IL-1ß in eosinophil-mediated small intestinal homeostasis and IgA production.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1ß (IL-1ß), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-ß, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-ß (transforming growth factor ß). GI eosinophils expressed a relatively high level of IL-1ß, and IL-1ß-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1ß in the small intestine.

Roles of basophils and mast cells infiltrating the lung by multiple antigen challenges in asthmatic responses of mice.

BACKGROUND AND PURPOSE: Mast cell hyperplasia has been observed in the lungs of mice with experimental asthma, but few reports have studied basophils. Here, we attempted to discriminate and quantify mast cells and basophils in the lungs in a murine asthma model, determine if both cells were increased by multiple antigen challenges and assess the roles of those cells in asthmatic responses. EXPERIMENTAL APPROACH: Sensitized Balb/c mice were intratracheally challenged with ovalbumin four times. Mast cells and basophils in enzymatically digested lung tissue were detected by flow cytometry. An anti-FcεRI monoclonal antibody, MAR-1, was i.p. administered during the multiple challenges. KEY RESULTS: The numbers of both mast cells (IgE(+) C-kit(+) ) and basophils (IgE(+) C-kit(-) CD49b(+) ) increased in the lungs after three challenges. Treatment with MAR-1 completely abolished the increases; however, a late-phase increase in specific airway resistance (sRaw), and airway eosinophilia and neutrophilia were not affected by the treatment, although the early-phase increase in sRaw was suppressed. MAR-1 reduced antigen-induced airway IL-4 production. Basophils infiltrating the lung clearly produced IL-4 after antigen stimulation in vitro; however, histamine and murine mast cell protease 1 were not increased in the serum after the challenge, indicating that mast cell activation was not evoked. CONCLUSION AND IMPLICATIONS: Both mast cells and basophils infiltrated the lungs by multiple intratracheal antigen challenges in sensitized mice. Neither mast cells nor basophils were involved in late-phase airway obstruction, although early-phase obstruction was mediated by basophils. Targeting basophils in asthma therapy may be useful for an early asthmatic response.

A myeloid cell-binding adenovirus efficiently targets gene transfer to the lung and escapes liver tropism.

Specific and efficient gene delivery to the lung has been hampered by liver sequestration of adenovirus serotype 5 (Ad5) vectors. The complexity of Ad5 liver tropism has largely been unraveled, permitting improved efficacy of Ad5 gene delivery. However, Kupffer cell (KC) scavenging and elimination of Ad5 still represent major obstacles to lung gene delivery strategies. KC uptake substantially reduces bioavailability of Ad5 for target tissues and compensatory dose escalation leads to acute hepatotoxicity and a potent innate immune response. Here, we report a novel lung-targeting strategy through redirection of Ad5 binding to the concentrated leukocyte pool within the pulmonary microvasculature. We demonstrate that this leukocyte-binding approach retargets Ad5 specifically to lung endothelial cells and prevents KC uptake and hepatocyte transduction, resulting in 165,000-fold enhanced lung targeting, compared with Ad5. In addition, myeloid cell-specific binding is preserved in single-cell lung suspensions and only Ad.MBP-coated myeloid cells achieved efficient endothelial cell transduction ex vivo. These findings demonstrate that KC sequestration of Ad5 can be prevented through more efficient uptake of virions in target tissues and suggest that endothelial transduction is achieved by leukocyte-mediated 'hand-off' of Ad.

Free radical-producing myeloid-derived regulatory cells: potent activators and suppressors of lung inflammation and airway hyperresponsiveness.

Levels of reactive free radicals are elevated in the airway during asthmatic exacerbations, but their roles in the pathophysiology of asthma remain unclear. We have identified subsets of myeloid-derived suppressor-like cells as key sources of nitric oxide and superoxide in the lungs of mice with evolving experimental allergic airway inflammation and established these cells as master regulators of the airway inflammatory response. The profiles of free radicals they produced depended on expression of inducible nitric oxide synthase (iNOS), arginase, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. These radicals controlled the pro- and anti-inflammatory potential of these cells, and also regulated the reciprocal pattern of their infiltration into the lung. The nitric oxide-producing cells were Ly-6C(+)Ly-6G(-) and they downmodulated T-cell activation, recruited T(reg) cells, and dramatically downregulated antigen-induced airway hyperresponsiveness. The superoxide-producing cells were Ly-6C(-)Ly-6G(+) and they expressed proinflammatory activities, exacerbating airway hyperresponsiveness in a superoxide-dependent fashion. A smaller population of Ly-6C(+)Ly-6G(+) cells also suppressed T-cell responses, but in an iNOS- and arginase-independent fashion. These regulatory myeloid cells represent important targets for asthma therapy.

Visualization of early APC/T cell interactions in the mouse lung following intranasal challenge.

We have used fluorescent latex beads, with or without covalently conjugated OVA, to facilitate study of Ag trafficking in the mouse lung and draining peribronchial lymph node (LN). At 6 h, and up to 48 h after intranasal administration, beads were observed as intracellular clusters in the tissue parenchyma. Flow cytometry of bead-positive (bead(+)) cells from the bronchoalveolar lavage demonstrated that a majority of these cells are CD11c(+), F4/80(+), and CD11b(-). Furthermore, fluorescent microscopy confirmed that a major subset of bead(+) cells in the lung tissue was also CD11c(+). In the draining peribronchial LNs, small numbers of beads were present in the subcapsular sinus as early as 6 h after inhalation. By 12 h and beyond, bead(+) cells had localized exclusively to the LN T zone. OVA-conjugated latex beads, in addition to stimulating brisk proliferation of naive, OVA-specific DO11.10 transgenic T cells in vitro, could also recruit OVA-specific T cells in vivo. In some cases, bead(+) APCs and CD4(+) Th1 cells were found adjacently localized in the lung tissue 6 h after airway challenge. Thus, interactions of bead(+) APCs with Ag-specific CD4(+) T cells occurred earlier in the peripheral airways than these same interactions occurred in the draining peribronchial LN. Lastly, after adoptive transfer, in vitro differentiated Th1 cells accumulated at peripheral sites in the lung tissue and airways before Ag challenge and therefore were ideally positioned to influence subsequent immune reactions of the airway.

IL-1beta is essential for langerhans cell activation and antigen delivery to the lymph nodes during contact sensitization: evidence for a dermal source of IL-1beta.

IL-1beta(-/-) mice manifest an impaired contact hypersensitivity response to the hapten trinitrochlorobenzene, with the principle defect expressed during the sensitization phase of this response. Following application of hapten to the skin, epidermal Langerhans cells of IL-1beta(-/-) mice failed to demonstrate the classical phenotype of activation. In addition, the delivery of epicutaneously applied fluorescein isothiocyanate to draining lymph nodes was decreased in IL-1beta(-/-) mice. Hapten delivery to draining lymph nodes could be restored by intradermal injection of recombinant IL-1beta. Reconstitution of lethally irradiated IL-1beta(-/-) mice by transfer of wild-type bone marrow restored hapten-stimulated IL-1beta mRNA expression, demonstrating that IL-1beta production was dependent on bone marrow-derived cells. In wild-type skin, IL-1beta expression was upregulated in a time- and dose-dependent fashion following hapten application. Interestingly, prominent IL-1beta expressing cells were found in the dermis, suggesting that dermal cells may contribute significantly to the contact hypersensitivity response.

Interleukin-1beta promotes repair of the CNS.

Interleukin-1beta (IL-1beta) is a proinflammatory cytokine associated with the pathophysiology of demyelinating disorders such as multiple sclerosis and viral infections of the CNS. However, we demonstrate here that IL-1beta appears to promote remyelination in the adult CNS. In IL-1beta(-/-) mice, acute demyelination progressed similarly to wild-type mice and showed parallel mature oligodendrocyte depletion, microglia-macrophage accumulation, and the appearance of oligodendrocyte precursors. In contrast, IL-1beta(-/-) mice failed to remyelinate properly, and this appeared to correlate with a lack of insulin-like growth factor-1 (IGF-1) production by microglia-macrophages and astrocytes and to a profound delay of precursors to differentiate into mature oligodendrocytes. Thus, IL-1beta may be crucial to the repair of the CNS, presumably through the induction of astrocyte and microglia-macrophage-derived IGF-1.

A role for IL-1 alpha in inducing pathologic inflammation during bacterial infection.

Infection with pathogenic microbes often results in a significant inflammatory response. A cascade of proinflammatory cytokines including tumor necrosis factor alpha (TNF-alpha) and IL-1 initiates this response. Although there is a clear role for IL-1 during infection, little is known to distinguish the role of IL-1 alpha from that of IL-1 beta during this process. With the use of Yersinia enterocolitica as a model enteric pathogen, we have identified a specific role for IL-1 alpha in inducing pathologic inflammation during bacterial infection. Depletion of IL-1 alpha in mice infected with wild-type Y. enterocolitica results in significantly decreased intestinal inflammation. Furthermore, a bacterial mutant that does not induce IL-1 alpha expression but induces normal levels of IL-1 beta, TNF-alpha, and IFN-gamma, causes greatly reduced intestinal inflammation and is attenuated by LD(50) analysis in the C57BL/6 mouse model. These results demonstrate a distinct and unrecognized role for IL-1 alpha in inducing intestinal inflammation that cannot be compensated for by the endogenous levels of IL-1 beta, TNF-alpha, or IFN-gamma that are produced in response to Y. enterocolitica. Additionally, these results suggest that IL-1 alpha-induced inflammation is a major contributor to the pathology of yersiniosis.

Signaling through TNF receptor p55 in TNF-alpha-deficient mice alters the CXCL13/CCL19/CCL21 ratio in the spleen and induces maturation and migration of anergic B cells into the B cell follicle.

The organization of secondary lymphoid tissues into distinct T and B cell compartments supports proper regulation of an immune response to foreign Ags. In the splenic white pulp, this compartmentalization is also thought to be important in the maintenance of B cell tolerance. Using lymphotoxin-alpha-(LT-alpha)-, TNF-alpha-, or TNFRp55-deficient mice, all with disrupted splenic architecture, we tested whether normal T/B segregation and/or intact follicular structure are necessary for the maintenance of anti-dsDNA B cell anergy. This study demonstrates that anti-dsDNA B cells remain tolerant in LT-alpha(-/-), TNF-alpha(-/-), and TNFRp55(-/-) mice; however, TNF-alpha or a TNF-alpha-dependent factor is required for their characteristic positioning to the T/B interface. Providing a TNF-alpha signal in TNF-alpha(-/-) mice by systemic administration of an agonist anti-TNFRp55 mAb induces the maturation of the anti-dsDNA B cells and their movement away from the T cell area toward the B cell area. Additionally, the agonist Ab induces changes in the follicular environment, including FDC clustering, up-regulation of the CXC chemokine ligand CXCL13, and down-regulation of the CC chemokine ligands CCL19 and CCL21. Therefore, this study suggests that a balance between B and T cell tropic chemokine signals may be an important mechanism for positioning anergic B cells at the T/B interface of the splenic white pulp.

Confocal fluorescent intravital microscopy of the murine spleen.

Intravital microscopy has provided many insights into cellular interactions in various secondary lymphoid tissues. Because this technique allows for the visualization of cellular movement in real-time, it has been very powerful. However, until now, it has been difficult to apply this technique to the spleen. We report a technique that utilizes the Nikon RCM-8000 scanning laser, confocal microscope that allows for visualization of cellular movement in real-time in the rodent spleen. Using fluorescently labeled high molecular weight dextran or monoclonal antibodies, we are able to visualize fluorescently labeled cells rolling, tethering, and adhering in the spleen. In addition, we show that the majority of blood flow to the spleen remains within the white pulp nodules, as do most transferred erythrocytes at early time points. This is the first report of intravital microscopy of the spleen using a method that allows for easy identification of transferred cells.

Induction, distribution and modulation of upper airway allergic inflammation in mice.

BACKGROUND: To further elucidate mechanisms of human allergic rhinosinusitis, we studied the induction, distribution and modulation of allergen-induced upper airway inflammation in a BALB/c mouse model. METHODS: Allergic inflammation induced with ovalbumin (OVA) by intraperitoneal (IP) injection in alum was compared to repeated intranasal instillation. The type and distribution of inflammatory cells was compared in the respiratory and olfactory epithelial compartments. Eosinophil distribution was assessed using Scarlet Red stain and a polyclonal antibody recognizing eosinophil major basic protein (MBP). The role of interleukin (IL)-5 in upper airway inflammation was tested by administration of polyclonal anti-IL-5 antibody during the sensitization protocol. RESULTS: Unsensitized control mice receiving saline failed to develop upper airway eosinophil infiltration. IP OVA-sensitized mice developed marked upper airway mucosal eosinophil infiltration after aerosol OVA challenge, whereas repeated intranasal instillation of OVA produced qualitatively similar, but less intense eosinophil infiltration. Using either sensitization protocol, eosinophil infiltration was seen in areas of the lower portion of the nasal septum, the floor and the lower lateral walls of the mid-caudal region of the nasal cavity. Immunofluorescence staining for MBP confirmed this distribution of eosinophils but also demonstrated some eosinophils in the maxillary sinuses and in circumscribed regions of the ethmoturbinates. All areas of eosinophil infiltration were lined by respiratory epithelium. The selective infiltration of respiratory but not olfactory epithelium by eosinophils was unassociated with a measurable induction of epithelial ICAM-1 or eotaxin expression. OVA-induced upper airway eosinophil infiltration was found to be IL-5 dependent, since administration of a polyclonal anti-IL-5 antibody (TRFK-5) during OVA sensitization resulted in a marked modulation (80% decrease) in eosinophil infiltration in response to subsequent OVA challenge. CONCLUSION: The mouse upper airway, specifically in areas containing respiratory epithelium, is a target for OVA-induced allergic inflammation. This selective infiltration of respiratory, but not olfactory, epithelium is, in part, dependent upon IL-5. This model is useful for further dissection of the inflammatory response with genetic manipulations and targeted immunological approaches.

Essential role of lymph nodes in contact hypersensitivity revealed in lymphotoxin-alpha-deficient mice.

Lymph nodes (LNs) are important sentinal organs, populated by circulating lymphocytes and antigen-bearing cells exiting the tissue beds. Although cellular and humoral immune responses are induced in LNs by antigenic challenge, it is not known if LNs are essential for acquired immunity. We examined immune responses in mice that lack LNs due to genetic deletion of lymphotoxin ligands or in utero blockade of membrane lymphotoxin. We report that LNs are absolutely required for generating contact hypersensitivity, a T cell-dependent cellular immune response induced by epicutaneous hapten. We show that the homing of epidermal Langerhans cells in response to hapten application is specifically directed to LNs, providing a cellular basis for this unique LN function. In contrast, the spleen cannot mediate contact hypersensitivity because antigen-bearing epidermal Langerhans cells do not access splenic white pulp. Finally, we formally demonstrate that LNs provide a unique environment essential for generating this acquired immune response by reversing the LN defect in lymphotoxin-alpha(-/)- mice, thereby restoring the capacity for contact hypersensitivity.

Analysis of isolated YAC clones.

This unit provides a series of protocols describing the analysis and manipulation of an isolated YAC clone. The procedures are based upon the use of the YAC vector pYAC4. Once an isolated YAC clone has been obtained from a core laboratory, the clone can be analyzed as described herein. Methods for analysis involve growing and storing YAC-containing yeast strains and purifying YAC DNA in a form suitable for assessing the size of the artificial chromosome and for conventional Southern blotting. Preparation of yeast chromosomes in agarose plugs for subsequent analysis by pulsed-field gel electrophoresis is also described. Additional protocols are provided for recovering DNA fragments from the ends of a YAC genomic insert to be used as probes for detecting chimerism and for chromosome walking. Finally, preparation of high-molecular-weight YAC DNA is described and a general method for subcloning YAC inserts into cosmid or lambda vectors for higher-resolution analysis is provided.

Overview of strategies for screening YAC libraries and analyzing YAC clones.

This unit provides an introduction to the use of yeast artificial chromosome-bearing yeast clones (hereafter referred to as YAC clones) in genome analysis. It describes criteria for designing a polymerase chain reaction (PCR) assay to be used in screening a YAC core library and discusses the rationale for verification and characterization of YAC clones obtained from these core laboratories. Protocols for maintaining YAC clones, analyzing YAC insert structure, preparing YAC DNA, and subcloning YAC inserts into other vectors are presented elsewhere in this volume.

Antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin.

Whether germinal centers (GC) with follicular dendritic cell (FDC) clusters are the essential sites for affinity maturation of immunoglobulin is still controversial. To re-evaluate the role of GC / FDC in affinity maturation and somatic mutation in a defined antigen system, lymphotoxin-alpha(- / -) and TNF receptor I(- / -) mice, lacking GC / FDC, were immunized with (4-hydroxy-3-nitrophenyl) acetyl-sheep RBC (NP-SRBC). In contrast to soluble hapten-carrier systems, NP-SRBC allows us to compare affinity maturation in the presence or absence of adjuvant. These mice showed a dramatically impaired ability to generate high-affinity IgG to NP, but retained the ability to produce low-affinity anti-NP IgG when NP-SRBC was used in the absence of adjuvant. In contrast to wild-type mice, somatic mutation of the expressed IgG heavy chain gene was rarely detected in these GC / FDC-deficient mice. This suggests that GC / FDC are essential for affinity maturation. Trapping antigen-specific B cells inside the T cell zone of TNFRI(- / -) mice may prolong the interaction between T and B cells, which allows class switching but no further affinity maturation of IgG. Interestingly, GC / FDC-deficient mice could be induced to generate high-affinity, somatically mutated IgG antibodies by immunization with the same amount of NP-SRBC antigen emulsified in incomplete Freund's adjuvant or repeated immunization with the antigen alone. Thus, these data support a model in which prolonged availability of antigen is required for somatic mutation and affinity maturation, and FDC or adjuvants facilitate such processes by slowly releasing antigens.

Lymphotoxin-alpha-dependent spleen microenvironment supports the generation of memory B cells and is required for their subsequent antigen-induced activation.

Lymphotoxin alpha-deficient (LTalpha-/-) mice show dramatically reduced IgG responses after either primary or secondary immunizations with sheep red blood cells (SRBC). When splenocytes from SRBC-primed wild-type donor mice were infused into irradiated naive wild-type recipient mice, they generated a robust memory IgG response, but not when infused into LTalpha-/- recipients, indicating that the microenvironment that develops in LTalpha-/- mice is incompetent to support the activation of this memory response. When irradiated wild-type mice were reconstituted with splenocytes from primed LTalpha-/- donors and then challenged with the same immunizing Ag, no memory response was observed, indicating further that memory cells could not be generated in the LTalpha-/- environment. To address which lymphocyte subsets were impaired in the LTalpha-/- mice, we performed reconstitution experiments using a hapten/carrier system and T cells and B cells from different primed donors. There was no detectable defect in either the generation or expression of memory T cells from LTalpha-/- donors. In contrast, B cells were not primed for memory in the microenvironment of LTalpha-/- mice. Additionally, primed wild-type memory B cells could not express a memory IgG response in the LTalpha-/- microenvironment. Thus, splenic white pulp structure, which depends on the expression of LTalpha for its development and maintenance, is needed to support the generation of memory B cells and to permit existing memory B cells to express an isotype switched memory Ig response following antigenic challenge.

The role of CCR7 in TH1 and TH2 cell localization and delivery of B cell help in vivo.

Subsets of murine CD4+ T cells localize to different areas of the spleen after adoptive transfer. Naïve and T helper 1 (TH1) cells, which express the chemokine receptor CCR7, are home to the periarteriolar lymphoid sheath, whereas activated TH2 cells, which lack CCR7, form rings at the periphery of the T cell zones near B cell follicles. Retroviral transduction of TH2 cells with CCR7 forces them to localize in a TH1-like pattern and inhibits their participation in B cell help in vivo but not in vitro. Thus, differential expression of chemokine receptors results in unique cellular migration patterns that are important for effective immune responses.

Cooperation between Th1 and Th2 cells in a murine model of eosinophilic airway inflammation.

We have studied the actions of helper T lymphocyte-1 and -2 (Th1 and Th2) cells in an acute model of eosinophilic airway inflammation by infusing chicken ovalbumin-specific (OVA-specific) Th1 cells, Th2 cells, or both into unsensitized mice and challenging the mice with an OVA aerosol. OVA challenge after infusion of Th1 cells alone resulted in airway inflammation with lymphocytes and monocytes. Challenge after the infusion of Th2 cells alone resulted in minimal inflammation. In contrast, when Th1 and Th2 cells were transferred together, they cooperated to promote a robust eosinophil-predominant inflammatory response. Th1 cells alone were readily recruited to the airways after challenge, but in the absence of Th1 cells, Th2 cells did not accumulate in the airways. When transferred together, both Th1 and Th2 cells, as well as endogenous eosinophils, were effectively recruited. This recruitment was correlated with increased VCAM-1 expression in the medium- and large-sized vessels of the lung and could be inhibited by treating the mice with neutralizing antibodies to TNF-alpha or VCAM-1. These data indicate that Th2 cells require signals in addition to antigen for their effective recruitment to the airways. Th1 cells can provide these signals.

Dysregulated myelopoiesis in mice lacking Jak3.

Jak3 is a cytoplasmic tyrosine kinase that associates with the common chain of the interleukin-2 (IL-2) receptor and is involved in the function of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15. Mice deficient in Jak3 have few T and B cells, and no natural killer cells. Herein we show that the myeloid lineages in these mice are also affected by the loss of Jak3. Mice lacking Jak3 exhibit splenomegaly by 4 months of age. Peripheral blood smears show an increase in the number of neutrophils and cells of the monocytic lineage. Flow cytometry of splenocytes and peripheral blood show a significant increase in FcgammaRII/III(FcgammaR)/Mac-1, FcgammaR/Gr-1, and FcgammaR/F4/80 double-positive cells in -/- and +/- mice compared to wild-type mice, consistent with an expansion of cells of the myeloid lineages. In addition, as the mice age, F4/80 and CD3 positive mononuclear cells infiltrate the kidneys, lungs, and liver of these mice. When Jak3-/- mice are crossed with a transgenic mouse expressing Jak3 in the T and NK cell compartments, the splenomegaly and myeloid expansion are accentuated. These data correlate with the constitutive activation of T cells in the periphery as the transgenic cells lose their expression of Jak3 with age. However, when Jak3-/- mice are crossed with RAG-1-deficient animals, no splenomegaly or myeloid expansion is apparent. These results indicate that the loss of Jak3 in the T-cell compartment drives the expansion of the myeloid lineages.