甲型流感病毒对肺泡上皮细胞作用的研究进展

流感是流感病毒引起的一种急性呼吸道病毒感染性疾病,能够引起较高的发病率和病死率,给人类健康带来重大威胁.肺泡上皮细胞是机体抵御甲型流感病毒的肺部关键组成部分,同时也是季节性和流行性甲型流感病毒最主要的靶向细胞和感染细胞之一,本文对甲型流感病毒对肺泡上皮细胞作用的研究进展进行简要阐述.     

Defense mechanisms against influenza virus infection in the respiratory tract mucosa

The respiratory tract mucosa is not only the site of infection for influenza viruses but also the site of defense against virus infection. Viruses are initially detected and destroyed non-specifically by innate immune mechanisms, but if the viruses escape the early defense mechanisms, they are detected and eliminated specifically by adaptive immune mechanisms. The major adaptive immune mechanisms are as follows. (i) Specific secretory-IgA (S-IgA) antibodies (Abs) and CTLs (CD8+ cytotoxic T lymphocytes) are involved in the recovery from influenza following viral infection of naive mice. (ii) Preexisting specific S-IgA and IgG Abs in the immunized animals are involved in viral elimination by forming virus-Ig complexes shortly after re-infection. By their polymeric nature, the S-IgA Abs, which are carried to the mucus by transepithelial transport used for dimeric IgA (dIgA) Abs, provide not only protection against homologous virus infection but also cross-protection against drift virus infection. The IgG Abs, which transude from the serum to the mucus by diffusion, provide protection against homologous virus infection. They are largely distributed on the alveolar epithelia to prevent influenza pneumonia. (iii) In the absence of Abs in the pre-immunized animals, the production of specific IgA and IgG Abs by B memory cells is accelerated after re-infection, and these antibodies play a role in viral elimination from day 3 onwards after re-infection. (iv) In epithelial cells of infected animals, specific dIgA Abs being trafficked through the epithelial cells may be involved in the prevention of viral assembly by binding to newly synthesized viral proteins. (v) In the pre-immunized animals, CTL production by memory T cells is also accelerated and these cells appear to participate in the killing of the host cells infected with different subtype viruses (within the same type) from day 3 onwards after re-infection. (vi) Similarly, memory Th1 cells that mediate an accelerated delayed-type hypersensitivity response are involved in blockade of virus replication by secreting IFN-gamma in mice challenged with different subtype viruses. These defense mechanisms suggest that the development of a mucosal vaccine, capable of inducing S-IgA Abs, which provide cross-protection against variant viruses within the same subtype, serum IgG Abs to prevent lethal influenza pneumonia and CTLs, which provide broad cross-protection against different subtype viruses, is strategically important to control influenza.     

H5N1-infected cells in lung with diffuse alveolar damage in exudative phase from a fatal case in Vietnam

Necropsied lung tissues of three fatal cases with avian influenza A virus (H5N1) infection in Vietnam were analyzed to detect H5N1 virus-infected cells. Formalin-fixed and paraffin-embedded lung tissue sections showed typical histological features of diffuse alveolar damage (DAD) in all cases. Immunohistochemistry for the influenza A virus nucleoprotein antigen revealed positive signals of bronchiolar and alveolar epithelial cells in only one patient, who exhibited DAD with an exudative phase and died on the 6th day after onset. However, no signal was detected in the other two cases of DAD with a proliferative phase. These patients died on day 16 and day 17 after onset, respectively. H5N1 virus antigens were detected predominantly in epithelial cells in terminal bronchioles and in alveoli, i.e., type I and type II alveolar pneumocytes, and in alveolar macrophages. The pathogenesis of exudative DAD caused by H5N1 infection is discussed.     

Analysis of in vivo dynamics of influenza virus infection in mice using a GFP reporter virus

Influenza A virus is being extensively studied because of its major impact on human and animal health. However, the dynamics of influenza virus infection and the cell types infected in vivo are poorly understood. These characteristics are challenging to determine, partly because there is no efficient replication-competent virus expressing an easily traceable reporter gene. Here, we report the generation of a recombinant influenza virus carrying a GFP reporter gene in the NS segment (NS1-GFP virus). Although attenuated when compared with wild-type virus, the NS1-GFP virus replicates efficiently in murine lungs and shows pathogenicity in mice. Using whole-organ imaging and flow cytometry, we have tracked the dynamics of influenza virus infection progression in mice. Imaging of murine lungs shows that infection starts in the respiratory tract in areas close to large conducting airways and later spreads to deeper sections of the lungs. In addition to epithelial cells, we found GFP-positive antigen-presenting cells, such as CD11b⁺CD11c⁻, CD11b⁻CD11c⁺, and CD11b⁺CD11c⁺, as early as 24 h after intranasal infection. In addition, a significant proportion of NK and B cells were GFP positive, suggesting active infection of these cells. We next tested the effects of the influenza virus inhibitors oseltamivir and amantadine on the kinetics of in vivo infection progression. Treatment with oseltamivir dramatically reduced influenza infection in all cell types, whereas, surprisingly, amantadine treatment more efficiently blocked infection in B and NK cells. Our results demonstrate high levels of immune cells harboring influenza virus antigen during viral infection and cell-type–specific effects upon treatment with antiviral agents, opening additional avenues of research in the influenza virus field.     

Expression of viral antigens after infection of human lymphocytes, monocytes, and macrophages with influenza virus

Previous studies demonstrated that in vitro infection with influenza A viruses altered several functions of human monocytes and macrophages, but did not detectably alter functions of human lymphocytes. For a determination of whether both types of leukocytes can be infected by the influenza viruses, human mononuclear leukocytes were infected in vitro and assayed for newly produced and cell surface-expressed viral antigens, with use of indirect immunofluorescence techniques and flow cytometry. Infected cells, including both monocytes and lymphocytes, expressed viral hemagglutinin, neuraminidase, and matrix protein on their surfaces. The expression of influenza virus antigens by such immunocompetent cells may be important in human antiviral defense.     

Chronic bronchitis: the role of viruses

Mucus is produced by the epithelial cells in the glands, gland ducts, and the cells lining the airway lumen in the lower airways.The chronic cough and sputum production that defines chronic bronchitis is associated with an inflammatory reaction involving this mucus-secreting apparatus. Respiratory viral infections target the epithelial cells of the lung producing desquamation, microvascular dilatation, edema, and an inflammatory cell infiltrate. These changes predispose the lower airways to bacterial infection by interfering with mucociliary clearance and reducing bacterial killing by macrophages. The exact role of those infections in the pathogenesis of chronic bronchitis has not been clearly determined but they probably play a critical role in inducing bacterial colonization and initiating acute exacerbations of COPD. This article reviews the classification of the viral agents responsible for respiratory tract infection and the nature of the changes they produce in the normal airways and in the airways of patients with chronic bronchitis during acute infections.     

Effects of Sendai virus infection on function of cultured mouse alveolar macrophages.

Cultured mouse alveolar macrophages supported the growth of Sendai virus (murine parainfluenza I virus), as measured by both a 10-fold increase in extracellular virus titers and development of viral antigens on most of the cells. Synthesis of virus continued for at least 1 month without cytopathic effects. Macrophage phagocytic activity for Candida, Staphylococcus epidermidis, and opsonized erythrocytes remained unaffected by the infection, and the ability of the cells to kill S. epidermidis and S. Aureus was also unchanged. The defects in alveolar macrophage function observed in Sendai-infected mouse lungs probably are not due to a direct effect of the virus on macrophage function.     

Influenza virus infection of human alveolar and peripheral blood-derived macrophages. Production of factors that alter fibroblast proliferation

Influenza virus infection of the lung has been shown, in animal models as well as in isolated human case reports, to cause a long-term alveolitis characterized by increased numbers of free lung macrophages. Recruitment of peripheral blood monocytes into the interstitial spaces, with subsequent maturation into tissue macrophages, accounts for most of the observed increase in cell numbers. The current studies examined the release of soluble factors by lavage-derived alveolar macrophages from healthy volunteers, and by peripheral blood-derived macrophages from the same subjects. Cell-free supernatant fluids generated by the macrophages after in vitro exposure or sham exposure to influenza virus were tested for effects on human fibroblast proliferation. The data indicated that autologous human alveolar macrophages and peripheral blood-derived macrophages both produce fibroblast-stimulating factor(s) in response to in vitro infection. The data suggest that macrophage release of fibroblast-stimulating factor(s) may play a role in the development of end-stage fibrotic lung disease occasionally seen as a sequela of influenza pneumonia.     

Differential interaction of virulent and attenuated influenza virus strains with ferret alveolar macrophages: possible role in pathogenicity

The ferret provides a unique model for the study of human influenza. The interaction between alveolar macrophages and virus strains with different levels of virulence was examined in vitro. The greater virulence of wild-type A strains over type B and C viruses was reflected in the higher production of infectious virus progeny and subsequent cytopathology, even though the expression of viral antigens was equivalent for all strains tested. These included A/Ann Arbor/6/60 (H2N2) and A/Rochester/1/82 (H3N2), B/Hong Kong/72, and C/Ann Arbor/1/50. The attenuated cold-adapted and temperature-sensitive variant of A/Ann Arbor/6/60 behaved like its parent except that a longer period was needed to reach peak viral release. In contrast, the avirulent host-range reassortant CR-43-3 did not productively replicate, though viral antigen expression was comparable to that of the other strains. Type C virus infected few cells and these continued to release low virus levels in the absence of detectable cytopathology. The results suggest that the ability of certain strains to infect and replicate in alveolar macrophages can be correlated to their in vivo virulence and may play a role in determining the course of viral pathogenesis.     

Therapeutic efficacy of liposome-encapsulated ribavirin and muramyl tripeptide in experimental infection with influenza or herpes simplex virus

Large, negatively charged, multilamellar liposomes were examined for their ability to improve the therapeutic activity of the broad-spectrum antiviral agent ribavirin (RIB) and the synthetic immunostimulant muramyl tripeptide (MTP-PE) in the treatment of viral pneumonitis. Liposome-encapsulated MTP-PE (L-MTP-PE) was superior to free MTP-PE in activating alveolar macrophages and in protecting mice against intranasal challenge with 10 LD50 (50% lethal dose) of herpes simplex virus type 1 (HSV-1). Mice treated with liposome-encapsulated or free MTP-PE had no detectable viremia and had lower pulmonary titers of virus than controls. Liposome-encapsulated RIB (L-RIB; 3 mg per mouse), administered several hours after infection, was more effective than was free RIB (10 mg per mouse) in protecting mice against intranasal challenge with 10 LD50 of influenza virus, but neither L-RIB nor free RIB protected mice against HSV-1 infection. In contrast, combination therapy with both L-RIB and L-MTP-PE was more effective than either agent used alone.     

Macrophage impairment underlies airway occlusion in primary respiratory syncytial virus bronchiolitis

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.     

Influenza virus infection in nude mice.

The role of T-cell function in influenza virus infection was studied by aerosol infection of nude mice with an influenza A virus (PR-8 strain). Nude mice died somewhat later than normal mice, and the antibody response of nude mice to the virus was minimal. Furthermore, nude mice did not eliminate the virus, which persisted for relatively long periods (two to three weeks).     

Sequential virus infections, bacterial superinfections, and fibrogenesis

Parainfluenza 1 (Sendai) and influenza A virus pneumonitis cause severe lung damage, which, upon resolution, is followed by persistent alveolitis and parenchymal changes characterized by patchy consolidation and collagen deposition in the affected areas. To determine whether these long-term sequelae of the virus pneumonias are cumulative, mice were infected by aerosol inhalation with Sendai virus, influenza A virus, or Sendai followed 30 days later by influenza virus infection. At 90 days after the initial infection, mice were killed for assay of long-term parenchymal changes as quantitated lung hydroxyproline (Hpr) content, morphometric analysis, and total and differential lavage cell counts. Sendai virus infection did not alter the proliferation of influenza virus in the lungs as quantitated by infectious virus titers on Day 1, 3, 5, 7, 9, and 11 of influenza infection. At Day 90, lung Hpr content was cumulative in dual-infected mice, with a concomitant increase in the persistent alveolitis. To determine whether bacterial infections played a similar role in these long-term pulmonary sequelae, mice were infected by aerosol inhalation with either Staphylococcus aureus or Klebsiella pneumoniae or, during the course of influenza virus infection, superinfected with each of the bacteria. Sixty days after infection with K. pneumoniae alone, lung Hpr levels were significantly increased over those in noninfected control mice. Infection with S. aureus had no effect on the quantitated parameters of long-term lung damage. In influenza-infected mice superinfected with K. pneumoniae, lung Hpr content was significantly increased over that of S. aureus did not elevate any quantitated parameter of lung damage when compared with the virus alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of T lymphocyte depletion on susceptibility to influenza virus infection and development of anti-viral immunity in lethally irradiated mice reconstituted with syngeneic bone marrow grafts

Lethally irradiated Balb/c mice reconstituted with syngeneic T cell-depleted or syngeneic untreated bone marrow (BM) were able to produce equal levels of hemagglutination inhibition (HI) antibodies at 4 weeks after bone marrow transplantation (BMT) in response to nasal infection with A/PR8 influenza virus given 1 week after BMT. Likewise, no differences in mortality rates could be observed following influenza virus infection 1 day after BMT. Antibody production was detected in 10-20% of BMT recipients. All animals responded to a secondary infection given 2 months later by production of secondary IgG-type HI antibodies. Mice reconstituted with BM enriched with spleen cells obtained from immune donors showed an improved survival rate as compared with recipients of na?ve BM, immune BM or T-depleted BM obtained from immune mice. Our results indicate that T cell depletion by itself does not increase susceptibility of syngeneic BMT recipients to virus infection. However, immune spleen cells may play a significant role in conveying protection against influenza virus infections. Although recipients of both immune and na?ve intact BM may mount better anti-influenza titers as compared with T-lymphocyte-depleted BMT recipients, it appears that the proportion of immune donor T cells in the marrow inoculum is insufficient for protection against influenza virus infection. Generation of memory cells to influenza viral antigens in the post-BMT period is not impaired in recipients of T-depleted marrow grafts, suggesting that memory cell precursors are unaffected by the T cell depletion procedure, or else that they were regenerated during the immediate post-BMT period from Thy 1.2-negative precursors.     

Discrimination of resident and infiltrated alveolar macrophages by flow cytometry in influenza A virus-infected mice

Laser flow cytometric analysis was used in conjunction with in vivo labeling with the lipophilic fluorescent dye DiIC18(5)-DS to discriminate resident alveolar macrophages from newly infiltrating monocytes/macrophages in mice with and without pulmonary influenza A virus infection. Leukocytes in bronchoalveolar lavage (BAL) and peripheral blood were analyzed by 2-color flow cytometry as a function of time following intravenous injection of DiIC18(5)-DS. At 4 hours, dye-positive leukocytes were present in both BAL and blood of normal mice, indicating that DiIC18(5)-DS rapidly crossed the pulmonary endothelial-epithelial barrier. At 4 days after dye injection, 98% of BAL cells were DiIC18(5)-DS positive, and almost all of these were monocytes/macrophages based on labeling with fluorescein isothiocyanate (FITC)-conjugated antibody to the Mac-3 marker. Only 3.2% +/- 0.3% of peripheral blood monocytes (approximately 0.16% of total peripheral blood leukocytes) were DiIC18(5)-DS positive at 6 days after injection, whereas > 95% of BAL leukocytes were strongly dye-positive on days 6 to 28. When DiIC18(5)-DS was injected in mice 6 days prior to intranasal challenge with influenza A, flow cytometry indicated that 57.8% 5.6% and 60.7% +/- 8.5% of macrophages/monocytes in BAL were newly infiltrated (i.e., DiIC18(5)-DS negative, Mac-3 positive) at 4 and 7 days, respectively, post viral infection. The discrimination of subpopulations of resident and newly recruited macrophages in BAL should facilitate future mechanistic studies on pulmonary infection and inflammatory lung injury.     

Ontogeny of T cell tolerance to peripherally expressed antigens

Transgenic expression of the influenza virus hemagglutinin (HA) in the pancreatic islet beta cells of InsHA mice leads to peripheral tolerance of HA-specific T cells. To examine the onset of tolerance, InsHA mice were immunized with influenza virus A/PR/8 at different ages, and the presence of nontolerant T cells was determined by the induction of autoimmune diabetes. The data revealed a neonatal period wherein T cells were not tolerant and influenza virus infection led to HA-specific beta cell destruction and autoimmune diabetes. The ability to induce autoimmunity gradually waned, such that adult mice were profoundly tolerant to viral HA and were protected from diabetes. Because cross-presentation of islet antigens by professional antigen-presenting cells had been reported to induce peripheral tolerance, the temporal relationship between tolerance induction and activation of HA-specific T cells in the lymph nodes draining the pancreas was examined. In tolerant adult mice, but not in 1-week-old neonates, activation and proliferation of HA-specific CD8(+) T cells occurred in the pancreatic lymph nodes. Thus, lack of tolerance in the perinatal period correlated with lack of activation of antigen-specific CD8(+) T cells. This work provides evidence for the developmental regulation of peripheral tolerance induction.     

Interferon production in rat type II pneumocytes and alveolar macrophages

The time course and magnitude of interferon production induced by influenza virus were determined in type II pneumocytes and alveolar macrophages isolated from rats (Sprague-Dawley). Although the peak of interferon production was approximately 20 h in both alveolar cell types, it was more than three- to fourfold higher in type II cells than in macrophages. Dose-response relationships were noted between the virus multiplicity of induction as well as population numbers of either alveolar cell type and interferon yields. The viral-induced cytokines produced by rat type II cells and alveolar macrophages exhibit physiochemical and biological properties characteristic of interferons and, with respect to type II pneumocytes, mark their heretofore unrecognized capability to produce interferon. The best cross-species antiviral protection afforded by these rat interferons to cells of different origin, expressed as percentage of homologous species (100%), was as follows: guinea pig 50, mouse 40%, and both human and monkey 0%. The heterologous antiviral activity by interferons from either rat alveolar macrophages or type II cells on alpha interferon-sensitive guinea pig cells suggests that these cytokines may be more appreciably related to the alpha-like interferon species. The growth of influenza and Sendai viruses was precluded in both rat alveolar macrophages and type II pneumocytes. The findings herein suggest that type II cells may be a major source of alveolar interferon for activating the antiviral state and modulating alveolar cell functions requisite for lung integrity.     

Respiratory syncytial virus infection of human cord and adult blood monocytes and alveolar macrophages

We studied the permissiveness of human leukocytes, blood monocytes, alveolar macrophages, and cord blood monocytes to infection with respiratory syncytial virus (RSV). Specific immunofluorescence was used to determine the percentage of infected leukocytes. The results indicated that monocytes were the most susceptible human leukocyte to in vitro infection with RSV. Polymorphonuclear leukocytes demonstrated no specific fluorescent staining after 24 h of exposure to RSV, whereas peripheral blood nonadherent mononuclear cells demonstrated a low percentage of positive cells, with a mean of 6 +/- 1% SE. In contrast, 37 +/- 5% of monocytes expressed RSV antigen after viral exposure. Exposure of monocytes to lipopolysaccharide (LPS) for 1 h prior to RSV increased the percentage of infected cells to 48 +/- 6% and stimulated their secretion of prostaglandin E2 (PGE2) and alpha tumor necrosis factor (TNF). Intrinsic mononuclear phagocytic factors influencing the permissiveness to RSV were studied by determining infection of adult and cord blood and alveolar mononuclear phagocytes (MP). Alveolar and blood MP simultaneously isolated from adult donors were similarly infected by RSV, which varied with the viral dose. Cord blood MP were more susceptible to RSV infection than were adult MP, 58 +/- 9% infected versus 37 +/- 5%, respectively (p less than 0.05). Treatment with LPS for 1 h prior to RSV exposure did not increase infection of cord blood MP as seen with adult blood MP. However, LPS can induce human monocytes to secrete cytokines with antiviral activity, and our results indicate that both gamma interferon and TNF, independently or in combination, prevented infection of monocytes in a dose-dependent manner.