Lisa M. Hodge

The Pulmonary Environment Promotes Th2 Cell Responses After Nasal-Pulmonary Immunization with Antigen Alone, but Th1 Responses Are Induced During Instances of Intense Immune Stimulation

The purpose of this study was to determine the nature of the CD4+ Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4+, and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-γ, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-γ) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4+ Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1α (MIP-1α) and MIP-1β mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1β expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.

Immunoglobulin A (IgA) responses and IgE-associated inflammation along the respiratory tract after mucosal but not systemic immunization.

ABSTRACT The purpose of the present study was to determine the extent of immunologic responses, particularly immunopathologic responses, within the upper and lower respiratory tracts after intranasal immunization using the mucosal adjuvant cholera toxin (CT). BALB/c mice were nasally immunized with influenza virus vaccine combined with CT. The inclusion of the mucosal adjuvant CT clearly enhanced generation of antibody responses in both the nasal passages and lungs. After nasal immunization, antigen-specific immunoglobulin A (IgA) antibody-forming cells dominated antibody responses throughout the respiratory tract. However, IgG responses were significant in lungs but not in nasal passages. Furthermore, parenteral immunization did not enhance humoral immunity in the upper respiratory tract even after a nasal challenge, whereas extrapulmonary lymphoid responses enhanced responses in the lung. After nasal immunization, inflammatory reactions, characterized by mononuclear cell infiltration, developed within the lungs of mice but not in nasal passages. Lowering dosages of CT reduced, but did not eliminate, these adverse reactions without compromising adjuvancy. Serum IgE responses were also enhanced in a dose-dependent manner by inclusion of CT. In summary, there are differences in the generation of humoral immunity between the upper respiratory tract and the lung. As the upper respiratory tract is in a separate compartment of the immune system from that stimulated by parenteral immunization, nasal immunization is an optimal approach to generate immunity throughout the respiratory tract. Despite the promise of nasal immunization, there is also the potential to develop adverse immunopathologic reactions characterized by pulmonary airway inflammation and IgE production.

Co-expression of granulocyte-macrophage colony-stimulating factor with antigen enhances humoral and tumor immunity after DNA vaccination

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to enhance humoral and tumor immunity resulting from DNA immunization. The genes encoding GM-CSF and antigen were cloned onto the same plasmid backbone, but separate promoters drove expression of each gene. beta-Galactosidase was used as the model antigen to generate antibody responses while the human tumor antigen, MAGE-1, was used to monitor tumor resistance. Immunization with a DNA vaccine co-expressing GM-CSF and beta-gal resulted in higher antigen-specific IgG responses than immunization with antigen encoding plasmid alone or co-inoculated with GM-CSF expressing plasmid. Similarly, DNA vaccines expressing both MAGE-1 antigen and GM-CSF were more effective in protecting against B16-MAGE-1 melanoma. However, both GM-CSF co-expressing DNA vaccines and co-inoculation with plasmids encoding the cytokine or antigen enhanced the generation antigen-specific IFN-gamma and IL-6 responses. These results demonstrate that co-expressing both GM-CSF and antigen on a DNA vaccine enhances humoral and tumor immune responses.

The Upper and Lower Respiratory Tracts Differ in Their Requirement of IFN-γ and IL-4 in Controlling Respiratory Mycoplasma Infection and Disease

The purpose of this study is to evaluate the significance of IFN-γ and IL-4 production in controlling mycoplasma infection and the pathogenesis of disease in the upper and lower respiratory tract. By using IFN-γ knockout and IL-4 knockout BALB/c mice, we were able to study the contribution of these cytokines in the development of pathogenesis and/or protection in response to mycoplasma respiratory infection, in both the upper and lower respiratory tracts. The loss of either IFN-γ or IL-4 does not affect disease pathogenesis or mycoplasma organism numbers in the upper respiratory tract. However, in the absence of IL-4, the nasal passages developed a compensatory immune response, characterized by higher numbers of macrophages and CD8+ T cells, which may be masking detrimental effects due to IL-4 deficiency. This is in contrast to the lower respiratory tract, where the loss of IFN-γ, but not IL-4, leads to higher mycoplasma numbers and increased disease severity. The loss of IFN-γ impacted the innate immune system’s ability to effectively clear mycoplasma, as the number of organisms was higher by day 3 postinfection. This higher organism burden most likely impacted disease pathogenesis; however, the development of Th2 cell-mediated adaptive immune response most likely contributed to lesion severity at later time points during infection. Our studies demonstrate that the upper and lower respiratory tracts are separate and distinct in their cytokine requirements for generating immunity against mycoplasma infection.

Cytokine and Chemokine Transcription Profile during Mycoplasma pulmonis Infection in Susceptible and Resistant Strains of Mice: Macrophage Inflammatory Protein 1β (CCL4) and Monocyte Chemoattractant Protein 2 (CCL8) and Accumulation of CCR5+ Th Cells

ABSTRACT The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1β (MIP-1β; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1β/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1β- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.

Role of Upper and Lower Respiratory Tract Immunity in Resistance to Mycoplasma Respiratory Disease

This study determined whether respiratory tract immunization protects against mycoplasma infection and compared preferential immunization of the upper respiratory tract (nasal) with upper and lower respiratory tract (nasal-pulmonary) immunization. Small volumes of inoculum preferentially deposited antigen and induced IgA responses in nasal passages. Larger inoculums deposited antigen in both the upper and lower respiratory tracts, generating corresponding IgA responses. Mice were given nasal or nasal-pulmonary immunizations with Mycoplasma pulmonis antigen alone or with cholera toxin (CT), and resistance to infection was determined. Generation of upper respiratory tract immunity reduced mycoplasma infection at this site, but CT was needed to elicit protective responses. In the lower respiratory tract, nasal-pulmonary immunization was most effective, but nasal immunization did confer some protection from pulmonary infection. In contrast, intraperitoneal immunization resulted in little protection. Thus, respiratory tract immunity plays a major role in resisting mycoplasma infection, and it should be considered during vaccine development.

Lymphatic Pump Treatment Mobilizes Leukocytes from the Gut Associated Lymphoid Tissue into Lymph

BACKGROUND Lymphatic pump techniques (LPT) are used clinically by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances lymphatic circulation and provides protection during infection are not understood. Rhythmic compressions on the abdomen during LPT compress the abdominal area, including the gut-associated lymphoid tissues (GALT), which may facilitate the release of leukocytes from these tissues into the lymphatic circulation. This study is the first to document LPT-induced mobilization of leukocytes from the GALT into the lymphatic circulation. METHODS AND RESULTS Catheters were inserted into either the thoracic or mesenteric lymph ducts of dogs. To determine if LPT enhanced the release of leukocytes from the mesenteric lymph nodes (MLN) into lymph, the MLN were fluorescently labeled in situ. Lymph was collected during 4 min pre-LPT, 4 min LPT, and 10 min following cessation of LPT. LPT significantly increased lymph flow and leukocytes in both mesenteric and thoracic duct lymph. LPT had no preferential effect on any specific leukocyte population, since neutrophil, monocyte, CD4+ T cell, CD8+ T cell, IgG+B cell, and IgA+B cell numbers were similarly increased. In addition, LPT significantly increased the mobilization of leukocytes from the MLN into lymph. Lymph flow and leukocyte counts fell following LPT treatment, indicating that the effects of LPT are transient. CONCLUSIONS LPT mobilizes leukocytes from GALT, and these leukocytes are transported by the lymphatic circulation. This enhanced release of leukocytes from GALT may provide scientific rationale for the clinical use of LPT to improve immune function.

Lymphatic pump manipulation mobilizes inflammatory mediators into lymphatic circulation

Lymph stasis can result in edema and the accumulation of particulate matter, exudates, toxins and bacteria in tissue interstitial fluid, leading to inflammation, impaired immune cell trafficking, tissue hypoxia, tissue fibrosis and a variety of diseases. Previously, we demonstrated that osteopathic lymphatic pump techniques (LPTs) significantly increased thoracic and intestinal duct lymph flow. The purpose of this study was to determine if LPT would mobilize inflammatory mediators into the lymphatic circulation. Under anesthesia, thoracic or intestinal lymph of dogs was collected at resting (pre-LPT), during four minutes of LPT, and for 10 min following LPT (post-LPT), and the lymphatic concentrations of interleukin-2 (IL-2), IL-4, IL-6, IL-10, interferon-γ, tissue necrosis factor α, monocyte chemotactic protein-1 (MCP-1), keratinocyte chemoattractant, superoxide dismutase (SOD) and nitrotyrosine (NT) were measured. LPT significantly increased MCP-1 concentrations in thoracic duct lymph. Further, LPT increased both thoracic and intestinal duct lymph flux of cytokines and chemokines as compared with their respective pre-LPT flux. In addition, LPT increased lymphatic flux of SOD and NT. Ten minutes following cessation of LPT, thoracic and intestinal lymph flux of cytokines, chemokines, NT and SOD were similar to pre-LPT, demonstrating that their flux was transient and a response to LPT. This re-distribution of inflammatory mediators during LPT may provide scientific rationale for the clinical use of LPT to enhance immunity and treat infection.

Lymphatic pump treatment augments lymphatic flux of lymphocytes in rats.

BACKGROUND Lymphatic pump techniques (LPT) are used by osteopathic practitioners for the treatment of edema and infection; however, the mechanisms by which LPT enhances the lymphatic and immune systems are poorly understood. METHODS AND RESULTS To measure the effect of LPT on the rat, the cisterna chyli (CC) of 10 rats were cannulated and lymph was collected during 4 min of 1) pre-LPT baseline, 2) 4 min LPT, and 3) 10 min post-LPT recovery. LPT increased significantly (p < 0.05) lymph flow from a baseline of 24 ± 5 μl/min to 89 ± 30 μl/min. The baseline CC lymphocyte flux was 0.65 ± 0.21 × 10⁶ lymphocytes/min, and LPT increased CC lymphocyte flux to 6.10 ± 0.99 × 10⁶ lymphocytes/min (p < 0.01). LPT had no preferential effect on any lymphocyte population, since total lymphocytes, CD4+ T cells, CD8+ T cells, and B cell numbers were similarly increased. To determine if LPT mobilized gut-associated lymphocytes into the CC lymph, gut-associated lymphocytes in the CC lymph were identified by staining CC lymphocytes for the gut homing receptor integrin α4β7. LPT significantly increased (p < 0.01) the flux of α4β7 positive CC lymphocytes from a baseline of 0.70 ± 0.03 × 10⁵ lymphocytes/min to 6.50 ± 0.10 × 10⁵ lymphocytes/min during LPT. Finally, lymphocyte flux during recovery was similar to baseline, indicating the effects of LPT are transient. CONCLUSIONS Collectively, these results suggest that LPT may enhance immune surveillance by increasing the numbers of lymphocytes released in to lymphatic circulation, especially from the gut associated lymphoid tissue. The rat provides a useful model to further investigate the effect of LPT on the lymphatic and immune systems.

Pyruvate-enriched cardioplegia suppresses cardiopulmonary bypass-induced myocardial inflammation.

BACKGROUND Cardiopulmonary bypass-induced oxidative stress initiates inflammation that can damage the myocardium. This study tested whether cardioplegia enriched with the intermediary metabolite and antioxidant pyruvate dampens postbypass myocardial inflammation. METHODS Pigs were maintained on cardiopulmonary bypass while their hearts were arrested for 60 minutes with 4:1 blood:crystalloid cardioplegia, in which the crystalloid contained 188 mM glucose ± 24 mM pyruvate. Pigs were weaned from bypass after 30 minutes of whole blood reperfusion and recovered for 4 hours. Glutathione (GSH) and glutathione disulfide (GSSG) were measured in coronary sinus plasma to indirectly monitor myocardial GSH redox state (GSH/GSSG). Left ventricular myocardium was sampled 4 hours after cardiopulmonary bypass for analyses of C-reactive protein, matrix metalloproteinases 2 and 9 and tissue inhibitor of metalloproteinase-2 (TIMP-2), and to assess neutrophil infiltration by histology and myeloperoxidase assay. RESULTS Coronary sinus GSH/GSSG fell 70% after cardiopulmonary bypass with control cardioplegia, but pyruvate cardioplegia produced a robust increase in coronary sinus GSH/GSSG that persisted for 4 hours after bypass. Myocardial C-reactive protein content increased 5.6-fold after control bypass, and neutrophil infiltration and myeloperoxidase activity also increased, but pyruvate-fortified cardioplegia prevented these inflammatory effects. Control cardioplegia lowered myocardial TIMP-2 content by 59% and increased matrix metalloproteinase-9 activity by 35% versus nonbypass sham values, but pyruvate cardioplegia increased TIMP-2 content ninefold versus control cardioplegia and prevented the increase in matrix metalloproteinase-9. Matrix metalloproteinase-2 was not affected by bypass ± pyruvate. CONCLUSIONS Pyruvate-enriched cardioplegia dampens cardiopulmonary bypass-induced myocardial inflammation. Increased GSH/GSSG and TIMP-2 may mediate pyruvates effects.