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Toll-like receptor (TLR)-mediated inflammation may contribute to neonatal sepsis, for which pentoxifylline (PTX), a phosphodiesterase inhibitor that raises intracellular cAMP, is a candidate adjunctive therapy. We characterized the anti-inflammatory effects of PTX toward TLR-mediated production of inflammatory (tumor necrosis factor (TNF) and interleukin (IL)-1β) and proresolution (IL-6 and IL-10) cytokines in human newborn and adult blood. Newborn cord and adult blood were treated with PTX (50–400 µmol/l) before, during or after stimulation with LPS (TLR4 agonist), R848 (TLR7/8 agonist) or LPS/ATP (inflammasome activation). Cytokines were measured by multiplex assay (supernatants), intracellular cytokines and signaling molecules by flow cytometry, and mRNA by quantitative real-time PCR. Whether added 2 h pre-, simultaneously to, or 2 h post-TLR stimulation, PTX inhibited TLR-mediated cytokine production in a concentration-dependent manner, with greater efficacy and potency in newborn blood, decreasing intracellular TNF and IL-1β with relative preservation of IL-10 and IL-6. PTX decreased TLR-mediated TNF mRNA while increasing IL-10 mRNA. Neonatal plasma factors contributed to the anti-inflammatory effects of PTX in newborn blood that were independent of soluble TNF receptor concentrations, p38 MAPK phosphorylation and IĸB degradation. PTX is a potent and efficacious inhibitor of TLR-mediated inflammatory cytokines in newborn cord blood and a promising neonatal anti-inflammatory agent.

Nitric oxide (NO) has been discovered to be a potent vasodilator. Endogenous NO is produced by many cell types and is important in regulating basal vasomotor tone. Exogenous NO has recently been used very successfully to treat pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen therapy. Unfortunately, there are few reports on the combined toxicity of NO and hyperoxia, either of which can be toxic alone. Together, the potential for the formation of peroxynitrite(ONOO˙) - an extremely reactive and toxic free radical - is increased. To directly test this potential toxicity, cultured human alveolar epithelial(A549) cells were grown in room air (control), hyperoxia (95% O2), 2 mM SNAP (S-nitroso-N-acetyl penicillamine - an NO donor) and hyperoxia + SNAP. Live cells were counted in each of the groups above daily for the next six days, and media and gasses were refreshed each day. Cells began to die in hyperoxia after 4 to 5 d, but cell counts were relatively unchanged in NO. However, cells exposed to both NO and hyperoxia began to die on day 2, and died rapidly thereafter. This cytotoxic effect was clearly synergistic, the death rate far exceeding any additive effect. Some cells were grown on cover slips for later immunocytochemistry for nitrotyrosine, which is a stable byproduct of peroxynitrite (which is too reactive to detect directly). Immunoreactive nitrotyrosine was most abundant only in cells exposed to NO + hyperoxia. Western blots indicate that a variety of proteins become nitrated under various conditions. However, a unique band appeared on blots only after culture in NO + hyperoxia. These experiments indicate that NO and hyperoxia have a synergistic cytotoxic effect on alveolar cells in culture, which is probably mediated by production of peroxynitrite.(Funded by grants from the NIH-NHLBI, ALA and Winthrop-University Hospital)