In this study, we have demonstrated the in vivo detection of lipid-derived carbon-centeredfree radical in the SEB-induced IP model using the ESR spin-trapping method with POBN. SEB also increased the numbers of total cells, macrophages, neutrophils, and lymphocytes in the BAL fluid. These results suggest that the manifestation of IP is accompanied by lipid-derived carbon-centered free radical production. Although the ESR spin trapping technique can not prove the cell compartment of the POBN-trapped lipid-derived-carbon- centered radicals, we assume that POBN have dissolved significantly in the cell membrane since the octanol-water partition coefficient of POBN is 0.15 [22
]. We found that GdCl3
significantly inhibited lipid radical formation and lung inflammation as indicated by the number of total cells in the BAL fluid and the manifestation of IP. SEB-induced lipid radical was unaffected by the genetic inactivation of Nox, whereas the radical generation was markedly decreased by the XO inhibitor allopurinol. Pretreatment with the iron chelator Desferal or the iNOS inhibitor 1400W significantly inhibited lipid radical generation, macrophage and neutrophil counts in the BAL fluid, and histological changes in the lung. These results suggest that activated alveolar macrophages, XO, and NO are potential sources of lipid radical and, perhaps, important in the pathogenesis of SEB-induced IP.
Alteration of membrane integrity by peroxidation is known to modify membrane fatty acid composition, disrupt permeability, decrease electrical resistance, and increase flip-flopping between monolayers and inactivated cross-linked proteins [24
]. These collective effects of lipid peroxidation on cellular processes have been implicated as the underlying mechanism for numerous pathological conditions [26
]. Others have shown that free radical-induced lipid peroxidation also has relevance to LPS-induced acute lung injury [28
]. We found that the intratracheal instillation of SEB significantly enhanced free radicals in the lung, primarily in the form of lipid-derived carbon-centered free radicals detected as POBN radical adducts in the present study, and the free radical production was clearly correlated with an increase in alveolar macrophages and neutrophils ().
It is well known that phagocytes produce superoxide radicals via Nox, and several reports show that Nox has a crucial role in tissue damage [29
]. In the pulmonary system, ROS generated by Nox play distinct physiological roles in airway and vascular remodeling [31
]. Other studies suggest that excessive phagocyte-derived ROS also may play a role in lung injury during asthma and inflammatory events [32
]. Our studies with Nox2 −/− mice demonstrated that the generation of free radicals did not require oxidants generated by Nox.
On the other hand, we found that the production of lipid radical in the lung decreased and the histological change was ameliorated by the administration of allopurinol, an inhibitor of XO, the other enzyme which produces superoxide. XO and xanthine dehydrogenase (XDH) are interconvertible forms of the same enzyme, and XDH/XO is detected in several cells such as endothelial cells, epithelial cells, neutrophils, and macrophages [15
]. Most investigators agree that XDH activity converts to an oxidase that produces superoxide and hydrogen peroxide [36
]. This generation of ROS is thought to be the basis of XDH/XO involvement in various pathologic conditions such as influenza virus infection and neutrophil-mediated lung injury [37
]. Our data show that XO activation may play a role in the radical generation by superoxide overproduction since an inhibitor of XO suppressed free radical generation in SEB induced IP.
Other studies have demonstrated that some cytokines or hypoxia upregulated XDH/XO generation at the translational and post-transcriptional levels [34
]. Tumor necrosis factor-α (TNF-α), interleukin-1, and IFN-γ were shown to lead to increased XDH/XO activity in epithelial cells [34
]. XO enzyme activity is also enhanced in endothelial cells by TNF-α and chemotactic peptide [41
]. These findings indicate that XDH/XO activity is regulated in a cell-specific manner and by inflammatory cytokines and physiologic events. However, we do not have data to confirm or reject these findings.
As alveolar macrophages play an essential role in the inflammatory response, we examined the effect of GdCl3
, a macrophage inhibitor, on the generation of free radicals. The GdCl3
pretreatment attenuated the number of alveolar macrophages after injection [42
]. Some studies have shown that GdCl3
depressed hepatic macrophage phagocytic activity and the production of reactive nitrogen and oxygen intermediates [43
]. Several papers reported the inhibition of neutrophilic infiltration by GdCl3
, which may be the result of enzyme blockage and factors released from macrophages that are related to neutrophil migration and adhesion [17
]. Here, GdCl3
pretreatment also indicated that the activation of alveolar macrophages was important for lipid radical generation, inflammation, and the development of IP. Although Nox was not responsible for the production of lipid-derivedcarbon-centered free radicals, nevertheless, proinflammatory cytokines, chemokines, and adhesion molecules released from phagocytes may play an important role in the free radical generation and IP by SEB.
Our experiment with Desferal pretreatment indicates that free iron plays a role in the formation of lipid radical in IP induced by SEB instillation. Desferal pretreatment significantly reduced the amplitude of the ESR signal generated after SEB instillation. Histologically, Desferal also decreased the alveolar septa thickened with inflammatory cells and fibroblasts in the lungs. Desferal pretreatment significantly reduced not only free radical generation but also total cell count in the BAL fluid. It has been reported that alterations in proinflammatory cytokines, adhesion molecules, and chemotactic gradients play an important role in the accumulation of neutrophils or lymphocytes in lung inflammation [46
]. Desferal has been shown to interfere with the adhesion functions [47
]. In addition, a previous report showed that iron regulated XO activity in the lung [48
]. These findings suggest that Desferal-induced anti-inflammatory activities may be important in its protective effects, in addition to the inhibition of iron’s catalytic production of hydroxyl radical.
The effect of iNOS inhibitors on SEB-induced POBN adduct formation was also tested, because we previously reported that SEB enhanced the NO production from macrophages and neutrophils in the lung, and the iNOS inhibitor tends to protect against the development of IP in this model [15
]. Pretreatment with iNOS inhibitors significantly reduced the amplitude of the signal generated by SEB and decreased the thickened alveolar septa in inflammatory cells and fibroblasts in the lungs. These results suggest that NO also plays a role in the generation of lipid-derived carbon-centered free radicals in SEB-induced IP.
In conclusion, this is the first demonstration that superantigens generate lipid radicals. We found that SEB-induced lipid radical was generated through xanthine oxidase activation with iron and NO induction, but NADPH oxidase was not involved. Moreover, macrophage toxicants, xanthine oxidase inhibitors, iron chelators, or inducible nitric oxide synthase inhibitors may be potential therapeutic agents against alveolitis and fibrosis in interstitial pneumonia.