Lee, James J.

Abstract Adenosine is a signaling molecule produced during conditions that cause cellular stress or damage. This signaling pathway is implicated in the regulation of pulmonary disorders through the selective engagement of adenosine receptors. The goal of this study was to examine the involvement of the A3 adenosine receptor (A3R) in a bleomycin model of pulmonary inflammation and fibrosis. Results demonstrated that A3R-deficient mice exhibit enhanced pulmonary inflammation that included an increase in eosinophils. Accordingly, there was a selective up-regulation of eosinophil-related chemokines and cytokines in the lungs of A3R-deficient mice exposed to bleomycin. This increase in eosinophil numbers was accompanied by a decrease in the amount of extracellular eosinophil peroxidase activity in lavage fluid from A3R-deficient mice exposed to bleomycin, an observation suggesting that the A3R is necessary for eosinophil degranulation in this model. Despite an increase in inflammatory metrics associated with A3R-deficient mice treated with bleomycin, there was little difference in the degree of pulmonary fibrosis. Examination of fibrotic mediators demonstrated enhanced transforming growth factor (TGF)-β1 expression, but not a concomitant increase in TGF-β1 activity. This was associated with the loss of expression of matrix metalloprotease 9, an activator of TGF-β1, in alveolar macrophages and airway mast cells in the lungs of A3R-deficient mice. Together, these results suggest that the A3R serves antiinflammatory functions in the bleomycin model, and is also involved in regulating the production of mediators that can impact fibrosis.

The current paradigm surrounding allergen-mediated T helper type 2 (Th2) immune responses in the lung suggests an almost hegemonic role for T cells. Our studies propose an alternative hypothesis implicating eosinophils in the regulation of pulmonary T cell responses. In particular, ovalbumin (OVA)-sensitized/challenged mice devoid of eosinophils (the transgenic line PHIL) have reduced airway levels of Th2 cytokines relative to the OVA-treated wild type that correlated with a reduced ability to recruit effector T cells to the lung. Adoptive transfer of Th2-polarized OVA-specific transgenic T cells (OT-II) alone into OVA-challenged PHIL recipient mice failed to restore Th2 cytokines, airway histopathologies, and, most importantly, the recruitment of pulmonary effector T cells. In contrast, the combined transfer of OT-II cells and eosinophils into PHIL mice resulted in the accumulation of effector T cells and a concomitant increase in both airway Th2 immune responses and histopathologies. Moreover, we show that eosinophils elicit the expression of the Th2 chemokines thymus- and activation-regulated chemokine/CCL17 and macrophage-derived chemokine/CCL22 in the lung after allergen challenge, and blockade of these chemokines inhibited the recruitment of effector T cells. In summary, the data suggest that pulmonary eosinophils are required for the localized recruitment of effector T cells.

CD38 controls the chemotaxis of leukocytes to some, but not all, chemokines, suggesting that chemokine receptor signaling in leukocytes is more diverse than previously appreciated. To determine the basis for this signaling heterogeneity, we examined the chemokine receptors that signal in a CD38-dependent manner and identified a novel “alternative” chemokine receptor signaling pathway. Similar to the “classical” signaling pathway, the alternative chemokine receptor pathway is activated by Gαi2-containing Gi proteins. However, unlike the classical pathway, the alternative pathway is also dependent on the Gq class of G proteins. We show that Gαq-deficient neutrophils and dendritic cells (DCs) make defective calcium and chemotactic responses upon stimulation with N-formyl methionyl leucyl phenylalanine and CC chemokine ligand (CCL) 3 (neutrophils), or upon stimulation with CCL2, CCL19, CCL21, and CXC chemokine ligand (CXCL) 12 (DCs). In contrast, Gαq-deficient T cell responses to CXCL12 and CCL19 remain intact. Thus, the alternative chemokine receptor pathway controls the migration of only a subset of cells. Regardless, the novel alternative chemokine receptor signaling pathway appears to be critically important for the initiation of inflammatory responses, as Gαq is required for the migration of DCs from the skin to draining lymph nodes after fluorescein isothiocyanate sensitization and the emigration of monocytes from the bone marrow into inflamed skin after contact sensitization.

The trafficking of leukocytes from the blood to sites of inflammation is the cumulative result of receptor-ligand-mediated signaling events associated with the leukocytes themselves as well as with the underlying vascular endothelium. Our data show that Gα i signaling pathways in the vascular endothelium regulate a critical step required for leukocyte diapedesis. In vivo studies using knockout mice demonstrated that a signaling event in a non-lymphohematopoietic compartment of the lung prevented the recruitment of proinflammatory leukocytes. Intravital microscopy showed that blockade was at the capillary endothelial surface and ex vivo studies of leukocyte trafficking demonstrated that a Gα i -signaling event in endothelial cells was required for transmigration. Collectively, these data suggest that specific Gα i2 -mediated signaling between endothelial cells and leukocytes is required for the extravasation of leukocytes and for tissue-specific accumulation.

AbstractThe heterotrimeric guanine nucleotide-binding protein Gαi2 is involved in regulation of immune responses against microbial and nonmicrobial stimuli. Gαi2−/− mice have a selectively impaired IgM response consistent with a disorder in B cell development yet have augmented T cell effector function associated with increased production of IFN-γ and IL-4. The goal of the present study was to determine if a deficiency in the Gαi2 protein in mice would affect the protective immune response against Strongyloides stercoralis, which is IL-4-, IL-5-, and IgM-dependent. Gαi2−/− and wild-type mice were immunized and challenged with S. stercoralis larvae and analyzed for protective immune responses against infection. Gαi2−/− mice failed to kill the larvae in the challenge infection as compared with wild-type mice despite developing an antigen-specific Th2 response characterized by increased IL-4, IL-5, IgM, and IgG. Transfer of serum collected from immunized Gαi2−/− mice to naïve wild-type mice conferred passive protective immunity against S. stercoralis infection thus confirming the development of a protective antibody response in Gαi2−/− mice. Differential cell analyses and myeloperoxidase assays for quantification of neutrophils showed a significantly reduced recruitment of neutrophils into the microenvironment of the parasites in immunized Gαi2−/− mice. However, cell transfer studies demonstrated that neutrophils from Gαi2−/− mice are competent in killing larvae. These data demonstrate that Gαi2 signaling events are not required for the development of the protective immune responses against S. stercoralis; however, Gαi2 is essential for the recruitment of neutrophils required for host-dependent killing of larvae.

AbstractThe identification and characterization of mouse basophils have historically been hampered by the extreme rarity of this cell type. Virtually no photomicrographs of hematologically stained (eg, Wright-Giemsa) examples of mouse basophils exist in the literature. However, 4 recent studies in the past 2 years have used flow cytometry and a defined set of cell-surface markers to identify and subsequently isolate mouse “basophils,” including the publication of stained cytospin preparations of these cells. Surprisingly, a reevaluation of the data from all 4 of the studies revealed several issues of concern that suggest that the cells under study are not necessarily basophils. Nonetheless, we propose that these studies do provide the foundation for a reevaluation of the defining characteristics of a basophil and/or provide support for the provocative conclusion that a new previously overlooked leukocyte subtype has been identified. The purpose of this commentary is to revisit these previously published studies, highlight the relevant issues, and provide a different perspective in the hope of developing a consensus within the research community as to the true identity of the “basophils” described in these studies.

ABSTRACTThe goal of this study was to determine the roles of eosinophils and neutrophils in innate and adaptive protective immunity to larvalStrongyloides stercoralisin mice. The experimental approach used was to treat mice with an anti-CCR3 monoclonal antibody to eliminate eosinophils or to use CXCR2−/−mice, which have a severe neutrophil recruitment defect, and then determine the effect of the reduction or elimination of the particular cell type on larval killing. It was determined that eosinophils killed theS. stercoralislarvae in naïve mice, whereas these cells were not required for the accelerated killing of larvae in immunized mice. Experiments using CXCR2−/−mice demonstrated that the reduction in recruitment of neutrophils resulted in significantly reduced innate and adaptive protective immunity. Protective antibody developed in the immunized CXCR2−/−mice, thereby demonstrating that neutrophils were not required for the induction of the adaptive protective immune response. Moreover, transfer of neutrophil-enriched cell populations recovered from either wild-type or CXCR2−/−mice into diffusion chambers containing larvae demonstrated that larval killing occurred with both cell populations when the diffusion chambers were implanted in immunized wild-type mice. Thus, the defect in the CXCR2−/−mice was a defect in the recruitment of the neutrophils and not a defect in the ability of these cells to kill larvae. This study therefore demonstrated that both eosinophils and neutrophils are required in the protective innate immune response, whereas only neutrophils are necessary for the protective adaptive immune response to larvalS. stercoralisin mice.

ABSTRACTEosinophils are a hallmark of allergic diseases and helminth infection, yet direct evidence for killing of helminth parasites by their toxic granule products exists only in vitro. We investigated the in vivo roles of the eosinophil granule proteins eosinophil peroxidase (EPO) and major basic protein 1 (MBP) during infection with the rodent filariaLitomosoides sigmodontis. Mice deficient for either EPO or MBP on the 129/SvJ background developed significantly higher worm burdens than wild-type mice. Furthermore, the data indicate that EPO or MBP is involved in modulating the immune response leading to altered cytokine production during infection. Thus, in the absence of MBP, mice showed increased interleukin-10 (IL-10) production after stimulation of macrophages from the thoracic cavity where the worms reside. In addition to elevated IL-10 levels, EPO−/−mice displayed strongly increased amounts of the Th2 cytokine IL-5 by CD4 T cells as well as a significantly higher eosinophilia. Interestingly, a reduced ability to produce IL-4 in the knockout strains could even be seen in noninfected mice, arguing for different innate propensities to react with a Th2 response in the absence of either EPO or MBP. In conclusion, both of the eosinophil granule products MBP and EPO are part of the defense mechanism against filarial parasites. These data suggest a hitherto unknown interaction between eosinophil granule proteins, defense against filarial nematodes, and cytokine responses of macrophages and CD4 T cells.

AbstractWe explore the controversial issue of the role of eosinophils in host defense against helminthic parasites using the established Schistosoma mansoni infection model in 2 novel mouse models of eosinophil lineage ablation (ΔdblGATA and TgPHIL). No eosinophils were detected in bone marrow of infected ΔdblGATA or TgPHIL mice, despite the fact that serum IL-5 levels in these infected mice exceeded those in infected wild type by approximately 4-fold. Liver granulomata from infected ΔdblGATA and TgPHIL mice were likewise depleted of eosinophils compared with those from their respective wild types. No eosinophil-dependent differences in granuloma number, size, or fibrosis were detected at weeks 8 or 12 of infection, and differential accumulation of mast cells was observed among the ΔdblGATA mice only at week 12. Likewise, serum levels of liver transaminases, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) increased in all mice in response to S mansoni infection, with no eosinophil-dependent differences in hepatocellular damage observed. Finally, eosinophil ablation had no effect on worm burden or on egg deposition. Overall, our data indicate that eosinophil ablation has no impact on traditional measures of disease in the S mansoni infection model in mice. However, eosinophils may have unexplored immunomodulatory contributions to this disease process.

ABSTRACTSeveral studies have demonstrated roles for eosinophils during innate and adaptive immune responses to helminth infections. However, evidence that eosinophils are capable of initiating an immune response to parasite antigens is lacking. The goal of the present in vitro study was to investigate the potential of eosinophils to serve as antigen-presenting cells (APC) and initiate an immune response to parasite antigens. Purified eosinophils were exposed to solubleStrongyloides stercoralisantigens, and the expression of various surface markers involved in cell activation was examined. Antigen-exposed eosinophils showed a sixfold increase in expression levels of CD69 and major histocompatibility complex (MHC) class II, a fourfold increase in levels of T-cell costimulatory molecule CD86, and a twofold decrease in levels of CD62L compared to eosinophils cultured in medium containing granulocyte-macrophage colony-stimulating factor. The ability of eosinophils to present antigen to T cells was determined by culturing them with T cells in vitro. Eosinophils pulsed with antigen stimulated antigen-specific primed T cells and CD4+T cells to increase interleukin-5 (IL-5) production. The blocking of MHC class II expression on eosinophils inhibited their ability to induce IL-5 production by CD4+T cells in culture. Antigen-pulsed eosinophils were able to prime naïve T cells and CD4+T cells in culture and polarized them into Th2 cells producing IL-5 similar to that induced by antigen-loaded dendritic cells. These results demonstrate that eosinophils are capable of activating antigen-specific Th2 cells inducing the release of cytokines and assist in the priming of naïve T cells to initiate Th2 responses against infection. This study highlights the potential of eosinophils to actively induce immune responses against infection by amplifying antigen-specific Th2-cell responses.