Macrophage inflammatory protein-1alpha (not T helper type 2 cytokines) is associated with severe forms of respiratory syncytial virus bronchiolitis.

It has been suggested that the pathogenesis of respiratory syncytial virus (RSV) infection is related to the development of T helper (Th) type 2 cytokine responses. The presence of Th1 and Th2 cytokines and the chemokines macrophage inflammatory protein (MIP)-1alpha and monocyte chemotactic protein (MCP)-1 were assessed by ELISA in nasopharyngeal secretions of infants with RSV infection. Infants with mild bronchiolitis had increased Th1 cytokines and reduced Th2 cytokines, compared with infants with upper respiratory tract illness alone. Severe bronchiolitis was characterized by a more balanced Th1-Th2 response that did not differ from that of infants with upper respiratory tract illness alone. In contrast, MIP-1alpha was markedly increased in infants with severe bronchiolitis. MIP-1alpha and MCP-1 levels also were inversely related to oxygen saturation (P<.005). Thus, the severity of RSV bronchiolitis appears to be related more to chemokine release than to Th2 cytokine production.

Mucosal immunity and viral infections.

The mucosal surfaces are the first portals of entry for most infectious agents, among which respiratory and intestinal viruses are of greatest epidemiological importance. To combat these infections, the immune system uses unspecific and specific mechanisms. Unspecific responses include the production of virus-induced cytokines, such as type 1 interferons and natural killer (NK) cell activity, while specific immune responses mainly depend on cytotoxic T cells, which are important especially in the early course of a viral infection, and on antibodies. At the mucosal sites, antiviral secretory IgA antibodies play a major role in clearing viral infections and preventing or modifying disease after re-exposure. Passive transfer of virus-specific antibodies has been used in experimental and clinical settings to prevent or treat viral mucosal infections. In the future, the development of new mucosal vaccines promises to have the strongest impact on the epidemiology of viral infections.

Vaccination strategies for mucosal immune responses.

Mucosal administration of vaccines is an important approach to the induction of appropriate immune responses to microbial and other environmental antigens in systemic sites and peripheral blood as well as in most external mucosal surfaces. The development of specific antibody- or T-cell-mediated immunologic responses and the induction of mucosally induced systemic immunologic hyporesponsiveness (oral or mucosal tolerance) depend on complex sets of immunologic events, including the nature of the antigenic stimulation of specialized lymphoid structures in the host, antigen-induced activation of different populations of regulatory T cells (Th1 versus Th2), and the expression of proinflammatory and immunoregulatory cytokines. Availability of mucosal vaccines will provide a painless approach to deliver large numbers of vaccine antigens for human immunization. Currently, an average infant will receive 20 to 25 percutaneous injections for vaccination against different childhood infections by 18 months of age. It should be possible to develop for human use effective, nonliving, recombinant, replicating, transgenic, and microbial vector- or plant-based mucosal vaccines to prevent infections. Based on the experience with many dietary antigens, it is also possible to manipulate the mucosal immune system to induce systemic tolerance against environmental, dietary, and possibly other autoantigens associated with allergic and autoimmune disorders. Mucosal immunity offers new strategies to induce protective immune responses against a variety of infectious agents. Such immunization may also provide new prophylactic or therapeutic avenues in the control of autoimmune diseases in humans.

Mucosal responses to parenteral and mucosal vaccines.

Most human pathogens are acquired through mucosal portals of entry, and replicate in the mucosal tissues. Subsequently, the infecting agent may invade the blood stream and produce disease at distant systemic sites. However, a large number of pathogenic organisms are limited to development of disease only at the site of initial mucosal replication. Studies carried out with naturally acquired infections and mucosally delivered vaccines have provided strong evidence for the existence of a common mucosal immune system in the organized lymphoid follicles in respiratory and intestinal epithelium, and in the mucosa of genital tract, mammary glands, conjunctiva, upper airways, and the middle ear cavity. Mucosal application of live attenuated oral poliovaccine (OPV), rubella virus vaccine (RA 27/3), adenoviruses, influenza A virus, rotavirus, salmonella, and cholera vaccines have demonstrated consistent development of secretory IgA, serum antibody, and cellular immune responses. Mucosal immunization appears to result in preferential expression of several integrins and cell adhesion molecules associated with homing of lymphocytes to mucosal sites of immunization. Induction of mucosal immune responses often result in specific protection against reinfection challenge and against illness. Replicating agents introduced via the parenteral route also result in the development of mucosal responses and protection against systemic illness. Parenteral immunization with non-replicating agents often fails to induce specific mucosal responses. Such immunization, however, is quite effective in mounting high levels of serum antibody with development of protection against systemic illness. Parenteral vaccines, such as enhanced potency inactivated polio vaccine (eIPV), Haemophilus influenzae type B (HIB), hepatitis B virus (HBV), and other non-mucosal vaccines, have been highly effective in preventing systemic disease during subsequent exposure to natural infection. Recent evidence has shown that parenteral immunization can also be quite effective in inducing varying degrees of functional mucosal antibody responses as detected by ELISA and less frequently by neutralization. Systemic illnesses such as poliomyelitis and Haemophilus influenzae meningitis and community circulation of these agents has been eliminated or significantly limited in many parts of the world with the exclusive use of inactivated vaccines. Based on these observations, it is suggested that development of serum immunological responses are effective in the prevention of systemic disease regardless of the types of vaccines or route of their administration. However, induction of pathogen-specific antibody or cellular immunity at the mucosal sites is best elicited by mucosal application of the antigen.

The role of the invariant chain in mucosal immunity.

The invariant chain (Ii) due to its intimate association with major histocompatibility complex (MHC) alpha and beta chains is a determining element in the development of immune responses. Ii plays a major role in the assembly, the intracellular transport and peptide selection by class II MHC. A segment of Ii designated as CLIP (class II-associated Ii peptide) binds into the antigen binding site of class II MHC molecules until class II MHC reach intracellular compartments that contain peptides from internalized antigens. This association limits the self endogenous peptides that can bind to class II MHC molecules. The removal of CLIP from class II MHC catalyzes the binding of antigenic peptides and their subsequent cell surface expression. An isoform of Ii, known as chondroitin sulfate-modified Ii (IiCS), that is surface-expressed enhances T cell activation while acting as a coreceptor for CD44. The expression of class II MHC molecules by mucosal epithelial cells has generated interest in the role that these cells may have in mucosal immunity. Since in classical antigen-presenting cells (APC) the biology of class II MHC is regulated by Ii, it is necessary to bring into perspective the known functions of Ii in conventional APC to understand the role that Ii may play in mucosal epithelial cells as potential regulators of local immune responses.

Rotavirus infection of cultured intestinal epithelial cells induces secretion of CXC and CC chemokines.

BACKGROUND & AIMS: Rotaviruses are the major cause of pediatric gastroenteritis worldwide. The target cell of rotavirus infection is the mature enterocyte of the small intestine. Recently, intestinal epithelial cells have been shown to produce chemoattractant mediators in response to cytokine stimulation and bacterial infection, suggesting a potentially important role of epithelial cells in initiating immune responses. In this study, the production of chemokines by cultured intestinal epithelial cells after rotavirus infection was investigated. METHODS: Two human intestinal epithelial cell lines (HT29 and Caco-2) were infected with sucrose-purified rotavirus (strain SA114F) and assayed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay for chemokine expression. Virus-like particles and inactivated rotavirus were used to test the importance of viral attachment and replication. RESULTS: Increased messenger RNA expression and secretion of immunoreactive interleukin 8, growth-related peptide alpha, and RANTES (regulated upon activation, normal T cell expressed and secreted) were detected in rotavirus-infected cells. Chemokine production was time and dose dependent and required viral replication. CONCLUSIONS: Rotavirus infection induces the expression of a subset of chemokines in intestinal epithelial cells. These data support the hypothesis that chemokine secretion by enterocytes may play a role in the initiation and modulation of the immune response to rotavirus infection.

Cell-specific expression of RANTES, MCP-1, and MIP-1alpha by lower airway epithelial cells and eosinophils infected with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the major cause of acute bronchiolitis in infancy, a syndrome characterized by wheezing, respiratory distress, and the pathologic findings of peribronchial mononuclear cell infiltration and release of inflammatory mediators by basophil and eosinophil leukocytes. Composition and activation of this cellular response are thought to rely on the discrete target cell selectivity of C-C chemokines. We demonstrate that infection in vitro of human epithelial cells of the lower respiratory tract by RSV induced dose- and time-dependent increases in mRNA and protein secretion for RANTES (regulated upon activation, normal T-cell expressed and presumably secreted), monocyte chemotactic protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha). Production of MCP-1 and MIP-1alpha was selectively localized only in epithelial cells of the small airways and lung. Exposure of epithelial cells to gamma interferon (IFN-gamma), in combination with RSV infection, induced a significant increase in RANTES production that was synergistic with respect to that obtained by RSV infection or IFN-gamma treatment alone. Epithelial cell-derived chemokines exhibited a strong chemotactic activity for normal human blood eosinophils. Furthermore, eosinophils were susceptible to RSV and released RANTES and MIP-1alpha as a result of infection. Therefore, the inflammatory process in RSV-induced bronchiolitis appears to be triggered by the infection of epithelial cells and further amplified via mechanisms driven by IFN-gamma and by the secretion of eosinophil chemokines.

The major component of IkappaBalpha proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells.

Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IkappaB inhibitor and can be released only following IkappaB degradation. In this study, we examined the spectrum of IkappaB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-alpha), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IkappaB inhibitory subunits IkappaBalpha, IkappaBbeta, and p105; however, following either stimulus, only the IkappaBalpha and IkappaBbeta steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IkappaBalpha in control cells was shortened to 5 min in TNF-alpha-stimulated cells and to 90 min in pRSV-infected cells. Although IkappaBalpha was resynthesized within 30 min following recombinant human TNFalpha treatment due to a robust 25-fold increase of IkappaBalpha mRNA expression (the RelA:IkappaBalpha positive feedback loop), following pRSV infection, there was no reaccumulation of IkappaBalpha protein, as infected cells produced only a 3-fold increase in IkappaBalpha mRNA at 24 h, indicating the RelA:IkappaBalpha positive feedback loop was insufficient to restore control IkappaBalpha levels. IkappaBalpha proteolysis induced by TNF-alpha occurred through the 26S proteasome, as both 26S proteasome activity and IkappaBalpha proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLF-CHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90% inhibited by the proteasome inhibitors; surprisingly, however, IkappaBalpha proteolysis was not. We conclude that RSV infection produces IkappaBalpha proteolysis through a mechanism primarily independent of the proteasome pathway.

Respiratory syncytial virus-infected pulmonary epithelial cells induce eosinophil degranulation by a CD18-mediated mechanism.

Respiratory syncytial virus (RSV)-induced bronchiolitis in infants is characterized by wheezing, respiratory distress, and the histologic findings of necrosis and sloughing of airway epithelium. High concentrations of eosinophil cationic protein (ECP), a cytotoxic protein contained in the granules of eosinophils, have been found in the airways of RSV-infected infants. The mechanisms of eosinophil degranulation in vivo remain largely unknown. Since RSV-infected respiratory epithelial cells are a rich source of cytokines with eosinophil-activating properties, our studies were designed to mimic in vitro the interaction between RSV, pulmonary epithelial cells (A549), and eosinophils in the airway mucosa. We report in this work that, in the absence of epithelial cells, neither RSV, in the form of purified virions, nor UV-irradiated culture supernatant of RSV-infected epithelial cells (RSV-CM) induced eosinophil degranulation. On the other hand, eosinophils released significant amount of ECP when cultured with RSV-infected A549 cells. Uninfected A549 cells, which failed to induce eosinophil degranulation, were equally effective in triggering ECP release if they were cultured with eosinophils in the presence of RSV-CM. Although RSV-CM induced the up-regulation of the beta2 integrin CD11b on eosinophils and the expression of ICAM-1 on A549 cells, release of ECP was inhibited significantly by anti-CD18 mAb, but not by anti-ICAM-1 mAb. These results suggest a novel mechanism by which respiratory viruses may trigger the detrimental release of eosinophil granule proteins in the airway mucosa.

Enhanced cytokine production by milk macrophages following infection with respiratory syncytial virus.

Paired samples of milk and serum collected 3 days postpartum from 20 women were tested for the presence and level of interleukin (IL)-1, IL-6, IL-12, tumor necrosis factor alpha (TNF-alpha), and interferon-gamma (IFN-gamma) by enzyme immunoassay. The expression of these cytokine mRNAs in milk macrophages from eight donors were semiquantitatively analyzed by reverse transcriptase-polymerase chain reaction. The effects of respiratory syncytial virus (RSV) infection on cytokine production were determined in five samples of milk macrophages. Over 90% of the milk samples tested exhibited detectable levels of IL-1beta, IL-6, and TNF-alpha. No IL-12 or IFN-gamma activity was detected in the milk. IL-6 activity was weakly detected in about 45%, and TNF-alpha activity in about 10% of the serum samples tested. However, no IL-1beta, IL-12, or IFN-gamma activity was demonstrated in any of the serum samples. Milk macrophages from eight subjects all exhibited mRNA for IL-1beta, TNF-alpha, and IL-6, and IFN-gamma mRNA in six of eight subjects, although no IFN-gamma was detected in any of the 20 samples of milk tested. RSV exposure resulted in a 2- to 100-fold increase in the expression of IL-1beta, IL-6, and TNF-alpha mRNA as well as cytokine protein. Although RSV infection enhanced the expression of IFN-gamma mRNA, no detectable IFN-gamma was produced by the milk macrophages. These observations suggest that the milk macrophages are actively engaged in the physiological production of IL-1beta, IL-6, TNF-alpha, and IFN-gamma in the mammary gland and continue to possess the capacity to increase production of these cytokines in response to RSV and possibly other viral infections.

Respiratory syncytial virus induces selective production of the chemokine RANTES by upper airway epithelial cells.

The presence of histamine and eosinophil cationic protein in nasopharyngeal secretions of infants with respiratory syncytial virus (RSV)-induced bronchiolitis implies the activation of basophil and eosinophil leukocytes, but the specific mechanism of their recruitment has not been elucidated. Chemokines are potent and selective leukocyte chemotactic molecules that are also expressed by airway epithelial cells. Therefore, the pattern of chemokines produced in response to RSV infection was investigated in primary cultures of human nose- and adenoid-derived epithelial cells. Interleukin-8, growth-related peptide-alpha, and monocyte chemotactic protein-1 were constitutively released by uninfected epithelial cells and were not further enhanced by infection with RSV. RANTES (regulated upon activation, normal T cell-expressed and -secreted), which was present in negligible concentrations in uninfected cultures, was strongly induced by RSV infection, in a dose- and time-dependent manner. Through the release of RANTES, epithelial cells may control the selective concentration and activation of basophils and eosinophils in RSV-infected airway mucosa.

Antigen presentation of mucosal pathogens: the players and the rules.

A vast number of infectious pathogens gain entry into the host through mucosal surfaces, which have a much greater total surface area than the skin. Since the mucosa is continuously exposed to those pathogens, the development of an effective local immune response is of utmost importance. An obligatory step in the development of most immune responses is the presentation of antigens by specialized accessory cells, termed antigen-presenting cells (APC) to T lymphocytes. The recognition of antigens by T cells is largely determined by how the antigens are handled by the APC. Complex antigen-processing events generate a selected set of peptides which ultimately become associated with MHC molecules. The type of MHC molecules that bind the peptides in turn determine what T lymphocyte subset recognizes the peptides. Thus, an understanding of the molecular and cellular processes preceding the T cell recognition event is a prerequisite for understanding how mucosal immune responses develop, as well as for investigating alternative approaches to vaccine development and therapeutic strategies to control autoimmune diseases. This review discusses the cell biology of antigen processing and how various APC populations may participate in mucosal responses.

Transcriptional activation of the interleukin-8 gene by respiratory syncytial virus infection in alveolar epithelial cells: nuclear translocation of the RelA transcription factor as a mechanism producing airway mucosal inflammation.

The most common cause of epidemic pediatric respiratory disease, respiratory syncytial virus (RSV), stimulates interleukin-8 (IL-8) synthesis upon infecting airway epithelium, an event necessary for the development of mucosal inflammation. We investigated the mechanism for enhanced IL-8 production in human A549 type II pulmonary epithelial cells. Infection with sucrose-purified RSV (pRSV) produced a time-dependent increase in the transcriptional initiation rate of the IL-8 gene. Transient transfection of the human IL-8 promoter mutated in the binding site for nuclear factor-kappaB (NF-kappaB) demonstrated that this sequence was essential for pRSV-activated transcription. Gel mobility shift assays demonstrated pRSV induction of sequence-specific binding complexes; these complexes were supershifted only by antibodies directed to the potent NF-kappaB transactivating subunit RelA. Both Western immunoblot and indirect immunofluorescence assays showed that cytoplasmic RelA in uninfected cells became localized to the nucleus after pRSV infection. RelA activation requires replicating RSV, because neither conditioned medium nor UV-inactivated pRSV was able to stimulate its translocation. We conclude that RelA undergoes changes in subcellular distribution in airway epithelial cells upon pRSV infection. The ability of replicating RSV to activate RelA translocation may play an important role in activating IL-8 and other inflammatory gene products necessary for airway mucosal inflammation seen in RSV disease.

Respiratory syncytial virus infection of human respiratory epithelial cells up-regulates class I MHC expression through the induction of IFN-beta and IL-1 alpha.

CD8+ T cells mediate some of the damage to the lung epithelium following respiratory syncytial virus (RSV) infection. Since CD8+ T cells recognize antigen-laden class I MHC molecules on the target cells, we examined in this study the expression of class I MHC by RSV-infected respiratory epithelial cells. Respiratory epithelial cell lines and bronchial epithelial cells from normal human tissue responded to RSV infection with an increased expression of class I MHC as determined by flow cytometry and immunoprecipitation of class I MHC from metabolically radiolabeled cells. The increase in class I MHC expression was dependent on infectious, replicating virus. UV-irradiated culture supernatants from RSV-infected A549 cells, when added to fresh A549 cell cultures, induced an increase in class I MHC expression by those cells. The class I MHC increasing activity within supernatants from A549 cells was due, in large part, to IFN-beta, and to a lesser extent to IL-1 alpha. The addition of neutralizing Abs to both cytokines completely blocked the increase in class I MHC expression by cells treated with the above-mentioned supernatants. These results demonstrate that RSV infection elicits IFN-beta production by respiratory epithelial cells, which in turn leads to an increase in their synthesis of class I MHC, which would facilitate their recognition and lysis by RSV-specific CD8+ T cells.