Respiratory challenge with murine gammaherpesvirus 68 (MHV-68) leads to an acute productive infection of the lung and a persistent latent infection in B lymphocytes, epithelia, and macrophages. The virus also induces splenomegaly and an increase in the number of activated CD8 T cells in the circulation. Lymphotoxin- α-deficient (LTα−/−) mice have no lymph nodes and have disrupted splenic architecture. Surprisingly, in spite of the severe defect in secondary lymphoid tissue, LTα−/− mice could clear a productive MHV-68 infection, although with delayed kinetics compared to wild-type mice, and could control latent infection. Cytotoxic T-cell activity was comparable in the lungs and spleens of LTα−/− and wild-type mice. However, splenic gamma interferon responses were substantially reduced in LTα−/− mice. Furthermore, LTα−/− mice failed to develop splenomegaly or lymphocytosis. Although germinal centers were absent, LTα−/− mice were able to class switch and showed significant virus-specific antibody titers. This work demonstrates that organized secondary lymphoid tissue is not an absolute requirement for the generation of immune responses to viral infections.
Although the essential role of tumor necrosis factor (TNF) in the control of intracellular bac-terial infection is well established, it is uncertain whether the related cytokines lymphotoxin-α (LTα3) and lymphotoxin-β (LTβ) have independent roles in this process. Using C57Bl/6 mice in which the genes for these cytokines have been disrupted, we have examined the relative contribution of secreted LTα3 and membrane-bound LTβ in the host response to aerosol Mycobacterium tuberculosis infection. To overcome the lack of peripheral lymph nodes in LTα−/− and LTβ−/− mice, bone marrow chimeric mice were constructed. LTα−/− chimeras, which lack both secreted LTα3 and membrane-bound LTβ (LTα1β2 and LTα2β1), were highly susceptible and succumbed 5 wk after infection. LTβ−/− chimeras, which lack only the membrane-bound LTβ, controlled the infection in a comparable manner to wild-type (WT) chimeric mice. T cell responses to mycobacterial antigens and macrophage responses in LTα−/− chimeras were equivalent to those of WT chimeras, but in LTα−/− chimeras, granuloma formation was abnormal. LTα−/− chimeras recruited normal numbers of T cells into their lungs, but the lymphocytes were restricted to perivascular and peribronchial areas and were not colocated with macrophages in granulomas. Therefore, LTα3 is essential for the control of pulmonary tuberculosis, and its critical role lies not in the activation of T cells and macrophages per se but in the local organization of the granulomatous response.
lymphotoxin; TNF; tuberculosis; granuloma; lung
Lymphotoxin (LT) is widely regarded as a proinflammatory cytokine with activities equivalent to tumor necrosis factor (TNF). The contribution of LT to experimental autoimmune encephalomyelitis (EAE) was examined using TNF/LTα−/− mice, TNF−/− mice, and a new LTα−/− line described here. All mice were generated directly in the C57BL/6 strain and used for the preparation of radiation bone marrow chimeras to reconstitute peripheral lymphoid organs and restore immunocompetence. This approach overcame the problems related to the lack of lymph nodes that results from LTα gene targeting. We show here that when LT is absent but TNF is present, EAE progresses normally. In contrast, when TNF is absent but LT is present, EAE is delayed in onset and inflammatory leukocytes fail to move normally into the central nervous system parenchyma, even at the peak of disease. In the absence of both cytokines, the clinical and histological picture is identical to that seen when TNF alone is deficient, including demyelination. Furthermore, the therapeutic inhibition of TNF and LTα with soluble TNF receptor in unmanipulated wild-type or TNF−/− mice exactly reproduces these outcomes. We conclude from these studies that TNF and LT are functionally distinct cytokines in vivo, and despite sharing common receptors, show no redundancy of function nor mutual compensation.
The importance of lymphotoxin α (LTα) in lymphoid organogenesis is well established. Although LTα has been implicated in the pathogenesis of T-cell-mediated immunopathologies, the requirement for LTα in T-cell activation and effector function in vivo is not well understood. To determine the role of LTα in T-cell activation in vivo, we compared the generation of antigen-specific T-cell responses between wild type (+/+) and LTα-deficient (LTα−/−) mice during an acute infection with lymphocytic choriomeningitis virus (LCMV). Our studies showed that LCMV-infected LTα−/− mice had a profound impairment in the activation and expansion of virus-specific CD8 T cells in the spleen, as determined by cytotoxicity assays, intracellular staining for gamma interferon, and staining with major histocompatibility complex class I tetramers. Further, the nonlymphoid organs of LTα−/− mice also contained substantially lower number of LCMV-specific CD8 T cells than those of +/+ mice. Greatly reduced virus-specific CD8 T-cell responses in LTα−/− mice led to a defect in LCMV clearance from the tissues. In comparison to that in +/+ mice, the activation of LCMV-specific CD4 T cells was also significantly attenuated in LTα−/− mice. Adoptive transfer experiments were conducted to determine if abnormal lymphoid architecture in LTα−/− mice caused the impairment in the activation of LCMV-specific T-cell responses. Upon adoptive transfer into +/+ mice, the activation and expansion of LCMV-specific LTα−/− T cells were restored to levels comparable to those of +/+ T cells. In a reciprocal cell transfer experiment, activation of +/+ T cells was significantly reduced upon transfer into LTα−/− mice. These results showed that impairment in the activation of LCMV-specific T cells in LTα−/− mice may be due to abnormal lymphoid architecture and not to an intrinsic defect in LTα−/− T cells.
To study the role of CD8+ T cells in allergic sensitization, we examined the effects of in vivo depletion of CD8+ T cells prior to sensitization on IgE production, immediate type cutaneous hypersensitivity and development of altered airway responsiveness. BALB/c mice were thymectomized and treated with anti-CD8 antibody resulting in depletion of CD8+ T cells (<1%) in spleen and lymphoid tissues. In these mice, sensitization to ovalbumin (OVA) via the airways still resulted in IgE anti-OVA responses and immediate cutaneous reactions to OVA, but the animals were unable to develop airway hyperresponsiveness, eosinophil infiltration of the lung parenchyma, or IL-5 production in the local lymph nodes of the airway. Transfer of CD8+ T cells from naive animals during sensitization (on day 8 of the 10-d protocol) fully restored the ability to develop airway hyperresponsiveness and this was accompanied by IL-5 production and eosinophil accumulation in the lung. These data indicate a critical role for CD8+ T cells in the production of IL-5 and the development of altered airway responsiveness after antigen sensitization through the airways.
Because eosinophils recruited into the airways in allergic diseases are exposed to inhaled allergens, we evaluated whether eosinophils within the endobronchial lumen can function in vivo as antigen-presenting cells for inhaled antigens. We recovered eosinophils from the airways after aerosol antigen challenge in sensitized mice or from the peritoneal cavities of IL-5 transgenic mice and fluorescently labeled these cells ex vivo. These labeled cells, instilled intratracheally into normal mice, migrated into draining paratracheal lymph nodes and localized to T cell–rich paracortical areas. The homing of airway eosinophils to lymph nodes was not governed by eotaxin, because CCR3–/– and CCR3+/+ eosinophils migrated identically. Airway eosinophils, recovered after inhalational antigen challenge in sensitized mice, expressed MHC class II and costimulatory CD80 and CD86 proteins and functioned in vitro as CD80- and CD86-dependent, antigen-specific, antigen-presenting cells. Moreover, when instilled into the airways of antigen-sensitized recipient mice, airway eosinophils recovered after inhalational antigen challenge stimulated antigen-specific CD4+ T cell proliferation within paratracheal lymph nodes. Thus, eosinophils within the lumina of airways can process inhaled antigens, traffic to regional lymph nodes, and function in vivo as antigen-presenting cells to stimulate responses of CD4+ T cells.
Mice sensitized to ovalbumin develop allergic airway disease (AAD) with short-term aerosol challenge; however, airway inflammation resolves with long-term aerosol challenge, referred to as local inhalational tolerance (LIT).
We sought to determine if resolution of airway inflammation correlated with increases in lymphocyte subsets in local lung compartments, including putative regulatory T cells.
At the AAD stage, total numbers of T and B lymphocytes in bronchoalveolar lavage (BAL) were significantly increased above controls; however, at LIT, T and B lymphocytes were significantly reduced compared to AAD. In the lung tissue, the only alteration was a significant increase in CD4+ CD25+ T cells at AAD. In the hilar lymph node (HLN), CD4+ and CD4+ CD25+ T cells were significantly increased at AAD and LIT. In addition, CD8+ T cells were significantly elevated in the HLN at LIT, and CD19+ B cells were significantly increased at AAD. Adoptive transfer of HLN lymphocytes to lymphopenic mice confirmed that AAD lymphocytes could induce airway inflammation in response to aerosol challenge, whereas LIT lymphocytes were unable to do so. Depletion of CD4+ CD25+ T cells in vivo resulted in exacerbation of inflammation at AAD and LIT. CD4+ CD25+ T cells in the HLN also displayed suppressive activity in vitro. Additionally, T cells expressing Foxp3 were increased in the BAL and HLN during LIT.
These results indicate that lymphocytes with regulatory functions are increased and sustained in local lung compartments at LIT and that their appearance correlates with the resolution of lung inflammation.
Adoptive transfer; Allergic airway inflammation; Foxp3; Lymphocytes; Mouse; Ovalbumin; Regulatory T cells
NF-kB is a critical transcription factor for the production of many inflammatory cytokines. It is activated in the airway epithelium of human asthmatics and in mice after allergic stimulation. To examine the role of NF-kB activation in allergic inflammation we generated transgenic mouse lines that allowed for the inducible stimulation of NF-kB in airway epithelial cells. After allergic sensitization with ovalbumin and alum, mice were challenged daily with ovalbumin aerosols and NF-kB was activated in airway epithelium by administration of doxycycline. Enhancement of airway epithelial NF-kB expression alone did not lead to increased airway responsiveness to methacholine. However induction of epithelial NF-kB during allergic inflammation caused airway hyperresponsiveness, increased airway neutrophilic and lymphocytic inflammation and goblet cell hyperplasia. Accompanying the exaggerated inflammation was an increase in the cytokines, G-CSF, IL-15, and KC. Interestingly, the counter regulatory interleukin, IL-10, was suppressed by NF-kB activation. The epithelial NF-kB dependent modulation of these cytokines provides a plausible explanation for the increased inflammation seen with overexpression of NF-kB. Modulation of airway epithelial NF-kB activation enhances the airway hyperresponsiveness and mucus secretion found in the mouse lung during allergic inflammation. NF-kB represents a potential target for pharmacologic intervention in human asthma.
The purpose of this study was to explore whether repeated exposure to aerosolized ovalbumin (OVA) in the context of local expression of GM-CSF can initiate a Th2-driven, eosinophilic inflammation in the airways. On day -1, Balb/c mice were infected intranasally with an adenovirus construct expressing GM-CSF (Ad/GM-CSF). From day 0 to day 9 mice were exposed daily to an OVA aerosol. Mice exposed to OVA alone did not show any evidence of airway inflammation. Mice receiving both Ad/GM-CSF and aerosolized OVA exhibited marked airway inflammation characterized by eosinophilia and goblet cell hyperplasia. Migration of eosinophils into the airway was preceded by a rise in IL-5 and IL-4. Both IL-5 and class II MHC were critically required to generate airway eosinophilia. After resolution, airway eosinophilia was reconstituted after a single OVA exposure. Flow cytometric analysis of dispersed lung cells revealed an increase in macrophages and dendritic cells expressing B7.1 and B7.2, and expansion of activated (CD69-expressing) CD4 and CD8 T cells in mice exposed to OVA and Ad/GM-CSF. Our data indicate that expression of GM-CSF in the airway compartment increases local antigen presentation capacity, and concomitantly facilitates the development of an antigen-specific, eosinophilic inflammatory response to an otherwise innocuous antigen.
This study examined the role of lymphotoxin (LT)-α in host defense against airborne infection with Francisella tularensis, a gram-negative facultative intracellular bacterium and the causative agent of tularemia. Following a low-dose aerosol infection with the highly virulent type A strain of F. tularensis, mice deficient in LTα (LTα−/−) consistently harbored approximately 10-fold fewer bacteria in their spleens at day 2 and 10-fold more bacteria in their lungs at day 4 than LTα+/+ mice. However, the mortality and median time to death were indistinguishable between the two mouse strains. In addition, the inflammatory responses to the infection, as reflected by the cytokine levels and leukocyte influx in the bronchoalveolar lavage fluid and histopathological analysis, were generally similar between LTα−/− and LTα+/+ mice. These data suggest that although LTα does not contribute significantly to the resistance and host responses of mice to airborne type A F. tularensis infection, it does play a subtle role in the multiplication/dissemination of F. tularensis.
The aim of this study was to investigate whether dendritic cells (DCs) can induce sensitization to aeroallergen in a mouse model of allergic asthma. Ovalbumin-pulsed (OVA-pulsed) or unpulsed myeloid DCs that were injected into the airways of naive mice migrated into the mediastinal lymph nodes. When challenged 2 weeks later with an aerosol of OVA, activated CD4 and CD8 lymphocytes, eosinophils, and neutrophils were recruited to the lungs of actively immunized mice. These CD4+ lymphocytes produced predominantly IL-4 and IL-5 but also IFN-γ, whereas CD8+ lymphocytes produced predominantly IFN-γ. Histological analysis revealed perivascular and peribronchial eosinophilic infiltrates and goblet cell hyperplasia. Studies in IL-4–/– and CD28–/– mice revealed that production of IL-4 by host cells and provision of costimulation to T cells by DCs were critical for inducing the response. Lung CD4+ T cells strongly expressed the Th2 marker T1/ST2, and signaling through this molecule via a ligand expressed on DCs was essential for the establishment of airway eosinophilia. These data demonstrate that DCs in the airways induce sensitization to inhaled antigen and that molecules expressed on the surface of these cells are critical for the development of Th2-dependent airway eosinophilia.
During pulmonary mycobacterial infection, there is increased trafficking of dendritic cells from the lungs to the draining lymph nodes. We hypothesized that ongoing mycobacterial infection would modulate recruitment and activation of antigen-specific naive CD4+ T cells after airway antigen challenge. BALB/c mice were infected by aerosol with Mycobacterium bovis BCG. At peak bacterial burden in the lungs (4 to 6 weeks postinfection), carboxy-fluorescein diacetate succinimidyl ester-labeled naive ovalbumin-specific DO11.10 T cells were adoptively transferred into infected and uninfected mice. Recipient mice were challenged intranasally with soluble ovalbumin (OVA), and OVA-specific T-cell responses were measured in the lungs, draining mediastinal lymph nodes (MLN), and spleens. OVA challenge resulted in increased activation and proliferation of OVA-specific T cells in the draining MLN of both infected and uninfected mice. However, only BCG-infected mice had prominent OVA-specific T-cell activation, proliferation, and Th1 differentiation in the lungs. BCG infection caused greater distribution of airway OVA to pulmonary dendritic cells and enhanced presentation of OVA peptide by lung CD11c+ cells. Together, these data suggest that an existing pulmonary mycobacterial infection alters the phenotype of lung dendritic cells so that they can activate antigen-specific naive CD4+ T cells in the lungs in response to airway antigen challenge.
Chronic helminth infections, such as schistosomes, are negatively associated with allergic disorders. Here, using B cell IL-10-deficient mice, Schistosoma mansoni-mediated protection against experimental ovalbumin-induced allergic airway inflammation (AAI) was shown to be specifically dependent on IL-10-producing B cells. To study the organs involved, we transferred B cells from lungs, mesenteric lymph nodes or spleen of OVA-infected mice to recipient OVA-sensitized mice, and showed that both lung and splenic B cells reduced AAI, but only splenic B cells in an IL-10-dependent manner. Although splenic B cell protection was accompanied by elevated levels of pulmonary FoxP3+ regulatory T cells, in vivo ablation of FoxP3+ T cells only moderately restored AAI, indicating an important role for the direct suppressory effect of regulatory B cells. Splenic marginal zone CD1d+ B cells proved to be the responsible splenic B cell subset as they produced high levels of IL-10 and induced FoxP3+ T cells in vitro. Indeed, transfer of CD1d+ MZ-depleted splenic B cells from infected mice restored AAI. Markedly, we found a similarly elevated population of CD1dhi B cells in peripheral blood of Schistosoma haematobium-infected Gabonese children compared to uninfected children and these cells produced elevated levels of IL-10. Importantly, the number of IL-10-producing CD1dhi B cells was reduced after anti-schistosome treatment. This study points out that in both mice and men schistosomes have the capacity to drive the development of IL-10-producing regulatory CD1dhi B cells and furthermore, these are instrumental in reducing experimental allergic inflammation in mice.
Chronic asthma is often associated with neutrophilic infiltration in the airways. Neutrophils contain elastase, a potent secretagogue in the airways, nonetheless the role for neutrophil elastase as well as neutrophilic inflammation in allergen-induced airway responses is not well defined. In this study, we have investigated the impact of neutrophil elastase inhibition on the development of allergic airway inflammation and airway hyperresponsiveness (AHR) in previously sensitized and challenged mice.
BALB/c mice were sensitized and challenged (primary) with ovalbumin (OVA). Six weeks later, a single OVA aerosol (secondary challenge) was delivered and airway inflammation and airway responses were monitored 6 and 48 hrs later. An inhibitor of neutrophil elastase was administered prior to secondary challenge.
Mice developed a two-phase airway inflammatory response after secondary allergen challenge, one neutrophilic at 6 hr and the other eosinophilic, at 48 hr. PAR-2 expression in the lung tissues was enhanced following secondary challenge, and that PAR-2 intracellular expression on peribronchial lymph node (PBLN) T cells was also increased following allergen challenge of sensitized mice. Inhibition of neutrophil elastase significantly attenuated AHR, goblet cell metaplasia, and inflammatory cell accumulation in the airways following secondary OVA challenge. Levels of IL-4, IL-5 and IL-13, and eotaxin in BAL fluid 6 hr after secondary allergen challenge were significantly suppressed by the treatment. At 48 hr, treatment with the neutrophil elastase inhibitor significantly reduced the levels of IL-13 and TGF-β1 in the BAL fluid. In parallel, in vitro IL-13 production was significantly inhibited in spleen cells from sensitized mice.
These data indicate that neutrophil elastase plays an important role in the development of allergic airway inflammation and hyperresponsiveness, and would suggest that the neutrophil elastase inhibitor reduced AHR to inhaled methacholine indicating the potential for its use as a modulator of the immune/inflammatory response in both the neutrophil- and eosinophil-dominant phases of the response to secondary allergen challenge.
Neutrophil; Elastase; Airway; Hyperresponsiveness; Asthma
Airways inflammation is thought to play a central role in the pathogenesis of asthma. However, the precise role that individual inflammatory cells and mediators play in the development of airways hyperreactivity and the morphological changes of the lung during allergic pulmonary inflammation is unknown. In this investigation we have used a mouse model of allergic pulmonary inflammation and interleukin (IL) 5-deficient mice to establish the essential role of this cytokine and eosinophils in the initiation of aeroallergen-induced lung damage and the development of airways hyperreactivity. Sensitization and aerosol challenge of mice with ovalbumin results in airways eosinophilia and extensive lung damage analogous to that seen in asthma. Aeroallergen-challenged mice also display airways hyperreactivity to beta-methacholine. In IL-5-deficient mice, the eosinophilia, lung damage, and airways hyperreactivity normally resulting from aeroallergen challenge were abolished. Reconstitution of IL-5 production with recombinant vaccinia viruses engineered to express this factor completely restored aeroallergen-induced eosinophilia and airways dysfunction. These results indicate that IL-5 and eosinophils are central mediators in the pathogenesis of allergic lung disease.
Although the essential role of tumor necrosis factor (TNF) in resistance to Listeria monocytogenes infection is well established, the roles of the related cytokines lymphotoxin alpha (LTα) and lymphotoxin beta (LTβ) are unknown. Using C57BL/6 mice in which the genes for these cytokines were disrupted, we examined the contributions of TNF, LTα, and LTβ in the host response to Listeria. To overcome the lack of peripheral lymph nodes in LTα−/− and LTβ−/− mice, bone marrow chimeras were constructed. TNF−/− and LTα−/− chimeras that lacked both secreted LTα3 and membrane-bound LTα1β2 and LTα2β1 were highly susceptible and succumbed 4.5 and 6 days, respectively, after a low-dose infection (200 CFU). LTβ−/− chimeras, which lacked only membrane-bound LT, controlled the infection in a manner comparable to wild-type (WT) chimeras. The Listeria-specific proliferative and gamma interferon T-cell responses were equivalent in all five groups of infected mice (LTα−/− and LTβ−/− chimeras, WT chimeras, and TNF−/− and WT mice). TNF−/− mice and LTα−/− chimeras, however, failed to generate the discrete foci of lymphocytes and macrophages that are essential for bacterial elimination. Rather, aberrant necrotic lesions comprised predominantly of neutrophils with relatively few lymphocytes and macrophages were observed in the livers and spleens of TNF−/− and LTα−/− chimeras. Therefore, in addition to TNF, soluble LTα3 plays a separate essential role in control of listerial infection through control of leukocyte accumulation and organization in infected organs.
Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα−/−) mice that lack Peyer's patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.
Both nature and induced regulatory T (Treg) lymphocytes are potent regulators of autoimmune and allergic disorders. Defects in endogenous Treg cells have been reported in patients with allergic asthma, suggesting that disrupted Treg cell-mediated immunological regulation may play an important role in airway allergic inflammation. In order to determine whether adoptive transfer of induced Treg cells generated in vitro can be used as an effective therapeutic approach to suppress airway allergic inflammation, exogenously induced Treg cells were infused into ovalbumin-sensitized mice prior to or during intranasal ovalbumin challenge. The results showed that adoptive transfer of induced Treg cells prior to allergen challenge markedly reduced airway hyperresponsiveness, eosinophil recruitment, mucus hyper-production, airway remodeling, and IgE levels. This effect was associated with increase of Treg cells (CD4+FoxP3+) and decrease of dendritic cells in the draining lymph nodes, and with reduction of Th1, Th2, and Th17 cell response as compared to the controls. Moreover, adoptive transfer of induced Treg cells during allergen challenge also effectively attenuate airway inflammation and improve airway function, which are comparable to those by natural Treg cell infusion. Therefore, adoptive transfer of in vitro induced Treg cells may be a promising therapeutic approach to prevent and treat severe asthma.
Lymphotoxin alpha (LTα) can exist in soluble form and exert tumor necrosis factor (TNF)-like activity through TNF receptors. Based on the phenotypes of knockout (KO) mice, the physiological functions of LTα and TNF are considered partly redundant, in particular, in supporting the microarchitecture of the spleen and in host defense. We exploited Cre-LoxP technology to generate a novel neomycin resistance gene (neo) cassette-free LTα-deficient mouse strain (neo-free LTα KO [LTαΔ/Δ]). Unlike the “conventional” LTα−/− mice, new LTαΔ/Δ animals were capable of producing normal levels of systemic TNF upon lipopolysaccharide (LPS) challenge and were susceptible to LPS/d-galactosamine (D-GalN) toxicity. Activated neutrophils, monocytes, and macrophages from LTαΔ/Δ mice expressed TNF normally at both the mRNA and protein levels as opposed to conventional LTα KO mice, which showed substantial decreases in TNF. Additionally, the spleens of the neo-free LTα KO mice displayed several features resembling those of LTβ KO mice rather than conventional LTα KO animals. The phenotype of the new LTαΔ/Δ mice indicates that LTα plays a smaller role in lymphoid organ maintenance than previously thought and has no direct role in the regulation of TNF expression.
Rationale: Statin use has been linked to improved lung health in asthma and chronic obstructive pulmonary disease. We hypothesize that statins inhibit allergic airway inflammation and reduce airway hyperreactivity via a mevalonate-dependent mechanism.
Objectives: To determine whether simvastatin attenuates airway inflammation and improves lung physiology by mevalonate pathway inhibition.
Methods: BALB/c mice were sensitized to ovalbumin over 4 weeks and exposed to 1% ovalbumin aerosol over 2 weeks. Simvastatin (40 mg/kg) or simvastatin plus mevalonate (20 mg/kg) was injected intraperitoneally before each ovalbumin exposure.
Measurements and Main Results: Simvastatin reduced total lung lavage leukocytes, eosinophils, and macrophages (P < 0.05) in the ovalbumin-exposed mice. Cotreatment with mevalonate, in addition to simvastatin, reversed the antiinflammatory effects seen with simvastatin alone (P < 0.05). Lung lavage IL-4, IL-13, and tumor necrosis factor-α levels were all reduced by treatment with simvastatin (P < 0.05). Simvastatin treatment before methacholine bronchial challenge increased lung compliance and reduced airway hyperreactivity (P = 0.0001).
Conclusions: Simvastatin attenuates allergic airway inflammation, inhibits key helper T cell type 1 and 2 chemokines, and improves lung physiology in a mouse model of asthma. The mevalonate pathway appears to modulate allergic airway inflammation, while the beneficial effects of simvastatin on lung compliance and airway hyperreactivity may be independent of the mevalonate pathway. Simvastatin and similar agents that modulate the mevalonate pathway may prove to be treatments for inflammatory airway diseases, such as asthma.
statin; asthma; physiology; HMG-CoA reductase; small G protein
Although elevated levels of IgE in asthmatic patients are strongly associated with lung infiltration by activated T helper (Th) 2 cells, the physiological role of immunoglobulin E (IgE) in the airway remains largely undefined. Lymphotoxin-deficient α (LTα−/−) mice exhibit increased airway inflammation, paradoxically accompanied by diminished levels of IgE and reduced airway hyperresponsiveness in response to both environmental and induced antigen challenge. The severe lung inflammation in LTα−/− mice is Th1 in nature and can be alleviated by IgE reconstitution. Conversely, depletion of IgE in wild-type mice recapitulates the lung pathologies of LTα−/− mice. Therefore, this work has revealed that lymphotoxin is essential for IgE production, and a physiological role of IgE in the airway may consist of maintaining the balance of Th1 and Th2 responses to prevent aberrant inflammation.
bronchial hyperresponsiveness; cytokines; T helper cells; infiltration; allergen
Eosinophilic inflammation is closely related to angiogenesis in asthmatic airway remodeling. In ovalbumin-sensitized mice, bone marrow-derived pro-angiogenic endothelial progenitor cells (EPCs) are rapidly recruited into the lungs after ovalbumin aerosol challenge, and promptly followed by mobilization and recruitment of eosinophils.
We hypothesized that bone marrow-derived EPCs initiate the recruitment of eosinophils through expression of eosinophil chemoattractant eotaxin-1.
EPCs were isolated from ovalbumin murine model of allergic airway inflammation and from asthma patients. Endothelial and smooth muscle cells were isolated from mice. Eotaxin-1 expression was analyzed by immunofluorescence, real-time PCR or by ELISA. In vivo recruitment of eosinophils by EPCs was analyzed in mice.
Circulating EPCs of asthmatic individuals had higher levels of eotaxin-1 as compared to controls. In the murine model, ovalbumin allergen exposure augmented eotaxin-1 mRNA and protein levels in EPCs. The EPCs from ovalbumin-sensitized and challenged mice released high levels of eotaxin-1 upon contact with lung endothelial cells from sensitized and challenged mice, but not from control animals, and not upon contact with cardiac or hepatic endothelial cells from sensitized and challenged mice. Intranasal administration of the eotaxin-rich media overlying cultures of EPCs caused recruitment into lungs, confirming functional chemoattractant activity.
Bone marrow-derived EPCs are early responders to environmental allergen exposures, and initiate a parallel switch to a pro-angiogenic and pro-eosinophilic environment in the asthmatic lungs.
eosinophils; allergy; airway inflammation; angiogenesis; bone marrow; eotaxin
The prevalence of allergic diseases such as asthma has increased markedly over the past few decades. To evaluate the possible mutual influence of helminth infection and allergy, the combined effects of experimental allergic airway inflammation and infection with Strongyloides venezuelensis on various parasitological and inflammatory indices were evaluated in the rat. A challenge of immunized rats with aerosolized ovalbumin (OVA) resulted in eosinophilic inflammation that peaked 48 h after the challenge and was accompanied by airway hyperresponsiveness (AHR) to an intravenous acetylcholine challenge. S. venezuelensis infection concomitant with an OVA challenge of immunized rats resulted in prolonged pulmonary inflammation with increased eosinophil infiltration in bronchoalveolar lavage fluid but not in the lung tissue. These rats also showed a significant parasite burden reduction, especially during parasite migration through the lungs. However, the fecundity rates of worms that reached the intestine were similar in allergic and nonallergic animals. Despite airway inflammation, the increased responsiveness of the airways in the experimental asthma model was suppressed during parasite migration through the lungs (2 days). In contrast, parasite-induced AHR was unchanged 5 days after infection in immunized and challenged rats. In conclusion, infection with S. venezuelensis interfered with the onset of AHR following an antigen challenge of immunized rats. The ability of parasites to switch off functional airway responses is therapeutically relevant because we may learn from parasites how to modulate lung function and, hence, the AHR characteristic of asthmatic patients.
Although several cytokines, including tumor necrosis factor (TNF), can promote the growth of dendritic cells (DCs) in vitro, the cytokines that naturally regulate DC development and function in vivo have not been well defined. Here, we report that membrane lymphotoxin (LT), instead of TNF, regulates the migration of DCs in the spleen. LTα−/− mice, lacking membrane LTα/β and LTα3, show markedly reduced numbers of DCs in the spleen. Unlike wild-type mice and TNF−/− mice that have densely clustered DCs in the T cell zone and around the marginal zone, splenic DCs in LTα−/− mice are randomly distributed. The reduced number of DCs in lymphoid tissues of LTα−/− mice is associated with an increased number of DCs in nonlymphoid tissues. The number of splenic DCs in LTα−/− mice is restored when additional LT-expressing cells are provided. Blocking membrane LTα/β in wild-type mice markedly diminishes the accumulation of DCs in lymphoid tissues. These data suggest that membrane LT is an essential ligand for the presence of DCs in the spleen. Mice deficient in TNF receptor, which is the receptor for both soluble LTα3 and TNF-α3 trimers, have normal numbers of DCs. However, LTβR−/− mice show reduced numbers of DCs, similar to the mice lacking membrane LT α/β. Taken together, these results support the notion that the signaling via LTβR by membrane LTα/β is required for the presence of DCs in lymphoid tissues.
membrane lymphotoxin; tumor necrosis factor; dendritic cells; lymphotoxin receptor; migration
Lymphotoxin α (LTα) signals via tumor necrosis factor receptors (TNFRs) as a homotrimer and via lymphotoxin β receptor (LTβR) as a heterotrimeric LTα1β2 complex. LTα-deficient mice lack all lymph nodes (LNs) and Peyer's patches (PPs), and yet LTβ-deficient mice and TNFR-deficient mice have cervical and mesenteric LN. We now show that mice made deficient in both LTβ and TNFR type 1 (TNFR1) lack all LNs, revealing redundancy or synergism between TNFR1 and LTβ, acting presumably via LTβR. A complete lack of only PPs in mice heterozygous for both ltα and ltβ, but not ltα or ltβ alone, suggests a similar two-ligand phenomenon in PP development and may explain the incomplete lack of PPs seen in tnfr1−/− mice.
lymphotoxin beta; tumor necrosis factor receptor 1; knockout mice; mesenteric lymph nodes; Peyer's patches