mRNA induction, adding further evidence that several distinct receptor systems are engaged during the process of bacterial attachment and induction of cytokines and chemokines, such as GM-CSF and MIP-2, respectively.     

Cytokine gene expression and protein production in peripheral blood mononuclear cells of children with acute dengue virus infections

Plasma leakage in dengue hemorrhagic fever (DHF) is associated with elevated plasma levels of cytokines. To define further the contribution of immune activation to DHF and the source of cytokines, we analyzed the production of cytokines in peripheral blood mononuclear cells (PBMC) obtained from children with dengue, using RT-PCR and immunostaining. Tumor necrosis factor-alpha (TNF-alpha) and TNF-beta expression was detected in all samples by PCR and in < 50% of samples by immunostaining. Interferon-gamma (IFN-gamma) expression was detected in < 50% of samples by either method. Interleukin-2 (IL-2) and IL-4 expression was detected in a few samples by immunostaining but was not detectable by PCR. We found greater expression of TNF-alpha and IL-4 in DHF than in dengue fever or other (non-dengue) febrile illnesses. These results support the model of immunopathogenesis of DHF. However, low levels of cytokine expression in PBMC suggest that cellular activation in tissues may contribute to high serum cytokine levels in DHF.     

流感病毒诱导小鼠胶质细胞的细胞因子级联反应

目的:探讨流感病毒感染星形胶质细胞后释放的细胞因子是否会诱导正常胶质细胞趋化因子及促炎细胞因子转录水平的变化,从而产生细胞因子级联效应。方法:从新生小鼠大脑皮质分离培养神经胶质细胞,并进一步纯化星形胶质细胞,经纯度鉴定后,用感染复数为2的流感病毒H1N1和H3N2进行体外感染星形胶质细胞,分别于6小时和24小时收获上清,采用超滤分子截留的方法,去除流感病毒颗粒。用不同时间点的条件上清,分别刺激星形胶质细胞和小胶质细胞,24小时后提取RNA并进行反转录,利用Real-Time PCR检测促炎因子TNF-α、IL-1β、IL-6,趋化因子IP-10、MCP-1、MIP-1转录水平的变化。结果:不同时间点的条件上清皆可诱导正常胶质细胞的促炎症因子TNF-α、IL-1β、IL-6和趋化因子IP-10、MCP-1、MIP-1的转录水平发生不同程度的上调,产生细胞因子级联效应。结论:流感病毒H1N1和H3N2感染星形胶质细胞后所引起的细胞因子风暴,可诱导正常胶质细胞的细胞因子转录水平显著上调,引发细胞因子级联反应,其可能与流感病毒感染中枢神经系统所引发的细胞因子风暴及免疫病理损伤存在一定关系。     

IL-1-induced tumor necrosis factor-alpha elicits inflammatory cell infiltration in the skin by inducing IFN-gamma-inducible protein 10 in the elicitation phase of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a typical inflammatory response against contact allergens. Inflammatory cytokines, including IL-1 and tumor necrosis factor (TNF)-alpha, are implicated in the reaction, although the precise roles of each cytokine have not been completely elucidated. In this report, we dissected the functional roles of IL-1 and TNF-alpha during CHS. CHS induced by 2,4,6-trinitorochlorobenzene as well as oxazolone was suppressed in both IL-1alpha/beta(-/-) and TNF-alpha(-/-) mice. Hapten-specific T cell activation, as examined by T cell proliferation, OX40 expression and IL-17 production, was reduced in IL-1alpha/beta(-/-) mice, but not in TNF-alpha(-/-) mice, suggesting that IL-1 but not TNF-alpha is required for hapten-specific T cell priming in the sensitization phase. On the other hand, TNF-alpha, induced by IL-1, was necessary for the induction of local inflammation during the elicitation phase. We also found that the expression of IFN-gamma-inducible protein 10 (IP-10) was augmented at the inflammatory site. Although IP-10 mRNA expression was abrogated in TNF-alpha(-/-) mice, both CHS development and TNF-alpha mRNA expression occurred normally in IFN-gamma(-/-) mice, indicating that the induction of IP-10 during CHS was primarily controlled by TNF-alpha. Interestingly, CHS was suppressed by treatment with anti-IP-10 mAb, suggesting a critical role for IP-10 in CHS. Reduced CHS in TNF-alpha(-/-) mice was reversed by IP-10 injection during the elicitation phase. Thus, it was shown that the roles for IL-1 and TNF-alpha are different, although both cytokines are crucial for the development of CHS.     

Tissue plasminogen activator induced by dengue virus infection of human endothelial cells

Dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) are severe complications of dengue virus (DV) infection. However, the pathogenesis of hemorrhage induced by dengue virus infection is poorly understood. Since endothelial cells play a pivotal role in the regulation of hemostasis, we studied the effect of DV infection on the production of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) in vitro using both primary isolated endothelial cells, human umbilical cord veins cells, and a human microvascular endothelial cell line. DV infection significantly induced the secretion of tPA but not PAI-1 of human endothelial cells. In addition, tPA mRNA of endothelial cells was induced by DV as demonstrated by RT-PCR. Antibody against IL-6 but not control antibody inhibited DV-induced tPA production of endothelial cells. Furthermore, a good correlation between sera levels of IL-6 and tPA was found in DHF but not DF patients. These results suggest that IL-6 can regulate DV-induced tPA production of endothelial cells, which may play important roles in the pathogenic development of DHF/DSS.     

A simple one-step real-time RT-PCR for diagnosis of dengue virus infection

Dengue is the most important arbovirus disease in tropical and sub-tropical countries, and can be caused by infection with any of the four-dengue virus (DENV) serotypes. Infection with DENV can lead to a broad clinical spectrum, ranging from sub-clinical infection or an influenza-like disease known as dengue fever (DF) to a severe, sometimes fatal, disease characterized by hemorrhage and plasma leakage that can lead to shock, known as dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The diagnosis of dengue is routinely accomplished by serologic assays, such as IgM and IgG ELISAs, as well as HI tests, analyzing serum samples obtained from patients with at least 7 days of symptoms onset. These tests cannot be used for diagnosis during the early symptomatic phase. In addition, antibodies against dengue are broad reactive with other flaviviruses. Therefore, a specific diagnostic method for acute DENV infection is of great interest. In that sense, the real-time RT-PCR has become an important tool that can be used for early and specific detection of dengue virus genome in human serum samples. This study describes a simple, specific, and sensitive real-time RT-PCR for early diagnosis of dengue virus infection.     

Interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia

Respiratory infection by Actinobacillus pleuropneumoniae causes a highly pathogenic necrotizing pleuropneumonia with severe edema, hemorrhage and fever. Acute infection is characterized by expression of inflammatory cytokines, including interleukin-1 (IL-1), IL-6 and IL-8. To determine if high level production of inflammatory cytokines contributed to disease pathogenesis, we investigated if inhibiting macrophage activation with adenovirus type 5-expressed IL-10 (Ad-5/IL-10) reduced the severity of acute disease. Porcine tracheal epithelial cells infected with Ad-5/IL-10 produced bioactive human IL-10. When pigs were intratracheally infected with A. pleuropneumoniae, pigs pretreated with Ad-5/IL-10 showed a significant reduction in the amount of lung damage when compared to adenovirus type 5-expressing beta-galactosidase (Ad-5/beta-Gal)-treated and untreated pigs. In addition, serum zinc levels were unchanged, the lung weight/body weight ratio (an indicator of vascular leakage) was significantly reduced, and lung pathology scores were reduced. Myeloperoxidase activity in lung lavage fluid samples, an indicator of neutrophil invasion, was decreased to levels similar to that seen in pigs not infected with A. pleuropneumoniae. Reduction in inflammatory cytokine levels in lung lavage fluid samples correlated with the clinical observations in that pigs pretreated with Ad-5/IL-10 showed a corresponding reduction of IL-1 and tumor necrosis factor (TNF) compared with untreated and Ad-5/beta-Gal-treated pigs. IL-6 levels were unaffected by pretreatment with Ad-5/IL-10, consistent with observations that IL-6 was not derived from alveolar macrophages. Since inflammatory cytokines are expressed at high levels in acute bacterial pleuropneumonia, these results indicate that macrophage activation, involving overproduction of IL-1 and TNF, is a prime factor in infection-related cases of massive lung injury.     

Development of a new technique for recovery of cytokines from inflammatory sites in situ.

We attempted to recover cytokines from nasal mucosal surface following allergen challenge. Repeated lavage of nasal mucosa of seven allergic patients was done, but we failed to detect IL-1 beta in the lavage samples even in ten-fold concentrated materials. Therefore, we developed a new technique to recover cytokines using filter strips. Small filter strips were placed on nasal turbinates for 10 min at different time points after allergen challenge. The strips were air-dried, and stored. For recovery of cytokines individual strips were washed with small volumes of Hepes buffer containing 0.3% human serum albumin. Eluates were assayed for the presence of IL-1 beta and GM-CSF using commercially available ELISA. We were able to detect IL-1 beta and GM-CSF in eluates. Both cytokines were consistently detectable in the late phase allergic reaction peaking at 5 h. Nasal challenge with saline failed to detect any cytokine during the 7 h observation period. In standardization experiments known quantities of IL-1 beta and GM-CSF were applied to filter strips and the recovery ranged from 67 to 89%. Thus, we developed a simple technique of recovery of cytokines from inflammatory mucosa in situ.