Vitamin A Deficiency Disrupts Vaccine-Induced Antibody-Forming Cells and the Balance of IgA/IgG Isotypes in the Upper and Lower Respiratory Tract

Abstract Vaccination by intranasal (IN) inoculation with a replication-competent virus forms the basis of licensed and novel candidate respiratory viral vaccines (e.g., the cold-adapted influenza virus vaccine). A positive global impact of vaccination depends on vaccine efficacy in developing countries where dietary deficiencies are commonplace. The current study was designed using Sendai virus (SeV) as a model respiratory viral vaccine to test antibody-forming cell (AFC) residence and isotype expression in the context of a vitamin A deficiency (VAD). Samples were taken 1?mo after vaccination when AFCs generally reach their peak in healthy animals. In control animals on a healthy diet, SeV induced an antibody response with a relative bias toward IgA in the upper respiratory tract (URT, as sampled by nasal wash), and IgG in the lower respiratory tract (LRT, as sampled by bronchoalveolar lavage [BAL]). In the context of VAD, the SeV-specific IgA antibodies in the nasal wash were significantly reduced in favor of enhanced IgG antibodies in the BAL. When AFCs were examined in diffuse nasal-associated lymphoid tissues (d-NALT), lungs, cervical lymph nodes (CLN), and mediastinal lymph nodes (MLN), a similar pattern emerged. AFCs were most frequent in the d-NALT and most expressed IgA in control mice. In the context of VAD, these IgA-producing AFCs were significantly reduced in number, skewing the natural balance of IgA and IgG. Taken together, the results show that the VAD diet, which is well known for its association with immune defects in the gut, significantly alters AFC induction and isotype expression in the respiratory tract.     

Production of IgA monoclonal antibodies against influenza A virus

Nineteen IgA monoclonal antibodies against influenza A virus X-31 were obtained following intranasal infection of mice with influenza A virus X-31. It was demonstrated that specificities of IgA monoclonal antibodies are similar to those of IgG monoclonal antibodies. These IgA antibodies might be useful for the study of mucosal immunity against influenza A viruses. Infection may be an easier and better way of producing monoclonal antibodies against some viral agents.     

Natural Killer Cells Accumulate in Lung‐Draining Lymph Nodes and Regulate Airway Eosinophilia in a Murine Model of Asthma

Increasing evidence suggests a key role for the innate immune system in asthma development. Although the role of Natural Killer (NK) cells in allergic asthma is poorly known, modifications of the blood NK cell populations have been found in asthmatic and/or allergic patients. Their repartition and activation status in the inflammatory (lungs) and the regulatory (draining lymph nodes) sites of the allergic reaction is unknown. The aim of our study was to monitor NK cell migration pattern and activation status and to investigate the consequences of NK cell depletion during allergic airway reaction in a mouse model. Ovalbumin sensitization and challenges of BALB/cByJ mice had no effect on the total number of lung NK cells but significantly decreased the number of most mature NK cells and increased the level of the activation marker CD86. In the lung‐draining mediastinal lymph nodes, ovalbumin sensitization and challenges led to increased number of NK cells, and more precisely, immature NK cells and increased expression of CD86. Ovalbumin‐sensitized mice also exhibited increased percentage of proliferating NK cells in lung‐draining mediastinal lymph nodes. Anti‐ASGM1 antibody treatment depleted most NK cells and decreased bronchoalveolar lavage eosinophilia but did not modify airway responsiveness. Altogether, our study shows that pulmonary allergic sensitization induces modification in the NK cell compartment at the inflammatory and regulatory sites and suggests that NK cells may participate in the regulation of the asthmatic response and, more particularly, to the allergic airway eosinophilia.     

Influenzal pneumonia: early appearance of cross-reactive T cells in lungs of mice primed with heterologous type A viruses.

Mice were first primed with a type A or a type B influenza virus and then challenged intranasally at least 1 month later with another type A virus. Potent cytotoxic T cell populations were found in lung, and effector T cell function was also demonstrated in blood, spleen and mediastinal lymph nodes. Lymphocytes isolated from all of these anatomical sites were active against target cells infected with the same, or with serologically different, type A influenza viruses. Also, prior exposure to another type A virus resulted in more rapid development of effector function than was seen in mice that had first been infected with B/Lee. Cytotoxic T cell populations generated in mice with influenza thus tend overall to be type-specific, and there is substantial localization of these effector lymphocytes in the pneumonic lung.     

Antibody response to influenza infection of mice: different patterns for glycoprotein and nucleocapsid antigens

Our previous studies of C57BL/6 mice intranasally infected with influenza virus (A/PR8) revealed a spike of virus-specific immunoglobulin A (IgA)-secreting antibody-forming cells (AFC) in the mediastinal lymph node (MLN) 7 days post-infection. Here we show that these AFC are directed only against viral glycoprotein, and not nucleocapsid antigens. The early IgA spike associates with a decline in glycoprotein-specific AFC during week 2 post-infection. In contrast to the glycoprotein-specific AFC, nucleocapsid-specific, IgA-secreting AFC develop gradually in the MLN and persist for more than 3 weeks post-infection. As peripheral lymph node reactions wane, the nucleocapsid-specific AFC appear as long-sustained populations in the bone marrow. Microanatomical examination of the respiratory tract in infected mice shows foci of infection established in the lung 2 days post-infection, from which virus spreads to infect the entire lining of the trachea by day 3. At this time, viral haemagglutinin can be seen within the MLN, probably on projections from infected dendritic cells. This feature disappears within a day, though viral antigen expression continues to spread throughout the respiratory tract. Total IgA- and IgG-secreting AFC appear histologically in large numbers during the first week post-infection, significantly preceding the appearance of germinal centres (revealed by peanut agglutinin staining in week 2). To explain these results, we suggest that the initial immunogenic encounter of B cells with viral antigens occurs about 3 days post-infection in the MLN, with antigens transported by dendritic cells from airway mucosa, the only site of viral replication. Viral glycoproteins expressed as integral membrane components on the surface of infected dendritic cells [probably in the absence of cognate T helper (Th) cells] promote members of expanding relevant B-cell clones to undergo an IgA switch and terminal local plasmacytoid differentiation. Anti-glycoprotein specificities are thus selectively depleted from progeny of activated B-cell clones which are channelled to participate in germinal centre formation (perhaps by cognate T helper cells when they become sufficiently frequent). One product of the germinal centre reaction is the long-sustained, bone marrow-resident population, which is accordingly rich in anti-nucleoprotein, but not anti-glycoprotein specificities. Of note, we find that AFC responses toward influenza virus and Sendai virus differ, even though viral replication is limited to the airway mucosa in each case. The response towards Sendai virus exhibits neither the early appearance of anti-glycoprotein AFC expressing IgA in draining lymph nodes, nor the subsequent relative deficit of this specificity from bone marrow AFC populations.     

APRIL affects antibody responses and early leukocyte infiltration, but not influenza A viral control

A proliferation inducing ligand (APRIL) is implicated in the regulation of class switch recombination to IgA in T-independent B cell responses. Since B cells play an important role in the immunity to influenza A virus and resistance against the virus is partly controlled by T-independent IgA B cell responses, we studied the role of APRIL during an influenza A infection in vivo. APRIL transgenic, wild-type and APRIL deficient mice were intranasally infected with a non-lethal dose of a mouse adapted strain of influenza A. Compared to wild-type mice, APRIL deficient mice showed a twofold reduction in the amount of macrophages in the lungs and a tendency towards decreased granulocyte influx in the early leukocyte recruitment phase. Although the T cell immune response against influenza was unaffected, APRIL Tg mice showed prolonged influenza-specific IgM production and differential class switching. Unexpectedly, the IgA B cell response was completely T helper cell dependent and also not affected by the absence or presence of APRIL. In addition, viral clearance and recovery from the infection was not influenced by APRIL. Combined these results indicate that APRIL affects specific aspects of the anti-influenza response, but plays a limited role in disease recovery.     

Targeting CD45RB alters T cell migration and delays viral clearance

CD45 is a receptor tyrosine phosphatase essential for TCR signaling. One isoform, CD45RB, is down-regulated in memory cells and targeting CD45RB with a specific antibody has been shown to inhibit graft rejection. Its role in immunity to infection, however, has not been tested. Here, we report the effect of anti-CD45RB antibody treatment on the induction of anti-influenza CD8+ T cells and viral clearance. Anti-CD45RB-treated mice had delayed pulmonary viral clearance compared with untreated mice whose infection was completely cleared by day 8 post-infection. In anti-CD45RB-treated mice, the total CD4+ and CD8+ T cell numbers in both the lungs and mediastinal nodes were substantially reduced at days 5 and 8; this effect was less marked for the spleen. CD8+ T cells specific for influenza virus were also reduced compared with the control group in all three organs at day 8. By day 11, when both treated and control groups showed no virus remaining in the lungs, specific CD8+ T cell numbers were at similar low levels. Homing to lymph nodes and lung of dye-labeled T cells was greatly inhibited (by >80%) by anti-CD45RB treatment. This reduced homing corresponded with reduced CD62L and beta1-integrin expression in both uninfected and infected mice. Since CD62L plays a critical role in homing lymphocytes to lymph nodes, and high levels of CD62L and alpha4beta1-integrin are expressed by lymphocytes that home to bronchus-associated lymphoid tissue, we suggest that reduced expression of these molecules is a key explanation for the delay in immune responses.     

Kinetics and Isotype Profile of Rheumatoid Factor Production During Viral Infection: Organ Distribution of Antibody Secreting Cells

The kinetics and isotype profile of influenza virus-specific IgG antibodies were studied in correlation with the serum titre of IgG-reactive autoantibodies. An increased level of IgG isotype-specific, rheumatoid factor-type autoantibody secretion was observed in the late phase of the virus-specific memory response. These rheumatoid factors were specific for the IgG2a and IgG1 subclasses which dominated the anti-viral antibody response. As revealed by a preparative immunosorbent technique combined with isotype quantitation the majority of IgG2a- or IgG1-bound immunoglobulins isolated from the serum of virus-infected mice belonged to the same subclass as the target antibody. Comparison of the kinetics of appearance and the number of IgM-, IgG- and IgA-type IgG2a-reactive autoantibody secreting cells during the primary and memory anti-viral antibody responses showed isotype switch of IgM rheumatoid factor secreting cells predominantly to IgA. Localization of IgM and IgA antibody secreting cells demonstrated the wide organ distribution of IgM-type rheumatoid factor secreting cells. On the contrary, IgA rheumatoid factor production was observed only in Peyer's patches and at the site of the local virus-specific immune response, i.e. in mediastinal lymph nodes and in the lung. These results demonstrate that B cells specific for self IgG are activated and differentiated in concert with the virus-specific antibody response in similar microenvironments. The predominant involvement of the mediastinal lymph nodes and the spleen in the production of IgG2a-specific IgM-type autoantibodies suggest a regulatory function of this type of autoantibodies in modulating IgG2a production in both systemic and local anti-viral immune responses. The results also suggest a strictly regulated rheumatoid factor production which, however, can be unbalanced by repeated viral infections resulting in the escape of high affinity, isotype-switched autoantibodies.     

Enumeration and characterization of virus-specific B cells by multicolor flow cytometry

To better characterize B cell responses induced to influenza virus, we developed an assay to directly quantify and characterize virus-specific B cells. We used purified and biotinylated whole virus as well as the major influenza virus surface antigen, hemagglutinin (HA) to label virus-specific B cells induced by immunization of mice with whole influenza virus in adjuvant. Immunization with adjuvant alone caused non-specific binding of whole virus to a large number of B cells in the draining lymph nodes as assessed by flow cytometry. This precluded the use of whole virus as a specific staining reagent. In contrast, staining with bromelain-cleaved purified and biotinylated influenza virus HA identified a small population of B cells (roughly 1%) only in the draining lymph nodes of virus-immunized mice. FACS-purification and subsequent ELISPOT analysis showed that HA-labeled B cells contained the vast majority of virus-specific antibody-secreting cells at day 10 after immunization. Overall, virus-specific antibody-secreting cells comprised roughly 10% of the HA-labeled cells. Using HA-staining in conjunction with 8-color flow cytometry we further demonstrated that close to 90% of the HA-labeled cells were CD19+ IgD- CD23- CD24high CD38low germinal center B cells, many of which had incorporated bromodeoxyuridine, indicating recent cell division in vivo. We conclude that viral HA can be used in conjunction with cell surface and intracytoplasmic stains in multicolor flow cytometry to provide detailed phenotypic and functional information on virus HA-specific B cells.     

Induction of Mucosal B-Cell Memory by Intranasal Immunization of Mice with Respiratory Syncytial Virus

The capacity of live or inactivated respiratory syncytial virus (RSV) to induce B-cell memory in respiratory-associated lymphoid tissues of mice was examined. Eight weeks after primary inoculation with either live or inactivated RSV, adult BALB/c mice were challenged with 4 × 10⁵ PFU of RSV. Protection from viral shedding and mucosal production of RSV-specific antibodies were examined at various time points after challenge. We found that primary immunization with live, but not inactivated, RSV induced complete and durable protection upon challenge within the upper and lower respiratory tract. Also, primary immunization with live, but not inactivated, RSV enhanced the production of mucosal RSV-specific immunoglobulin A (IgA) upon challenge. Secondary mucosal IgA responses were characterized by (i) the early production of mucosal IgA by B cells that reside in organized nasal-associated lymphoid tissues, cervical lymph nodes, and bronchial lymph nodes, and (ii) the subsequent production of RSV-specific IgA by mucosal effector tissues, such as the tracheal lamina propria and lung. These findings suggest that primary infection of mice with live RSV might induce mucosal IgA-committed memory B cells. A greater understanding of the characteristics of RSA-specific mucosal memory B cells may facilitate the development of an RSV vaccine.     

Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules.

Mice that were intranasally immunized with different influenza A virus hemagglutinins (HA), derived from PR8 (H1N1), A/Yamagata (H1N1) or A/Fukuoka (H3N2) virus, together with cholera toxin B subunit as an adjuvant, were examined for protection against PR8 infection; PR8 HA and A/Yamagata HA immunization conferred complete protection, while A/Fukuoka HA immunization failed to confer protection. In parallel with protection, PR8 HA-, A/Yamagata HA-, and A/Fukuoka HA-immunized mice produced a high, a moderate and a low level of PR8 HA-reactive IgA in the respiratory tract, respectively. These IgA antibodies were not only higher in content in the nasal secretions, but also more cross-reactive than IgG. The purified IgA antibodies from respiratory tract washings of PR8 HA-immunized mice, which contained the HA-specific IgA corresponding to the amount detected in the nasal wash, were able to protect mice from PR8 challenge when transferred to the respiratory tract of naive mice. The transfer of IgA from A/Yamagata HA-immunized mice also afforded cross-protection against PR8 infection, whereas the IgA from A/Fukuoka HA-immunized mice failed to provide protection. The ability of transferred IgA to prevent viral infection was dependent on the amount of HA-reactive IgA remaining in the respiratory tract of the host at the time of infection. These experiments directly demonstrate that IgA antibodies to influenza A virus HA by themselves play a pivotal role in defence not only against homologous virus infection, but also against heterologous drift virus infection at the respiratory mucosa, the portal of entry for the viruses.     

Secretory IgA antibodies provide cross-protection against infection with different strains of influenza B virus

This study examined whether secretory IgA (S-IgA) antibodies (Abs) could confer cross-protective immunity against infection with influenza B viruses of antigenically distinct lineages. Wild-type or polymeric Ig receptor (pIgR)-knockout (KO) mice were immunized by infection with different B viruses or by intranasal (i.n.) administration with different inactivated vaccines. Four weeks later mice were challenged with either the B/Ibaraki/2/85 virus, representative of the B/Victoria/2/87 (B/Victoria)-lineage, or B/Yamagata/16/88 virus, representative of the B/Yamagata-lineage. Three days after challenge, nasal wash and serum specimens were assayed for IgA and IgG Abs specific for challenge viral antigens and for protection against challenge viruses. In wild-type mice, B/Ibaraki (or B/Yamagata) cross-reactive IgA Abs were detected at higher levels when infected or immunized with homologous-lineage viruses and at lower levels when infected or immunized with heterologous-lineage viruses. There was a correlation between the amount of nasal cross-reactive IgA Ab and the efficacy of cross-protection with a homologous-lineage virus. In mice lacking the pIgR, nasal cross-protective IgA Abs were only marginally detected in vaccinated mice and an accumulation of IgA in the serum was observed. This reduction of nasal IgA was accompanied by inefficient cross-protection against the B/Ibaraki (or B/Yamagata) virus infection. These results suggest that challenge viral-antigen cross-reactive S-IgA in nasal secretions induced by i.n. infection or vaccination is involved in providing cross-protection against challenge infection with virus within either the B/Victoria- or B/Yamagata-lineage.     

Residual antigen presentation after influenza virus infection affects CD8 T cell activation and migration

Activated virus-specific CD8 T cells remain in the lung airways for several months after influenza virus infection. We show that maintenance of this cell population is dependent upon the route of infection and prolonged presentation of viral antigen in the draining lymph nodes (DLN) of the respiratory tract. The local effects on T cell migration have been examined. We show retention of virus-specific CD8 T cells in the mediastinal lymph node (MLN) and continuing recruitment of blood-borne migrants into the lung airways during antigen presentation. These data show that antigen that is retained after pulmonary influenza virus infection controls the migratory pattern and activation state of virus-specific CD8 T cells near the site of virus amplification.     

Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.     

Immunoglobulin A-deficient mice exhibit altered T helper 1-type immune responses but retain mucosal immunity to influenza virus

We have previously demonstrated that immunoglobulin A (IgA)(-/-) knockout (KO) mice exhibit levels of susceptibility to influenza virus infection that are similar to those of their normal IgA(+/+) littermates. To understand the mechanism of this apparent mucosal immunity without IgA, immunoglobulin isotype and T helper 1 (Th1)-type [interferon-gamma (IFN-gamma)] and Th2-type [interleukin (IL)-4, IL-5)] cytokine responses to influenza vaccine were evaluated. Intranasal immunization with influenza virus subunit vaccine plus cholera toxin/cholera toxin B subunit (CT/CTB) induced significant influenza virus-specific immunoglobulin G (IgG) antibody in the serum and nasal passages of both IgA(-/-) and IgA(+/+) mice, while IgA antibodies were induced only in IgA(+/+) mice. IgA KO mice exhibited an IgG1 subclass haemagglutinin (HA)-specific response but no detectable IgG2a and IgG2b responses. In contrast, IgA(+/+) mice exhibited significant IgG1 as well as IgG2a responses. This indicates a predominant Th2-type response in IgA KO mice compared to normal mice. Following stimulation with influenza virus in vitro, splenic lymphocytes from immunized IgA(-/-) mice produced significantly lower levels of IFN-gamma than IgA(+/+) mice (P < 0.001), but elaborated similar levels of IL-4 and IL-5. This was true at both protein and mRNA levels. Immunized mice were challenged intranasally with a small inoculum of influenza virus to allow deposition of virus in the nasal mucosal passages. Compared to non-immunized mice, immunized IgA(-/-) and IgA(+/+) mice exhibited significant, but similar levels of reduction in virus titres in the nose and lung. These results demonstrate that in addition to IgA deficiency, IgA gene deletion also resulted in down-regulated Th1-type immune responses and confirm our previous data that IgA antibody is not indispensable for the prevention of influenza virus infection.     

In Vivo Anti-Influenza Virus Activity of Kampo (Japanese Herbal) Medicine “Sho-Seiryu-To”—Stimulation of Mucosal Immune System and Effect on Allergic Pulmonary Inflammation Model Mice

When BALB/c mice were treated with a Kampo (Japanese herbal) medicine “Sho-seiryu-to (SST)” (1 g/kg, 10 times) orally from 7 days before to 5 days after the infection and infected with mouse-adapted influenza virus A/PR/8/34 by nasal-site restricted infection, SST caused increment of the influenza virus hemagglutinin-specific IgA antibody secreting cells in nasal lymphocyte but not in Peyer's patch lymphocyte at 6 days after infection in comparison with water-treated mice. Oral administration of SST also augmented IL-2 receptor β chain⁺ (activated) T-cell in Peyer's patch lymphocyte, but not in the nasal lymphocyte. We previously reported that SST showed potent anti-influenza virus activity through augmentation of the antiviral IgA antibody titer in the nasal and broncho-alveolar cavities of the mice (T. Nagai and H. Yamada, 1994, Int. J. Immunopharmacol. 16, 605-613). These results suggest that oral administration of SST shows anti-influenza virus activity in the nasal cavity by activatation of T-cell in Peyer's patch lymphocyte and stimulation of production of anti-influenza virus IgA antibody in nasal lymphocyte. When ovalbumin-sensitized allergic pulmonary inflammation model mice were administered orally with SST (1 g/kg) from 8 days before (11 times) or from 2 h after (4 times) to 4 days after the infection and infected with mouse-adapted influenza virus A/PR/8/34, replications of the virus in the both nasal and broncho-alveolar cavities or only nasal cavity were significantly inhibited at 5 days after infection in comparison with water-treated control by augmenting antiviral IgA antibody, respectively. These results suggest that SST is useful for both prophylaxis and treatment of influenza virus infection on patients with allergic pulmonary inflammation, such as bronchial asthma.     

Influenza A facilitates sensitization to house dust mite in infant mice leading to an asthma phenotype in adulthood

The origins of allergic asthma, particularly in infancy, remain obscure. Respiratory viral infections and allergen sensitization in early life have been associated with asthma in young children. However, a causal link has not been established. We investigated whether an influenza A infection in early life alters immune responses to house dust mite (HDM) and promotes an asthmatic phenotype later in life. Neonatal (8-day-old) mice were infected with influenza virus and 7 days later, exposed to HDM for 3 weeks. Unlike adults, neonatal mice exposed to HDM exhibited negligible immune responsiveness to HDM, but not to influenza A. HDM responsiveness in adults was associated with distinct Ly6c+ CD11b+ inflammatory dendritic cell and CD8α+ plasmacytoid (pDC) populations that were absent in HDM-exposed infant mice, suggesting an important role in HDM-mediated inflammation. Remarkably, HDM hyporesponsiveness was overcome when exposure occurred concurrently with an acute influenza infection; young mice now displayed robust allergen-specific immunity, allergic inflammation, and lung remodeling. Remodeling persisted into early adulthood, even after prolonged discontinuation of allergen exposure and was associated with marked impairment of lung function. Our data demonstrate that allergen exposure coincident with acute viral infection in early life subverts constitutive allergen hyporesponsiveness and imprints an asthmatic phenotype in adulthood.     

Role of IgM antibodies versus B cells in influenza virus-specific immunity

We have compared the role of IgM antibodies with the role of B cells in control of primary influenza virus infection. Mice deficient in IgM (IgM(-/-)), but capable of producing other Igisotypes, exhibited increased pulmonary virus titers compared to wild-type mice. However, IgM(-/-) mice were less susceptible compared to B cell-deficient micro MT) mice. CD4(+) T cells from spleen and lung draining lymph nodes of infected micro MT mice showed reduced proliferation upon virus re-stimulation in vitro. Furthermore, numbers of IFN-gamma-producing CD4(+) effector T cells were reduced in the alveolar lavage (BAL) of micro MT mice but not IgM(-/-) mice. In contrast, total number of virus-specific CTL was almost comparable in BAL of micro MT and wild-type mice. Pulmonary recruitment of inflammatory macrophages and neutrophils occurred normally in both micro MT and IgM(-/-) mice. Interestingly, virus-specific IgG2a and IgG2b antibody responses were affected locally in the BAL and in the serum of IgM(-/-) mice, while IgG1 responses remained largely normal. Taken together, our data suggest a role for B cells to promote effector T cell responses and a role of both IgM and IgG antibodies in the defense against acute influenza virus infection.     

Cellular Changes in Lungs of Mice Infected with Influenza Virus: Characterization of the Cytotoxic Responses

Transpleural lavage of lungs from uninfected C3H mice yielded an average of 300,000 leukocytes per mouse. This number increased eightfold within 6 days after intranasal inoculation with virulent influenza A/Hong Kong/68 (H3N2) virus. Macrophages and lymphocytes in approximately equal numbers comprised 90% or more of the leukocytes both before and during infection. B, T, and null lymphocytes comprised, respectively, 9, 21, and 18% of the leukocytes before infection and 7, 26, and 5% by day 6. In absolute numbers, macrophages and T lymphocytes provided the major increments during infection. Cytotoxic activity of mononuclear cells from lung lavages was compared in a chromium release assay using syngeneic L929 target cells with the activity of mediastinal lymph nodes, spleens, and peripheral blood of uninfected and infected C3H mice. Nonspecific cytotoxicity for target cells infected with H3hkNeq1 or B/Lee influenza virus was found with mononuclear cells from uninfected mice. This activity tended to be highest with lavage leukocytes and was associated with adherent cells, presumably macrophages. Increased virus-specific cytotoxicity was detected with lavage cells by day 6 and persisted through day 9, the period of maximal pneumonia. Similar cytotoxic activity also appeared in cells from the nodes and spleen at this same time but was not detected in peripheral blood cells. The virus-specific cytotoxicity of lavage cells was due largely to a nonadherent cell possessing Fc receptors and theta antigen but lacking C3 receptors; these properties are compatible with actively cytotoxic T lymphocytes. The cytological characteristics of the infiltrating leukocytes and the cytotoxicity data suggest that the local T cell response to influenza virus infection in the lung is a major contributor to the pneumonia observed in this mouse model.     

Influenza virus infection of the murine uterus: a new model for antiviral immunity in the female reproductive tract

Secretory IgA (S-IgA) mediates local immunity to influenza virus in the murine upper respiratory tract and may play an important role in local immunity to various microorganisms in the female reproductive tract as well. Although the presence of IgA in cervicovaginal or uterine secretions has been correlated with immunity to a number of pathogens, there has been no direct demonstration of the mediation of uterine antiviral immunity by S-IgA. Influenza virus, although not a normal pathogen of the reproductive tract, was used to develop a model for the investigation of mucosal immunity in the uterus. PR8 (H1N1) influenza virus injected into the ovarian bursa of BALB/c mice grew well, with peak titers between days 3 and 5. Intravenous injection of polymeric IgA anti-influenza virus monoclonal antibody before or 30 min after viral challenge protected mice against viral infection. We believe this work to be the first direct demonstration of S-IgA-mediated antiviral uterine immunity. It provides a model for further investigation of immunity in the female reproductive tract.