Citations

Abstract Chronic asthma is characterized by pulmonary remodeling and sustained lung dysfunction. The expression of IL-13 has been demonstrated as sufficient and necessary to induce these pathologies. Despite the common assumption that T cells are the primary source of IL-13 in the lung following allergen provocation, the mechanistic events leading to IL-13 expression are unclear, including the potential importance of other cells (i.e., non-T cells). Methods: Co-expression of IL-5 and eotaxin-2 induces pulmonary histopathologies and lung dysfunction that are eosinophil-dependent (i.e., they are absent in triple transgenic I5/hE2/PHIL) animals. IL-13 knockout and CD4 knockout mice were crossed with I5/hE2 mice to assess the effects of these deficiencies on the induced pathologies occurring in this double transgenic model. Conclusions: The Th2 pulmonary pathologies associated with I5/hE2 mice included recruitment of pulmonary effector T cells and increased brochoalveolar Th2 cytokine levels. Conversely, I5/hE2/CD4-/- had only a ~50% reduction in IL-13 levels as compared to I5/hE2 mice, suggesting that a significant portion of IL-13 is non-T cell derived in these mice. I5/hE2/IL-13-/- mice are improved in lung dysfunction and mucus production as compared to I5/hE2 mice. All mice had similar levels of eosinophil degranulation. These results demonstrate a role for non-T cell derived IL-13 that contributes to mucus production and lung dysfunction in a mouse model of chronic severe asthma.

Abstract Eosinophil infiltration into tumors has been associated with positive and negative growth human cancers and in mouse models of tumorigenesis. Our previous studies have shown that eosinophils infiltrate tumors and differentially accumulate in the necrotic and capsule regions of tumors. Using various eosinophil-deficient mouse models, we demonstrate that in the absence of eosinophils tumor growth is attenuated. Methods: Tumor growth kinetics were assessed using a B16F10 melanoma subcutaneous cell injection model. Eosinophil chemotaxis was assessed using an ex vivo transwell insert assay system. We compared tumor growth in wild type animals vs. various eosinophil-deficient mouse models that are transgenic line of mice (PHIL) and MBP-/-/EPO-/- double knockout mice that are deficient in eosinophil-specific proteins (major basic protein (MBP) and eosinophil peroxide (EPO)) resulting in a blockade of eosinophilpoiesis. Conclusions: The migration of eosinophils was inhibited through blocking the sphingosine-1-phosphate pathway, suggesting recruitment and accumulation is primarily an inflammatory response. Tumor growth was decreased by ~40-60% in PHIL and in MBP-/-/EPO-/- double knockout mice relative to tumors in wild type animals. In addition, EPO-/- mice, and not MBP-/-mice, replicate this ~40% reduction in tumor size implicating a significant role for EPO in tumor growth kinetics. These data suggest eosinophils respond to inflammatory mediators and mediate augment tumor growth.

Abstract Products of the 5-lipoxygenase (5-LO) pathway (i.e., cys-leukotrienes and LTB4), are contributors to lung inflammation, airway remodeling, and hyperresponsiveness associated with allergic respiratory inflammation. Eosinophils are capable of leukotriene synthesis/secretion and chemotactically respond to these compounds. However, the in vivo role of eosinophils in leukotriene mediated events during asthmatic airway inflammation is not defined. To study the link between eosinophils and leukotrienes we used the I5/E2 double transgenic mouse model of severe asthma, over-expressing IL-5 systemically and eotaxin-2 in airways, in which lung pathologies are entirely dependent on the presence of eosinophils. We show that airway levels of cysteinyl leukotrienes and LTB4 are significantly elevated in I5/E2 mice. Crosses of I5E2 mice with 5-LO knockout animals abolished AHR in the I5/E2 parental model without significant effects on lung histopathology. Administration of a cys-leukotriene receptor antagonist (Montelukast®) to I5/E2 mice replicates the effects of the total leukotriene loss in I5/E2/5-LO-/- animals. In contrast, I5/E2 mice crossed with BLT-1-/- mice which are deficient of the LTB4 receptor fail to display effects on the pulmonary pathologies occurring in the I5/E2 parental model. These data demonstrate a unique role for cys-leukotrienes in the development of AHR independent of lung remodeling events occurring in this transgenic model of severe asthma.

Abstract Atopic dermatitis and allergic contact dermatitis are two of the most common skin diseases. In both diseases, eosinophils have been suggested as potential contributors of the pathologies linked with these skin conditions. Our goal was to establish a causative relationship between the inflammatory response of chemically induced contact hypersensitivity and eosinophil-mediated activities. Wild type and eosinophil-less PHIL mice were sensitized and challenged with 2,4-dinitrochlorobenzene (DNFB) or alternatively trimellitic anhydride (TMA). Time lapsed videography was used to capture the number of itching events associated with treatment. Immunohistochemistry for eosinophils and nerves was performed to correlate the effect of dermatitis on innervation and eosinophil accumulation in these mice. Our studies showed that in both the DNFB and TMA models a robust eosinophil infiltrate occurred that was accompanied by copious levels of eosinophil degranulation. More importantly, these studies demonstrated that metrics of inflammation (e.g., induced ear swelling (i.e., thickness)) was reduced in PHIL mice relative to wild type. Our observations were also directly correlative with a loss of nerve growth and branching in PHIL mice. These data implicate eosinophils as contributors to the pathologies linked with chemically-induced contact hypersensitivity, including the induced itch responses associated with these models.

Abstract Eosinophilia is a hallmark of worm infection and allergy. The parasitic nematode Trichinella spiralis establishes chronic infection in skeletal muscle. We have shown previously that in eosinophil-ablated, transgenic mice, T. spiralis larvae die in large numbers in the muscle. Death is promoted by inducible nitric oxide synthase (iNOS). We report here that transcription of iNOS was upregulated in the context of an established alternative activation phenotype in local leukocytes. Infiltrating macrophages, dendritic cells, and neutrophils produced iNOS. These changes were preceded by a reduced T cell recruitment to infected muscle that was coincident with a lag in larval growth, suggesting compromise of the parasite’s habitat. The results further define the cellular interactions that occur at a site of chronic nematode infection in which the eosinophil functions as a pivotal regulator of immunity.

Abstract Eosinophil effector functions have been hypothesized to be mediated in part by the release of proteins stored in the secondary granules of mature cells of this leukocyte lineage. Major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO) comprise the majority of protein in these granules. No role has been speculated for these proteins in eosinophilopoiesis and/or the accumulation of mature cells in circulation. Methods: Eosinophilopoiesis and eosinophil accumulation in the periphery were assessed in single and double knockout (MBP-1-/- and/or EPO-/-) mice. Bone marrow and peripheral blood leukocytes were assessed by IHC, FACS and in vitro cell culture assays. Mice were subjected to an acute OVA protocol assessing induced pulmonary pathologies in each of the strains of granule protein knockout mice. Results: We demonstrate that, unlike the single deficiency of MBP-1 or EPO, the absence of both granule proteins resulted in the loss of peripheral blood eosinophils. IHC assessments of bone marrow and spleen demonstrate that eosinophil lineage commitment occurs in these mice. However, these assessments and FACS studies demonstrated a blockade in the maturation of eosinophil-lineage committed cells. This blockade is not rescued by ex vivo culture or by bone marrow engraftment into wild type recipient mice. Similar to other eosinophil-less mouse models, MBP-1-/-/EPO-/- mice also fail to develop pulmonary inflammation in an OVA protocol.

Abstract Eosinophil recruitment and accumulation in the lungs of asthmatics is a hallmark feature of asthma. The causative relationship between eosinophils and induced Th2 pulmonary pathologies remains unclear. Our data suggest a novel role for eosinophils as immune modulators that drive Th2-mediated pulmonary pathologies through modulation of dendritic cell function leading to the suppression of Th1/Th17 immune responses. Methods: Eosinophils and ovalbumin (OVA)-pulsed myeloid dendritic cells were adoptively transferred into IL-5-/- (eosinophil-low) or PHIL mice (eosinophil-null) at the time of allergen challenge using an established acute OVA protocol. Allergen-induced pulmonary changes were assessed as changes in lung and lung draining lymph node (LDLN) cells, activation/polarization of T cells, and induced lung histopathologies. Conclusions: We demonstrate that a threshold level of eosinophils must migrate to the lung draining lymph node to promote dendritic cell recruitment and thus activation of antigen-specific memory T cells. Expression of MHC II or CCR7 on eosinophils is not necessary for proliferation of T cells in the LDLN. These lymphatic eosinophils are necessary to suppress Th1/Th17 neutrophilic polarized immune responses initiated by OVA-pulsed dendritic cells during allergen challenge. These results imply an important function for eosinophils in modulating the balance between Th2 and Th1/Th17 pathways in the development of allergic respiratory inflammation.

Abstract The human eosinophil granule contains a series of cationic proteins. Two of these, eosinophil-derived neurotoxin (EDN) and eosinophil protein X (EPX), are reported to have similar m.w. and both possess neurotoxic and helminthotoxic activities. Therefore, the properties of these molecules were analyzed to determine whether they differ. EDN was purified from eosinophils of patients with the hypereosinophilic syndrome and EPX from the buffy coat cells of normal individuals. By SDS-PAGE, both proteins showed a major band at 18.7 kDa and a minor band at 21.4 kDa. By two-dimensional non-equilibrium gel electrophoresis the proteins migrated identically. With radiolabeled proteins in reverse phase HPLC, both proteins eluted at the same concentration of acetonitrile and showed identical tryptic maps. Both proteins possessed comparable ribonuclease activity and both were comparably neurotoxic in the rabbit. By immunodiffusion the two proteins showed a reaction of identity; by RIA, with both polyclonal and monoclonal antibodies, the proteins had very similar inhibitory activities. These results indicate that EDN and EPX have virtually identical properties and are probably the same protein.

Abstract The eosinophil granule contains a series of basic proteins, including major basic protein, eosinophil peroxidase, eosinophil-derived neurotoxin (EDN), and eosinophil cationic protein (ECP). Both EDN and ECP are neurotoxins and helminthotoxins. Comparison of the partial N-terminal amino acid sequences of EDN and ECP showed 67% identity; surprisingly, they also showed structural homology to pancreatic ribonuclease (RNase). Therefore, we determined whether EDN and ECP possess RNase enzymatic activity. By spectrophotometric assay of acid soluble nucleotides formed from yeast RNA, purified EDN showed RNase activity similar to bovine pancreatic RNase, whereas ECP was 50 to 100 times less active. The RNase activity associated with ECP was not significantly inhibited after exposure of ECP to polyclonal or monoclonal antibody to EDN. These results indicate that EDN and ECP both possess RNase activity, the RNase activity of EDN and ECP is specific, and EDN and ECP have maintained not only structural but also functional homology to pancreatic RNase.

AbstractBackgroundPrevious studies have demonstrated low first‐time donor return rates (DRR) following catastrophic events. Little is known, however, about the influence of demographic factors on the DRR of first‐time donors during the COVID‐19 pandemic, including the unique motivation of COVID‐19 convalescent plasma (CCP) donors as compared to non‐CCP donors.Study Design and MethodsThirteen blood collection organizations submitted deidentified data from first‐time CCP and non‐CCP donors returning for regular (non‐CCP) donations during the pandemic. DRR was calculated as frequencies. Demographic factors associated with returning donors: race/ethnicity, gender, and generation (Gen Z: 19–24, Millennial: 25–40, Gen X: 41–56, and Boomer: ≥57 years old), within the CCP and non‐CCP first‐time cohorts were compared using chi‐square test at p < .05 statistical significance.ResultsFrom March 2020 through December 2021, there were a total of 44,274 first‐time CCP and 980,201 first‐time non‐CCP donors. DRR were 14.6% (range 11.9%–43.3%) and 46.6% (range 10.0%–76.9%) for CCP and non‐CCP cohorts, respectively. Age over 40 years (Gen X and Boomers), female gender, and White race were each associated with higher return in both donor cohorts (p < .001). For the non‐CCP return donor cohort, the Millennial and Boomers were comparable.ConclusionThe findings demonstrate differences in returning donor trends between the two donor cohorts. The motivation of a first‐time CCP donor may be different than that of a non‐CCP donor. Further study to improve first‐time donor engagement would be worthwhile to expand the donor base with a focus on blood donor diversity emphasizing engagement of underrepresented minorities and younger donors.