A

Fig. 7. H. pylori induces mitochondrial membrane depolarization, translocation of cytochrome c from mitochondria to cytosol, and activation of caspase-3 that is inhibited by MDL 72527. RAW 264.7 cells were treated with HPL in the absence and presence of MDL 72527 (250 ^M). (A) Mitochondrial membrane potential was measured by flow cytometry using MitoCapture dye at the times indicated. Summary data of mean relative fluorescence, expressed as negative values to indicate depolarization is shown. **p < 0.01 vs control, §§p < 0.01 vs HPL + MDL 72527. (B) Western blot analysis for cytochrome c (15 kDA protein). Upper panel: mito-chondrial fraction; middle panel: cytosolic fraction. Equal amounts of protein were loaded per lane, and equal loading was verified by staining of membranes with Ponceau S and immunoblot-ting for ß-actin (lower panel). (C) Cells were treated with HPL and caspase-3 activity measured by colorimetric assay at the times indicated. (D) In cells assessed at 24 h after stimulation, MDL 72527 (250 ^M) blocked the caspase-3 activation. For (C) and (D), **p < 0.01 vs control, §§p < 0.01 vs HPL. (Data shown are from ref. 41.)

Fig. 7. H. pylori induces mitochondrial membrane depolarization, translocation of cytochrome c from mitochondria to cytosol, and activation of caspase-3 that is inhibited by MDL 72527. RAW 264.7 cells were treated with HPL in the absence and presence of MDL 72527 (250 ^M). (A) Mitochondrial membrane potential was measured by flow cytometry using MitoCapture dye at the times indicated. Summary data of mean relative fluorescence, expressed as negative values to indicate depolarization is shown. **p < 0.01 vs control, §§p < 0.01 vs HPL + MDL 72527. (B) Western blot analysis for cytochrome c (15 kDA protein). Upper panel: mito-chondrial fraction; middle panel: cytosolic fraction. Equal amounts of protein were loaded per lane, and equal loading was verified by staining of membranes with Ponceau S and immunoblot-ting for ß-actin (lower panel). (C) Cells were treated with HPL and caspase-3 activity measured by colorimetric assay at the times indicated. (D) In cells assessed at 24 h after stimulation, MDL 72527 (250 ^M) blocked the caspase-3 activation. For (C) and (D), **p < 0.01 vs control, §§p < 0.01 vs HPL. (Data shown are from ref. 41.)

Macrophage Apoptosis

Fig. 8. Proposed mechanism of H. pylori-induced macrophage apoptosis in macrophages by polyamines. H. pylori stimulates expression of ODC, generating polyamines, and the increased spermine is acted upon by SMO(PAO1) that is also upregulated by H. pylori. The H2O2 so generated causes mitochondrial membrane depolarization, release of cytochrome c from mitochondria to cytosol, and activation of caspase-3, leading to apoptosis.

Fig. 8. Proposed mechanism of H. pylori-induced macrophage apoptosis in macrophages by polyamines. H. pylori stimulates expression of ODC, generating polyamines, and the increased spermine is acted upon by SMO(PAO1) that is also upregulated by H. pylori. The H2O2 so generated causes mitochondrial membrane depolarization, release of cytochrome c from mitochondria to cytosol, and activation of caspase-3, leading to apoptosis.

and additional studies are in progress in our laboratory to determine the response to other pathogenic stimuli.

5.2. Further Insights Into the Induction of Macrophage Apoptosis by Polyamines

Previous work from our laboratory has implicated specific H. pylori-derived factors in the activation of macrophages, including urease (3). This enzyme serves a biochemically essential function for the bacterium of converting urea to ammonia and carbon dioxide, creating an alkaline microenvironment required for the organism to survive in the acid milieu of the stomach. However, urease is also an immunologically active protein, and we have found that urease appears to mediate the induction of ODC in macrophages because isogenic mutant strains of H. pylori lacking urease exhibited significant attenuation of induction of ODC expression and activity, and recombinant urease stimulated ODC activation (43).

ODC expression has been shown to be enhanced by binding of the transcription factor c-Myc to the ODC promoter (6). We have substantial evidence that H. pylori and its urease induces ODC by a c-Myc-dependent process, because H. pylori induces c-Myc expression and inhibition of c-Myc binding prevents activation of the ODC promoter, ODC mRNA expression, ODC activity, and apoptosis (43). Additionally, we have recently found that the activation of c-Myc and induction of ODC in response to H. pylori is dependent on phosphorylation of extracellular signalregulated kinase (ERK) 1/2 (M. Asim, R. Chaturvedi, and K.T. Wilson, manuscript in preparation).

6. Involvement of SMO(PAO1) in the Pathogenic Aspects of Other Model Systems

We have conducted additional studies that indicate that SMO(PAO1) activity is important in other disease states. Specifically, we have recently reported that as in macrophages, SMO(PAO1) expression is upregulated in gastric epithelial cells exposed to H. pylori and that inhibition with MDL72527 or SMO(PAO1) siRNA significantly attenuated the induction of apoptosis (44). Importantly, we also demonstrated that DNA damage induced by H. pylori in these cells was mediated by H2O2 derived from induction of SMO(PAO1), which may have direct relevance to H. pylori-associated gastric cancer (44). Additionally, we reported that polyamines are beneficial in mouse colitis (45), and we now have evidence that SMO(PAO1) correlates with the severity of disease in several colitis models, suggesting that oxidative stress derived from spermine oxidation may play an important role either from apoptosis or other effects that we are now investigating.

7. The Balance of Spermine and its Oxidation Products, Determined by ODC and SMO(PAO1) is Important in the Regulation of Innate Immune Response

We have previously reported that in a coculture model in which H. pylori is separated from macrophages by a filter support the bacteria are killed by a NO-dependent mechanism (46). However, in the stomach, the bacteria persists for the life of the host, despite the expression of iNOS in the gastric mucosa (39). Because spermine has been shown to downregulate cytokine production by lipopolysaccharide-stimulated macrophages (47), we have assessed the effect of spermine on iNOS (48). Spermine inhibited NO production from H. pylori-stimulated macrophages in a concentration-dependent manner. Intriguingly, there was no effect on the induction of iNOS mRNA, but rather there was potent inhibition of iNOS protein translation. Consistent with this, knockdown of ODC by siRNA increased iNOS protein expression and NO production in response to H. pylori. This enhanced killing of the bacterium, whereas spermine inhibited killing (48). Recently, we have found that knockdown of SMO(PAO1) in H. pylori-stimulated macrophages prevents depletion of spermine, and thus inhibits iNOS protein levels, NO production, and bacterial killing (R. Chaturvedi and K.T. Wilson, manuscript in preparation).

8. Summary

Evidence has been presented in this review indicating that polyamines can have either a pro- or antiapoptotic effect in immune cells. This most likely depends greatly on the inducing stimuli, the cell type, and other factors in the intracellular environment. We have presented our work in the H. pylori model that has identified a pathway of macrophage apoptosis in which the activation of arginase, ODC, and SMO(PAO1) is required, with the latter resulting in generation of H2O2 that activates the mitochondr-ial pathway of apoptosis. We suggest that the balance of spermine and its oxidation is a critical aspect of innate immune response to pathogens, in that spermine accumulation inhibits responses such as iNOS-derived NO and cytokine production, whereas spermine catabolism leads to oxidative stress, apoptosis, and DNA damage.

9. Acknowledgments

Supported by grants to K. Wilson from the National Institutes of Health (R01

DK53620 and R21 DK63626), the Office of Medical Research, Department of Veterans

Affairs, and the Crohn's and Colitis Foundation of America.

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