Chronic airway infection/inflammation induces a Ca2+i-dependent hyperinflammatory response in human cystic fibrosis airway epithelia

Hyperinflammatory responses to infection have been postulated as a component of cystic fibrosis (CF) lung disease. Studies have linked intracellular calcium (Ca(2+)(i)) mobilization with inflammatory responses in several systems. We have reported that the pro-inflammatory mediator bradykinin (BK) promotes larger Ca(2+)(i) signals in CF compared with normal bronchial epithelia, a response that reflects endoplasmic reticulum (ER)/Ca(2+) store expansion induced by chronic luminal airway infection/inflammation. The present study investigated whether CF airway epithelia were hyperinflammatory and, if so, whether the hyperinflammatory CF phenotype was linked to larger Ca(2+) stores in the ER. We found that DeltaF508 CF bronchial epithelia were hyperinflammatory as defined by an increased basal and mucosal BK-induced interleukin (IL)-8 secretion. However, the CF hyperinflammation expressed in short-term (6-11-day-old) primary cultures of DeltaF508 bronchial epithelia was lost in long-term (30-40-day-old) primary cultures of DeltaF508 bronchial epithelia, indicating this response was independent of mutant cystic fibrosis transmembrane conductance regulator. Exposure of 30-40-day-old cultures of normal airway epithelia to supernatant from mucopurulent material (SMM) from CF airways reproduced the increased basal and mucosal BK-stimulated IL-8 secretion of short-term CF cultures. The BK-triggered increased IL-8 secretion in SMM-treated cultures was mediated by an increased Ca(2+)(i) mobilization consequent to an ER expansion associated with increases in protein synthesis (total, cytokines, and antimicrobial factors). The increased ER-dependent, Ca(2+)(i)-mediated hyperinflammatory epithelial response may represent a general beneficial airway epithelial adaptation to transient luminal infection. However, in CF airways, the Ca(2+)(i)-mediated hyperinflammation may be ineffective in promoting the eradication of infection in thickened mucus and, consequently, may have adverse effects in the lung.     

Innate immune response to viral infection

In viral infections the host innate immune system is meant to act as a first line defense to prevent viral invasion or replication before more specific protection by the adaptive immune system is generated. In the innate immune response, pattern recognition receptors (PRRs) are engaged to detect specific viral components such as viral RNA or DNA or viral intermediate products and to induce type I interferons (IFNs) and other pro-inflammatory cytokines in the infected cells and other immune cells. Recently these innate immune receptors and their unique downstream pathways have been identified. Here, we summarize their roles in the innate immune response to virus infection, discrimination between self and viral nucleic acids and inhibition by virulent factors and provide some recent advances in the coordination between innate and adaptive immune activation.     

Role of epithelial nitric oxide in airway viral infection

The airway mucosal epithelium is the first site of virus contact with the host, and the main site of infection and inflammation. Nitric oxide (NO) produced by the airway epithelium is vital to antiviral inflammatory and immune defense in the lung. Multiple mechanisms function coordinately to support high-level basal NO synthesis in healthy airway epithelium and further induction of NO synthesis in the infected airway of normal hosts. Hosts deficient in NO synthesis, such as those patients with cystic fibrosis, have impaired antiviral defense and may benefit from therapies to augment NO levels in the airways.     

Pendrin function in airway epithelia

The expression and function of the anion exchanger pendrin (SLC26A4) was thought to be limited mainly to the inner ear, kidney and thyroid. Recent data indicates that pendrin is also expressed in the bronchial epithelium following exposure to the T(H)2-type cytokines, interleukin (IL)-4 and IL-13. Expression of the transporter is also upregulated in bronchial asthma and chronic obstructive pulmonary disease. Both diseases involve respiratory inflammation leading to tissue destruction/remodeling and decreased airway function, and data so far indicate that increased pendrin expression and/or activity might contribute to their pathogenesis. In this review, we summarize data that have emerged within the past years aimed at revealing the role for pendrin in the airway epithelia.     

Eicosanoids mediate Galleria mellonella cellular immune response to viral infection

Nodulation is the predominant insect cellular immune response to bacterial and fungal infections and it can also be induced by some viral infections. Treating seventh instar larvae of greater wax moth Galleria mellonella with Bovine herpes simplex virus-1 (BHSV-1) induced nodulation reactions in a dose-dependent manner. Because eicosanoids mediate nodulation reactions to bacterial and fungal infection, we hypothesized that eicosanoids also mediate nodulation reactions to viral challenge. To test this idea, we injected G. mellonella larvae with indomethacin, a nonsteroidal anti-inflammatory drug immediately prior to intrahemocoelic injection of BHSV-1. Relative to vehicle-treated controls, indomethacin-treated larvae produced significantly reduced numbers of nodules following viral infection (down from approximately 190 nodules/larva to <50 nodules/larva). In addition to injection treatments, increasing dietary indomethacin dosages (from 0.01% to 1%) were associated with decreasing nodulation (by 10-fold) and phenoloxidase activity (by 3-fold) reactions to BHSV-1 injection. We infer from these findings that cyclooxygenase products, prostaglandins, mediate nodulation response to viral infection in G. mellonella.     

Influence of influenza viral infection on airway smooth muscle activity

The contractility of airway smooth muscle (ASM) plays an important role in pathophysiology of several bronchial disorders. Increased contraction of ASM during asthma and respiratory viral infection has been attributed to the release of mediators acting through different receptors. In the present study, influence of influenza type A virus (H1N1) infection has been examined on ASM responsiveness to various bronchoactive agents e.g. adenosine, histamine, 5-hydroxytryptamine (5-HT) and isoproterenol in an organ bath set up for isolated tissue preparation. The contractile effect of adenosine, histamine and 5-HT was enhanced, however, relaxant response of isoproterenol was attenuated with the duration following viral exposure. The most prominent response was observed 48 to 72 hr after infection and tissues from multiple exposure to virus infected animals showed the maximum contractile response. Results demonstrated the deleterious effect of viral infection on ASM function and the findings will be helpful in understanding the mechanism of influenza virus induced bronchoconstriction.     

Severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 as an important cause of severe lower respiratory tract infection in humans, and in vitro models of the lung are needed to elucidate cellular targets and the consequences of viral infection. The SARS-CoV receptor, human angiotensin 1-converting enzyme 2 (hACE2), was detected in ciliated airway epithelial cells of human airway tissues derived from nasal or tracheobronchial regions, suggesting that SARS-CoV may infect the proximal airways. To assess infectivity in an in vitro model of human ciliated airway epithelia (HAE) derived from nasal and tracheobronchial airway regions, we generated recombinant SARS-CoV by deletion of open reading frame 7a/7b (ORF7a/7b) and insertion of the green fluorescent protein (GFP), resulting in SARS-CoV GFP. SARS-CoV GFP replicated to titers similar to those of wild-type viruses in cell lines. SARS-CoV specifically infected HAE via the apical surface and replicated to titers of 10(7) PFU/ml by 48 h postinfection. Polyclonal antisera directed against hACE2 blocked virus infection and replication, suggesting that hACE2 is the primary receptor for SARS-CoV infection of HAE. SARS-CoV structural proteins and virions localized to ciliated epithelial cells. Infection was highly cytolytic, as infected ciliated cells were necrotic and shed over time onto the luminal surface of the epithelium. SARS-CoV GFP also replicated to a lesser extent in ciliated cell cultures derived from hamster or rhesus monkey airways. Efficient SARS-CoV infection of ciliated cells in HAE provides a useful in vitro model of human lung origin to study characteristics of SARS-CoV replication and pathogenesis.     

Basolateral chloride current in human airway epithelia

Electrolyte transport by airway epithelia regulates the quantity and composition of liquid covering the airways. Previous data indicate that airway epithelia can absorb NaCl. At the apical membrane, cystic fibrosis transmembrane conductance regulator (CFTR) provides a pathway for Cl(-) absorption. However, the pathways for basolateral Cl(-) exit are not well understood. Earlier studies, predominantly in cell lines, have reported that the basolateral membrane contains a Cl(-) conductance. However, the properties have varied substantially in different epithelia. To better understand the basolateral Cl(-) conductance in airway epithelia, we studied primary cultures of well-differentiated human airway epithelia. The basolateral membrane contained a Cl(-) current that was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The current-voltage relationship was nearly linear, and the halide selectivity was Cl(-) > Br(-) > I(-). Several signaling pathways increased the current, including elevation of cellular levels of cAMP, activation of protein kinase C (PKC), and reduction of pH. In contrast, increasing cell Ca(2+) and inducing cell swelling had no effect. The basolateral Cl(-) current was present in both cystic fibrosis (CF) and non-CF airway epithelia. Likewise, airway epithelia from wild-type mice and mice with disrupted genes for ClC-2 or ClC-3 all showed similar Cl(-) currents. These data suggest that the basolateral membrane of airway epithelia possesses a Cl(-) conductance that is not due to CFTR, ClC-2, or ClC-3. Its regulation by cAMP and PKC signaling pathways suggests that coordinated regulation of Cl(-) conductance in both apical and basolateral membranes may be important in controlling transepithelial Cl(-) movement.     

Histidine phosphorylation of annexin I in airway epithelia

Although [Cl(-)](i) regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe(3+)-Sepharose) and anion exchange (POROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca(2+)-dependent phospholipid-binding proteins. Phosphate on [(32)P]annexin I, phosphorylated using both [gamma-(32)P]ATP and [gamma-(32)P]GTP, was labile under acidic but not alkaline conditions. Phosphoamino acid analysis showed the presence of phosphohistidine. The site of phosphorylation was localized to a carboxyl-terminal fragment of annexin I. Our data suggest that cAMP and AMP (but not cGMP) may regulate annexin I histidine phosphorylation. We propose a role for annexin I in an intracellular signaling system involving histidine phosphorylation.     

Humoral antibody response to individual viral proteins after murine cytomegalovirus infection

The purpose of this study was to identify viral proteins that played an important role in the humoral immune response to murine cytomegalovirus (MCMV). Viral proteins were separated from a purified virus preparation on polyacrylamide gels, were blotted onto nitrocellulose strips, and were reacted with antisera collected from mice on various days post infection. No antibody response was detected in serum obtained 5 days post infection, but by 10 days there was a faint response to five different proteins. Thereafter, the number of proteins eliciting an antibody response, as well as the intensity of the response, increased with time so that by 42 days post infection a response to 13 major antigens was detected. This method provides a means of separating out important immunogens from the more than 30 different MCMV proteins originally identified by polyacrylamide gel electrophoresis. Such information may improve our understanding of the pathogenesis of MCMV infection as well as host immune responses to the virus.     

Cytotoxic macrophages: a rapid nonspecific response to viral infection.

In vitro and in vivo assays have been developed to study the relative contributions of various types of immune cytolysis in the destruction of infected cells after Semliki Forest virus infection of BALB/c mice. Highly cytotoxic activated macrophages, not specific for the infecting virus, appear on day 1, peak on day 2 to 3, and disappear within a week. Specifically sensitized T cells appear around day 3, peak on day 6, and disappear within a month. Cytotoxic antibody appears on day 4 and reaches high titers by day 8. Immune spleen cells greatly reduce the yield of virus from cultured cells. Infected cells rapidly disappear after transfer to infected animals.     

Melon phloem-sap proteome: developmental control and response to viral infection

In addition to small molecules such as sugars and amino acids, phloem sap contains macromolecules, including mRNA and proteins. It is generally assumed that all molecules in the phloem sap are on the move from source to sink, but recent evidence suggests that the macromolecules' direction of movement can be controlled by endogenous plant mechanisms. To test the hypothesis that the phloem-sap protein profile is affected by local metabolic activities, we analyzed the phloem-sap proteome in young and mature tissues of melon plants. We also examined the effect of cucumber mosaic virus (CMV) infection and expression of CMV movement protein in transgenic melon plants on the phloem protein profile. Sap collected from cut sections of young stems or petioles contained specific proteins that were absent from sap collected from mature stems or petioles. Most of these proteins were involved in defense response and protection from oxidative stress, suggesting that they play a role in maintaining safe activity of the sieve tubes in young tissues. Phloem sap collected from CMV-infected plants and transgenic plants expressing the CMV movement protein contained only a few additional proteins with molecular masses of 18 to 75 kDa. Here again, most of the additional proteins were associated with stress responses. Our study indicated that the proteome of phloem sap is dynamic and under developmental control. Entry and exit of proteins from the sieve tube can be regulated at the tissue level. Moreover, the plant can maintain regulation of protein trafficking from companion cells to sieve elements under viral infection or other perturbations in plasmodesmal function.     

Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of airway epithelia

Recent identification of two receptors for the adenovirus fiber protein, coxsackie B and adenovirus type 2 and 5 receptor (CAR), and the major histocompatibility complex (MHC) Class I alpha-2 domain allows the molecular basis of adenoviral infection to be investigated. Earlier work has shown that human airway epithelia are resistant to infection by adenovirus. Therefore, we examined the expression and localization of CAR and MHC Class I in an in vitro model of well differentiated, ciliated human airway epithelia. We found that airway epithelia express CAR and MHC Class I. However, neither receptor was present in the apical membrane; instead, both were polarized to the basolateral membrane. These findings explain the relative resistance to adenovirus infection from the apical surface. In contrast, when the virus was applied to the basolateral surface, gene transfer was much more efficient because of an interaction of adenovirus fiber with its receptors. In addition, when the integrity of the tight junctions was transiently disrupted, apically applied adenovirus gained access to the basolateral surface and enhanced gene transfer. These data suggest that the receptors required for efficient infection are not available on the apical surface, and interventions that allow access to the basolateral space where fiber receptors are located increase gene transfer efficiency.     

Latent adenoviral infection induces production of growth factors relevant to airway remodeling in COPD

Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD). The present study focuses on how the adenoviral E1A gene could alter expression of growth factors by human bronchial epithelial (HBE) cells. The data show that connective tissue growth factor (CTGF) and transforming growth factor (TGF)-beta 1 mRNA and protein expression were upregulated in E1A-positive HBE cells. Upregulation of CTGF in this in vitro model was independent of TGF-beta secreted into the growth medium. Comparison of E1A-positive with E1A-negative HBE cells showed that both expressed cytokeratin but only E1A-positive cells expressed the mesenchymal markers vimentin and alpha-smooth muscle actin. We conclude that latent infection of epithelial cells by adenovirus E1A could contribute to airway remodeling in COPD by the viral E1A gene, inducing TGF-beta 1 and CTGF expression and shifting cells to a more mesenchymal phenotype.     

Adenosine promotes IL-6 release in airway epithelia

In the airway epithelia, extracellular adenosine modulates a number of biological processes. However, little is known about adenosine's role in the inflammatory responses of airway epithelial cells. Recent studies suggest that the chronic elevation of extracellular adenosine in mice leads to pulmonary inflammation and fibrosis. Yet, the underlying molecular mechanism has not been well understood and little attention has been paid to the role of airway epithelia in adenosine-triggered inflammation. In the present work, we examined the role of adenosine in releasing IL-6 from airway epithelia. In Calu-3 human airway epithelial cells, apical but not basolateral adenosine elicited robust, apically polarized release of IL-6, along with proinflammatory IL-8. Both protein kinase A and protein kinase C mediated the adenosine-induced IL-6 release, at least partly via phosphorylation of CREB. Protein kinase C appeared to phosphorylate CREB through activating ERK. In addition, A2A but not A2B adenosine receptors were specifically required for the adenosine-induced IL-6 release. Furthermore, in rat bronchoalveolar lavage fluid, adenosine triggered the release of IL-6 as well as proinflammatory IL-1beta. Adenosine also mediated the release of a considerable portion of the LPS-induced IL-6 in rat bronchoalveolar lavage fluid. Our findings provide a possible molecular link between extracellular adenosine elevation and lung inflammation and fibrosis.     

Altered response to viral infection by tobacco plants perturbed in ubiquitin system.

Tobacco plants can be perturbed in their ubiquitin system by expression of a ubiquitin variant with a lys to arg change at position 48. Plants expressing this ubiquitin variant have a tendency to form necrotic lesions resembling those occurring as defense reactions against some pathogens. Using the interaction between tobacco and tobacco mosaic virus (TMV), it was investigated whether the ubiquitin variant influences plant responses to pathogen infection. Expression of the ubiquitin variant leads to changes in the response of both resistant and susceptible tobacco varieties. Resistant plants form fewer, but darker lesions. In sensitive plants, TMV replication is inhibited, but not completely abolished. Moreover, susceptible plants expressing the ubiquitin variant induce PR-1 mRNA in response to virus, whereas controls do not express PR-1 under these conditions.