and Specific Aims.) The long-term objective of this project is to investigate mechanisms whereby immunosuppression alters host defenses against pulmonary infection. The current application proposes to study the effect of depletion of CD4-positive lymphocytes on alveolar macrophage release of nitric oxide in vivo. The hypothesis is that a deficiency of CD4- positive lymphocytes impairs the release of cytokines involved in the upregulation of nitric oxide synthase in alveolar macrophages. This defect in generation of nitric oxide then leads to decreased host resistance to pulmonary infection.
Specific Aim 1 proposes to examine whether depletion of CD4-positive lymphocytes suppresses the release of cytokines into bronchoalveolar lavage fluid. Mice will be depleted of CD4- positive lymphocytes by weekly administration of a monoclonal anti- CD4 antibody and then will be challenged with intratracheal LPS as a stimulus for the release of host cytokines into bronchoalveolar lavage fluid. The proposed studies will focus on cytokines important in the regulation of macrophage nitric oxide production, including tumor necrosis factor, interferon-gamma, interleukin-2, and interleukin-6.
Specific Aim 2 proposes to examine whether depletion of CD4-positive lymphocytes suppresses cytokine-induced release of nitric oxide by alveolar macrophages. Proposed experiments will assay lavaged alveolar macrophages for production of reactive nitrogen intermediates with a sensitive chemiluminescence assay and intratracheal challenge with LPS. Additional experiments will investigate transcriptional regulation of nitric oxide synthase in CD4-depleted mice using PCR amplification of mRNA for inducible nitric oxide synthase. Based upon the results of experiments proposed for Specific Aim 1, experiments will be performed to identify the specific cytokine(s) responsible for suppressed release of nitric oxide in the CD4-depleted mice by systemic cytokine administration and in vivo neutralization with anti-cytokine antibodies.
Specific Aim 3 proposes to examine whether decreased release of nitric oxide by alveolar macrophages important in the altered host defenses against infection associated with CD4 lymphocyte deficiency. Protocols will be developed to upregulate nitric oxide synthase activity in vivo in the CD4-depleted mice infected with P. carinii. This will be accomplished by local and systemic administration of recombinant cytokines identified during experiments proposed for Specific Aims 1 and 2. The goal of these experiments will be to attenuate the intensity of P. carinii infection in the treated mice. Additional experiments will investigate whether inhibition of nitric oxide synthase in normal mice using oral arginine analogues will increase host susceptibility to P. carinii infection. The results of these experiments may provide novel information as to how a deficiency of CD4+ lymphocytes impacts on cytokine networks within the lung important in regulating nitric oxide production by alveolar macrophages and host resistance to infection. Information gained from these investigations may lead to extension of this work to the study of humans with HIV infection and others forms of immunosuppression.
