Studies comparing endogenous and recombinant serum amyloid A (SAA) have generated conflicting data on the proinflammatory function of these proteins. In exploring this discrepancy, we found that in contrast to commercially sourced recombinant human SAA1 (hSAA1) proteins produced in , hSAA1 produced from eukaryotic cells did not promote proinflammatory cytokine production from human or mouse cells, induce Th17 differentiation, or stimulate TLR2. Proteomic analysis of-derived hSAA1 revealed the presence of numerous bacterial proteins, with several being reported or probable lipoproteins. Treatment of hSAA1 with lipoprotein lipase or addition of a lipopeptide to eukaryotic cell-derived hSAA1 inhibited or induced the production of TNF-α from macrophages, respectively. Our results suggest that a function of SAA is in the binding of TLR2-stimulating bacterial proteins, including lipoproteins, and demand that future studies of SAA employ a recombinant protein derived from eukaryotic cells.

Airway epithelial NF-κB activation is observed in asthmatic subjects and is a cause of airway inflammation in mouse models of allergic asthma. Combination therapy with inhaled short-acting β2-agonists and corticosteroids significantly improves lung function and reduces inflammation in asthmatic subjects. Corticosteroids operate through a number of mechanisms to potently inhibit NF-κB activity. Since β2-agonists can induce expression of 11β-HSD1, which converts inactive 11-keto corticosteroids into active 11-hydroxy corticosteroids, thereby potentiating the effects of endogenous glucocorticoids, we examined whether this mechanism is involved in the inhibition of NF-κB activation induced by the β-agonist albuterol in airway epithelial cells. Treatment of transformed murine Club cells (MTCC) with (R)-albuterol (levalbuterol), but not with (S)- or a mixture of (R + S)- (racemic) albuterol, augmented mRNA expression of 11β-HSD1. MTCC were stably transfected with luciferase (luc) reporter constructs under transcriptional regulation by NF-κB (NF-κB/luc) or glucocorticoid response element (GRE/luc) consensus motifs. Stimulation of NF-κB/luc MTCC with lipopolysaccharide (LPS) or tumor necrosis factor-α (TNFα) induced luc activity, which was inhibited by pretreatment with (R)-, but not (S)- or racemic albuterol. Furthermore, pretreatment of GRE/luc MTCC with (R)-, but not with (S)- or racemic albuterol, augmented 11-keto corticosteroid (cortisone) induced luc activity, which was diminished by the 11β-HSD inhibitor glycyrrhetinic acid (18β-GA), indicating that there was a conversion of inactive 11-keto to active 11-hydroxy corticosteroids. LPS- and TNFα-induced NF-κB/luc activity was diminished in MTCC cells treated with a combination of cortisone and (R)-albuterol, an effect that was inhibited by 18β-GA. Finally, pretreatment of MTCC cells with the combination of cortisone and (R)-albuterol diminished LPS- and TNFα-induced pro-inflammatory cytokine production to an extent similar to that of dexamethasone. These results demonstrate that levalbuterol augments expression of 11β-HSD1 in airway epithelial cells, reducing LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production through the conversion of inactive 11-keto corticosteroids into the active 11-hydroxy form in this cell type.

Nitrogen dioxide (NO2) is an environmental air pollutant and endogenously-generated oxidant that contributes to the exacerbation of respiratory disease and can function as an adjuvant to allergically sensitize to an innocuous inhaled antigen. Since uric acid has been implicated as a mediator of adjuvant activity, we sought to determine whether uric acid was elevated and participated in a mouse model of NO2-promoted allergic sensitization. We found that uric acid was increased in the airways of mice exposed to NO2 and that administration of uricase inhibited the development of ovalbumin (OVA)-driven allergic airway disease subsequent to OVA challenge as well as the generation of OVA-specific antibodies. However, uricase was itself immunogenic, inducing a uricase-specific adaptive immune response that occurred even when the enzymatic activity of uricase had been inactivated. Inhibition of the OVA-specific response was not due to the capacity of uricase to inhibit OVA uptake or processing and presentation by dendritic cells, but at a later step that inhibited OVA-specific CD4+ T cell proliferation and cytokine production. Whereas blocking uric acid formation by allopurinol did not affect outcomes, administration of ultra-clean human serum albumin at protein concentrations equivalent to that of uricase inhibited NO2-promoted allergic airway disease. These results implicate that whereas uric acid levels are elevated in the airways of NO2-exposed mice, the powerful inhibitory effect of uricase administration on allergic sensitization is mediated more through antigen-specific immune deviation than on suppression of allergic sensitization, a mechanism to be considered in the interpretation of results from other experimental systems.