Elaine M. Castilow

Comparison of Histochemical Methods for Murine Eosinophil Detection in an RSV Vaccine-enhanced Inflammation Model:

A comparative study of histochemical detection of eosinophils in fixed murine tissue is lacking. Five histochemical methods previously reported for eosinophil detection were quantitatively and qualitatively compared in an established murine RSV vaccine–enhanced inflammation model. Nonspecific neutrophil staining was evaluated in tissue sections of neutrophilic soft tissue lesions and bone marrow from respective animals. Eosinophils had granular red to orange-red cytoplasmic staining, depending on the method, whereas neutrophils had, when stained, a more homogenous cytoplasmic pattern. Nonspecific background staining of similar coloration was variably seen in vascular walls and erythrocytes. Astra Blue/Vital New Red, Congo Red, Luna, Modified Hematoxylin and Eosin, and Sirius Red techniques were all effective in detecting increased eosinophil recruitment compared to controls; however, differences in eosinophil quantification varied significantly between techniques. Astra Blue/Vital New Red had the best specificity for differentiating eosinophils and neutrophils but had a reduced ability to enumerate eosinophils and was the most time intensive. The Luna stain had excessive nonspecific staining of tissues and a reduced enumeration of infiltrating eosinophils, which made it suboptimal. For multiple parameters such as eosinophil detection, specificity, and contrast with background tissues, the Sirius Red followed by Congo Red and Modified Hematoxylin and Eosin methods were useful, each with their own staining qualities.

IL-13 Is Required for Eosinophil Entry into the Lung during Respiratory Syncytial Virus Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.

Respiratory Syncytial Virus Synergizes with Th2 Cytokines to Induce Optimal Levels of TARC/CCL17

Respiratory syncytial virus (RSV) is a ubiquitous virus that preferentially infects airway epithelial cells, causing asthma exacerbations and severe disease in immunocompromised hosts. Acute RSV infection induces inflammation in the lung. Thymus- and activation-regulated chemokine (TARC) recruits Th2 cells to sites of inflammation. We found that acute RSV infection of BALB/c mice increased TARC production in the lung. Immunization of BALB/c mice with individual RSV proteins can lead to the development of Th1- or Th2-biased T cell responses in the lung after RSV infection. We primed animals with a recombinant vaccinia virus expressing either the RSV fusion (F) protein or the RSV attachment (G) protein, inducing Th1- and Th2-biased pulmonary memory T cell responses, respectively. After RSV infection, TARC production significantly increased in the vaccinia virus G-primed animals only. These data suggest a positive feedback loop for TARC production between RSV infection and Th2 cytokines. RSV-infected lung epithelial cells cultured with IL-4 or IL-13 demonstrated a marked increase in the production of TARC. The synergistic effect of RSV and IL-4/IL-13 on TARC production reflected differential induction of NFκB and STAT6 by the two stimuli (both are in the TARC promoter). These findings demonstrate that RSV induces a chemokine TARC that has the potential to recruit Th2 cells to the lung.

Differential Role of Gamma Interferon in Inhibiting Pulmonary Eosinophilia and Exacerbating Systemic Disease in Fusion Protein-Immunized Mice Undergoing Challenge Infection with Respiratory Syncytial Virus

ABSTRACT Secondary exposure to respiratory syncytial virus (RSV) can lead to immunopathology and enhanced disease in vaccinated individuals. Vaccination with individual RSV proteins influences the type of secondary RSV-specific immune response that develops upon challenge RSV infection, as well as the extent of immunopathology. RSV-specific memory CD4 T cells can directly contribute to immunopathology through their cytokine production. Immunization of BALB/c mice with a recombinant vaccinia virus (vv) expressing the attachment (G) protein of RSV results in pulmonary eosinophilia upon RSV challenge, whereas immunization of mice with a vv expressing the fusion (F) protein does not. We analyzed the CD4 T-cell response to an I-Ed-restricted CD4 T-cell epitope within the F protein of RSV corresponding to amino acids 51 to 66 in an effort to better understand the similarities and differences in the immune response elicited by the G versus the F protein. Vaccination with the G protein induces a mixture of RSV G-specific Th1 and Th2 cells with a restricted T-cell receptor repertoire. In contrast, we demonstrate here that immunization with the F protein elicits a broad repertoire of RSV F-specific CD4 T cells that predominantly exhibit a Th1 phenotype. However, in the absence of gamma interferon (IFN-γ), RSV F51-66-specific CD4 T cells secreted interleukin-5, and mice developed pulmonary eosinophilia after RSV challenge. IFN-γ-deficient mice exhibited decreased weight loss compared to wild-type controls, suggesting that IFN-γ exacerbates systemic disease. These data demonstrate that IFN-γ can have both beneficial and detrimental effects during a secondary RSV infection.

Cutting Edge: Eosinophils Do Not Contribute to Respiratory Syncytial Virus Vaccine-Enhanced Disease

Respiratory syncytial virus (RSV) infection of BALB/c mice previously immunized with a recombinant vaccinia virus (vacv) expressing the attachment (G) protein of RSV (vacvG) results in pulmonary eosinophilia, which mimics the response of formalin-inactivated RSV-vaccinated children, as well as increased weight loss, clinical illness, and enhanced pause (Penh). We show that RSV infection of eosinophil-deficient mice previously immunized with vacvG results in the development of increased weight loss, clinical illness, and Penh similar to that in wild-type controls. These measures of RSV vaccine-enhanced disease are dependent upon STAT4. Interestingly, neither IL-12 nor IL-23, the two most common STAT4-activating cytokines, proved necessary for the development of disease. We demonstrate that IFN-γ, which is produced following STAT4 activation, contributes to clinical illness and increased Penh, but not weight loss. Our results have important implications for future RSV vaccine design, suggesting that enhancing a Th1 response may exacerbate disease.

Overcoming T-cell-mediated immunopathology to achieve safe respiratory syncytial virus vaccination

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in young children. Premature infants, immunocompromised individuals, and the elderly exhibit an increased risk for the development of severe disease after RSV infection. Currently, there is not a safe and effective RSV vaccine available, in part due to our incomplete understanding of how severe immunopathology was induced following RSV infection of children previously immunized with a formalin-inactivated RSV vaccine. Much of our current understanding of RSV vaccine-enhanced disease can be attributed to the establishment of multiple mouse models of RSV vaccination. Studies analyzing the RSV-specific immune response in mice have clearly demonstrated that both CD4 and CD8 memory T cells contribute to RSV-induced immunopathology. In this review we will focus our discussion on data generated from the mouse models of RSV immunization that have advanced our understanding of how virus-specific T cells mediate immunopathology and RSV vaccine-enhanced disease.

Alcian Blue and Pyronine Y Histochemical Stains Permit Assessment of Multiple Parameters in Pulmonary Disease Models

Utilization of a combined Alcian Blue and Pyronine Y histochemical method for the assessment of multiple parameters in the respiratory tract of various species is described. Acidic mucins were deep blue (sialylated mucins), red (sulfated mucins), or variably purple (mixture of sialylated/sulfated mucins), and differential mucus production was readily detected in a murine respiratory syncytial virus vaccine model of pulmonary inflammation. Elastic fibers stained red in the walls of pulmonary arteries, connecting airways, alveolar septa, and subpleural interstitium. Mast cells had red to red-purple granular cytoplasmic staining. Nuclei were ubiquitously counterstained pale blue. Representative staining was detected in tissues from multiple species, including inbred mice, rats, ferrets, cats, dogs, sheep, and pigs. The fluorescent property of the stained tissues offers additional modalities with which to analyze tissue sections. This histochemical technique detects multiple critical parameters in routine paraffin sections of lung tissue, reduces the need for repeated serial sectioning and staining, and is cost-effective and simple to perform.