A replication-deficient recombinant adenovirus encoding a chimeric protein capable of binding tumor necrosis factor (TNF) and lymphotoxin was given to mice. Administration of this virus (10(9) pfu intravenously) yielded high levels of the recombinant protein in plasma and afforded significant protection to a lethal challenge with lipopolysaccharide with or without D-galactosamine. However, this protein inhibitor was readily detectable in the lung and was associated with decreased neutrophil recruitment and bacterial killing after intratracheal LPS or Pseudomonas aeruginosa, respectively. These data reflect the dual role of many proinflammatory cytokines. This model of TNF inhibition is similar to the homozygous 55-kDa TNF receptor deletion; thus, adenovirus-mediated gene transfer of cytokine inhibitors in vivo is a useful tool to abrogate the function of single or multiple cytokines for investigational or therapeutic purposes.

Alcohol (EtOH) has been shown to suppress lipopolysaccharide (LPS)-induced nitric oxide (NO) generation and tumor necrosis factor (TNF) production in the lung in vivo. We have previously reported that EtOH suppressed gene expression for inducible nitric oxide synthase (iNOS) with a subsequent decrease in release of reactive nitrogen intermediates by alveolar macrophages and recruited lung neutrophils. We hypothesized that a similar mechanism may be involved in EtOH-induced suppression of LPS-stimulated TNF production. In contrast to what we found with iNOS, EtOH had no effect on TNF mRNA in alveolar macrophages or recruited lung neutrophils. However, immunoreactive and bioactive TNF was reduced by 72%. EtOH treatment resulted in an increased level of the membrane-bound 26-kDa form of TNF, which suggested that proteolytic cleavage of this prohormone was affected by EtOH. Experiments with t-butyl alcohol, a tertiary alcohol that is not metabolized to acetaldehyde, yielded similar results. Thus EtOH appears to be the active substance in suppression of TNF in the lung in vivo. Pretreatment with intratracheal interferon-gamma 24 h before intratracheal LPS increased TNF bioactivity partly due to increased TNF mRNA and by increasing TNF processing, as evidenced by a decrease in the 26-kDa TNF prohormone and an increase in immunoreactive and bioactive TNF.

E1-deleted adenoviral vectors are increasingly being utilized for in vivo gene transfer. The potential use of these vectors is limited by transient expression of the transgene and a markedly reduced rate of transduction following readministration, presumably due to a host immune response to the vector. We hypothesized that CD4+ lymphocytes are necessary to generate an immune response to these vectors and that administration of a depleting anti-CD4 antibody (GK1.5) might prolong transgene expression in vivo. We found that pretreatment of mice with a single injection (transient depletion) or weekly injections of GK1.5 (persistent depletion), markedly prolonged expression of an adenovirus-encoded tumor necrosis factor (TNF) inhibitor or luciferase gene compared to controls. Moreover, mice treated with GK1.5 showed no antiadenoviral antibody response to repeat administration of the vector and a second adenoviral transgene could be expressed in these animals. However, control mice developed a significant neutralizing antibody response that prevented transgene expression with administration of a second adenovirus. These findings demonstrate that manipulation of the host immune response may expand potential applications of gene transfer utilizing adenoviral vectors.

Interferon-gamma (IFN-gamma) is a critical cytokine in pulmonary host defenses against both intracellular and extracellular pathogens. To investigate whether this cytokine could be used therapeutically, we constructed an E1-deleted recombinant adenovirus encoding murine IFN-gamma. After intratracheal inoculation in rats, this vector resulted in prolonged expression of functional cytokine in vivo, as demonstrated by increased alveolar macrophage class II major histocompatibility complex expression, enhanced release of tumor necrosis factor in response to lipopolysaccharide, and enhanced host defenses against Pseudomonas aeruginosa. We postulate that this vector may be useful to study the role of exogenous IFN-gamma in a variety of pulmonary intracellular and extracellular pathogens.

The role of mononuclear phagocytes and their cytokine products in host defense against Pneumocystis carinii (PC) remains unclear. The cytokine tumor necrosis factor (TNF) has been proposed as critical for host defense against this pathogen. To investigate the role of this cytokine in PC infection, we treated immunocompetent mice (CD4+) or mice depleted of CD4 lymphocytes (CD4-) with a recombinant adenovirus encoding a TNF inhibitor gene (AdTNF-R). AdTNF-R treated CD4+ animals displayed delayed clearance of PC after intratracheal inoculation, whereas AdTNF-R treated CD4 animals developed more severe chronic infection. Moreover, AdTNF-R treated CD4- animals, in contrast to control CD4- mice, failed to show any interleukin-6 (IL-6) gene induction in the lung after PC challenge. The results firmly implicate TNF in host defense against PC, and support a role for TNF in orchestrating the intrapulmonary cytokine cascade in PC infection.