Importance of Arginase

We have demonstrated that H. pylori upregulates expression of inducible nitric oxide (NO) synthase (iNOS) in gastritis tissues in vivo (39) and in macrophages in vitro (3), and iNOS-derived NO had been implicated as a causal factor in macrophage apoptosis in response to lipopolysaccharide plus interferon-y (40). When we identified that H. pylori also upregulated the arginase II isoform of arginase, which competes with iNOS for the same substrate, L-arginine, we determined the respective roles of arginase II and iNOS in H. pylori-induced macrophage apoptosis (35). We were initially surprised to find that iNOS played no role because use of iNOS inhibitors or peritoneal macrophages from iNOS-/- mice had no effect on the apoptosis, whereas two different inhibitors of arginase effectively inhibited apoptosis (35).

Arginase Cancer

Fig. 1. ODC activity and polyamines enhance macrophage apoptosis. (A) Helicobacter pylori-stimulated macrophage apoptosis is ODC dependent. RAW 264.7 cells were cocultured with H. pylori, with or without DFMO (5 mM) and 5 ^Mputrescine (put), spermidine (spd), or spermine (sp). After 24 h, macrophage apoptosis levels were determined by enzyme-linked immunosorbent assay. **p < 0.01 vs control; §§p < 0.01 vs H. pylori-stimulated macrophages; ##p < 0.01 vs macrophages stimulated with H. pylori in the presence of DFMO. (B) Stimulation of macrophage apoptosis by polyamines alone. RAW 264.7 cells were treated for 24 h with different concentrations of putrescine, spermidine, or spermine. *p < 0.05, **p < 0.01 vs control macrophages. (Data shown are from ref. 35, Copyright 2002, The American Association of Immunologists, Inc.)

Fig. 1. ODC activity and polyamines enhance macrophage apoptosis. (A) Helicobacter pylori-stimulated macrophage apoptosis is ODC dependent. RAW 264.7 cells were cocultured with H. pylori, with or without DFMO (5 mM) and 5 ^Mputrescine (put), spermidine (spd), or spermine (sp). After 24 h, macrophage apoptosis levels were determined by enzyme-linked immunosorbent assay. **p < 0.01 vs control; §§p < 0.01 vs H. pylori-stimulated macrophages; ##p < 0.01 vs macrophages stimulated with H. pylori in the presence of DFMO. (B) Stimulation of macrophage apoptosis by polyamines alone. RAW 264.7 cells were treated for 24 h with different concentrations of putrescine, spermidine, or spermine. *p < 0.05, **p < 0.01 vs control macrophages. (Data shown are from ref. 35, Copyright 2002, The American Association of Immunologists, Inc.)

4.2. Implication of Polyamines in Macrophage Apoptosis

In considering the likely mechanism of the latter effect, because arginase generates L-ornithine, and the latter is the substrate for ODC and the synthesis of polyamines, we determined if polyamines mediated the apoptosis. As shown in Fig. 1, inhibition of polyamine synthesis with DFMO completely abrogated the induction of apoptosis by H. pylori (35). The apoptosis could be restored by add-back of spermidine or spermine, but not putrescine, and similarly addition of spermidine or spermine alone, but not putrescine, induced macrophage apoptosis. This led us to hypothesize that both arginase II and ODC induction was required for the H. pylori-induced macrophage apoptosis. Because macrophage apoptosis in response to a pathogen would be undesirable for the host, by leading to depletion of cells necessary for the innate immune response, we pursued additional studies to determine how polyamines could be causing the apoptosis.

4.3. Induction of ODC and SMO(PAO1) and the Relationship to Apoptosis

Because polyamine oxidation had been implicated in the apoptosis of other cell types, we hypothesized that H. pylori was capable of inducing this phenomenon in macrophages. Because H. pylori is a noninvasive organism in vivo, we established that

Helicobacter Pylori Macrophage

Fig. 2. Helicobacter pylori induces mRNA expression of ornithine decarboxylase, polyamine oxidase 1, and spermidine/spermine W'-acetyltransferase in RAW 264.7 macrophages. Cells were stimulated with lysate of H. pylori at a multiplicity of infection of 100 for 6 h. (A) Reverse transcriptase polymerase chain reaction; (B) Real-time polymerase chain reaction analysis, using SYBR Green, data are shown on a logarithmic scale. **p < 0.01, ***p < 0.001 vs unstimulated cells. Data shown are from ref. 41. HPL, lysate of H. pylori; ODC, ornithine decarboxylase; PAO1, polyamine oxidase; SSAT, spermidine/spermine W-acetyltransferase.

Fig. 2. Helicobacter pylori induces mRNA expression of ornithine decarboxylase, polyamine oxidase 1, and spermidine/spermine W'-acetyltransferase in RAW 264.7 macrophages. Cells were stimulated with lysate of H. pylori at a multiplicity of infection of 100 for 6 h. (A) Reverse transcriptase polymerase chain reaction; (B) Real-time polymerase chain reaction analysis, using SYBR Green, data are shown on a logarithmic scale. **p < 0.01, ***p < 0.001 vs unstimulated cells. Data shown are from ref. 41. HPL, lysate of H. pylori; ODC, ornithine decarboxylase; PAO1, polyamine oxidase; SSAT, spermidine/spermine W-acetyltransferase.

it could induce macrophage apoptosis in vitro when separated from the cells by a filter support (35) or when bacterial supernatants or lysed bacteria were used (41). Figure 2 demonstrates that H. pylori lysate (HPL) induced increased messenger RNA (mRNA) expression of ODC, SMO(PAO1), and SSAT, but not APAO (41). ODC activity peaked at 6 h after HPL stimulation and exhibited elevation to 24 h (Fig. 3A). As shown in Fig. 3B, SMO(PAO1) activity was significantly increased at 6 h and continued to increase until its peak at 18 h. In contrast, APAO activity is not induced, and SSAT activity is not detectable until 18 h. The polyamine profile after HPL stimulation (Fig. 3C) indicated that

Fig. 3

in the first 12 h, all of the putrescine was likely converted to spermidine. The increase in spermidine was significant at 6 and 12 h, and the spermine was increased at 12 h, but less so than spermidine; from 12-24 h, there was a clear decline in spermine and sper-midine that was inversely proportional to the increase in putrescine. These data are consistent with the initial induction of SMO(PAO1) followed by the subsequent induction of SSAT activity.

By using annexin V plus PI labeling of live cells and flow cytometry, we measured both early and late apoptosis (41). Figure 3D indicates that early apoptosis, representing annexin V+/PI- cells, was significantly increased at 6 and 12 h after HPL stimulation, and as the early apoptosis decreased from 12 to 24 h, there was a concomitant increase in late apoptosis that peaked at 18 h, an indication that early apoptosis represented a progressive apoptotic process. The presence of apoptosis beginning at 6 h was consistent with the initial spike in ODC activity at 6 h and the increase in PAO1 activity at this point, and argues against an important role of the SSAT-APAO pathway in the apoptosis, because SSAT activity did not increase until 18 h after stimulation.

4.4. H. pylori-Induced Macrophage Apoptosis is Dependent on Spermine Oxidation by SMO(PAO1)

Because the time course of induction of gene expression and enzyme activity of SMO(PAO1) and polyamine back conversion correlated with apoptosis, we determined the effect of an inhibitor of PAO, MDL 72527. As shown in Fig. 4, the increase in apoptosis and reduction in cell viability with HPL stimulation was inhibited in a concentration-dependent manner by MDL 72527. Similar results were observed in non-transformed mouse peritoneal macrophages (41). Because MDL 72527 inhibits both APAO and SMO(PAO1), we transiently transfected RAW 264.7 cells with a small interfering RNA (siRNA) specific for SMO(PAO1). As shown in Fig. 5, PAO1 siRNA markedly decreased the HPL-stimulated PAO1 mRNA expression (Fig. 5A) and completely blocked the induction of PAO1 enzyme activity (Fig. 5B). In parallel, the PAO1 siRNA completely abolished the HPL-induced apoptosis (Fig. 5C), and restored the cell viability (Fig. 5D). Because knockdown of stimulated PAO1 expression completely eliminated HPL-induced macrophage apoptosis, we have provided direct evidence that SMO(PAO1), specifically, is responsible for the apoptosis (41).

Fig. 3. Time course of induction of enzyme activities of ODC, PAO1, SSAT, and associated changes in polyamine levels and apoptosis. RAW 264.7 macrophages were stimulated with HPL at multiplicity of infection of 100. In (A), ODC activity was measured by conversion of [14C]l-ornithine to [14C]O2. In (B), PAO1 and APAO activities were measured by the luminol method, assessing liberation of H2O2 from either spermine in the case of PAO1, or A^-acetylspermine, for APAO. SSAT activity was determined by incorporation of l-[14C]acetylCoA into spermidine and formation of l-[14C]acetylspermidine. In (C), polyamine levels were determined by HPLC from macrophage lysates. In (D), live cells were stained with annexin V (FITC-labeled) and propidium iodide (PI) and flow cytometry performed. Summary data demonstrating peak early apoptosis (annexin V positive only) at 12 h, and peak late apoptosis (both annexin V and PI positive) at 24 h. *p < 0.05, **p < 0.01 vs time 0, §p < 0.05, §§p < 0.01 vs peak level. (Data shown are from ref. 41.)

Fig. 4. Inhibition of H. pylori-induced apoptosis in macrophages by the PAO inhibitor MDL 72527. (A) Summary data of apoptosis in RAW 264.7 cells stimulated with H. pylori lystate (HPL), determined by propidium iodide (PI) staining of fixed cells followed by flow cytometry and quantification of apoptosis by ModFit-LT software. (B) Demonstration that HPL reduces cell viability, determined by XTT assay, with HPL, and inhibition of this effect with MDL 72527 in concordance with data in (A). (C) Representative histogram plots of PI stained cells (data summarized in [A]) showing the increase in cells in the sub-G0/Gj fraction, indicative of apoptosis, with HPL stimulation, and inhibition with MDL 72527. **p < 0.01 vs unstimulated cells, §§p < 0.01 vs HPL only. (Data shown are from ref. 41.)

Fig. 4. Inhibition of H. pylori-induced apoptosis in macrophages by the PAO inhibitor MDL 72527. (A) Summary data of apoptosis in RAW 264.7 cells stimulated with H. pylori lystate (HPL), determined by propidium iodide (PI) staining of fixed cells followed by flow cytometry and quantification of apoptosis by ModFit-LT software. (B) Demonstration that HPL reduces cell viability, determined by XTT assay, with HPL, and inhibition of this effect with MDL 72527 in concordance with data in (A). (C) Representative histogram plots of PI stained cells (data summarized in [A]) showing the increase in cells in the sub-G0/Gj fraction, indicative of apoptosis, with HPL stimulation, and inhibition with MDL 72527. **p < 0.01 vs unstimulated cells, §§p < 0.01 vs HPL only. (Data shown are from ref. 41.)

4.5. Overexpression of SMO(PAO1) Causes Macrophage Apoptosis

When RAW 264.7 cells were transfected with a full-length complimentary DNA for SMO(PAO1), there was a 7.3 ± 0.9-fold increase in SMO(PAO1) activity that was paralleled by a 7.2 ± 0.5-fold increase in apoptosis; the level of enzyme activity and apoptosis so generated was essentially identical to that induced by HPL in the neo-transfected cells (41).

4.6. H. pylori-/nc/ucec/ SMO(PAO1) Results in H2O2 Generation That Causes Apoptosis in Macrophages

To determine if H2O2 released by SMO(PAO1) activation was responsible for the apoptosis, we measured intracellular H2O2 levels in response to HPL by flow cytometry.

Fig. 5. Transfection with PAO1 siRNA inhibits H. pylori-stimulated mRNA expression, enzyme activity, and apoptosis in macrophages. RAW 264.7 cells were transiently transfected with duplex small interfering RNA (siRNA) targeted against nt 467-487 in the coding sequence for murine PAO1 or with scrambled siRNA control. (A) Reverse transcriptase polymerase chain reaction analysis of cells transfected as indicated, in the absence and presence of HPL for 6 h. (B) PAO1 enzyme activity measured as in Fig. 2B, after 24 h. (C) apoptosis, measured by PI staining and flow cytometry of fixed cells. (D) Cell viability, measured by XTT assay. **p < 0.01 vs scrambled siRNA alone without HPL; §§p < 0.01 for PAO1 siRNA vs scrambled siRNA. (Data shown are from ref. 41)

Fig. 5. Transfection with PAO1 siRNA inhibits H. pylori-stimulated mRNA expression, enzyme activity, and apoptosis in macrophages. RAW 264.7 cells were transiently transfected with duplex small interfering RNA (siRNA) targeted against nt 467-487 in the coding sequence for murine PAO1 or with scrambled siRNA control. (A) Reverse transcriptase polymerase chain reaction analysis of cells transfected as indicated, in the absence and presence of HPL for 6 h. (B) PAO1 enzyme activity measured as in Fig. 2B, after 24 h. (C) apoptosis, measured by PI staining and flow cytometry of fixed cells. (D) Cell viability, measured by XTT assay. **p < 0.01 vs scrambled siRNA alone without HPL; §§p < 0.01 for PAO1 siRNA vs scrambled siRNA. (Data shown are from ref. 41)

As shown in Fig. 6A, HPL induced a significant increase that was attenuated by MDL 72527 or the H2O2-detoxifying enzyme, catalase. We also used the Amplex Red® assay that specifically measures H2O2 in solution to demonstrate that supernatant levels of

Production H2o2 Live Cell

Fig. 6. H. pylori induces H2O2 production in RAW 264.7 macrophages, that is inhibited by MDL 72527, and catalase, and inhibition of apoptosis with catalase. Cells were stimulated with HPL and live macrophages were treated with CM-H2DCFDA; representative fluorescence data are shown. The thick line represents the fluorescence tracing from unstimulated cells. The thin line represents the fluorescence tracing from the cells stimulated with HPL alone or with HPL plus MDL 72527 or catalase. Note the shift of the curve to the right with HPL, indicating more fluorescence intensity, which is prevented by MDL 72527 or catalase. (B) Catalase inhibits apoptosis in RAW 264.7 macrophages, as assessed by staining with PI and annexin V. **p < 0.01 vs control, §p < 0.05, §§p < 0.01 vs HPL alone. (Data shown are from ref. 41.)

Fig. 6. H. pylori induces H2O2 production in RAW 264.7 macrophages, that is inhibited by MDL 72527, and catalase, and inhibition of apoptosis with catalase. Cells were stimulated with HPL and live macrophages were treated with CM-H2DCFDA; representative fluorescence data are shown. The thick line represents the fluorescence tracing from unstimulated cells. The thin line represents the fluorescence tracing from the cells stimulated with HPL alone or with HPL plus MDL 72527 or catalase. Note the shift of the curve to the right with HPL, indicating more fluorescence intensity, which is prevented by MDL 72527 or catalase. (B) Catalase inhibits apoptosis in RAW 264.7 macrophages, as assessed by staining with PI and annexin V. **p < 0.01 vs control, §p < 0.05, §§p < 0.01 vs HPL alone. (Data shown are from ref. 41.)

H2ü2 induced by HPL were markedly inhibited by MDL 72527 or catalase. Consistent with these findings, catalase inhibited apoptosis in a concentration-dependent manner (Fig. 6B). Cell-permeable polyethylene glycolated-catalase was able to inhibit the apoptosis by 100% (41). These studies indicate that H2O2 generation is a major cause of HPL-induced apoptosis.

4.7. SMO(PAO1) Induction Results in Mitochondrial Membrane Depolarization, Cytochrome c Release, and Caspase-3 Activation

Because depolarization of mitochondrial membrane potential (Aym) has been implicated in apoptosis, we determined the ability of H. pylori to cause this event (41). As shown in Fig. 7A, there was a significant decrease in Ay that was significantly inhibited by MDL 72527. To confirm activation of the mitochondrial apoptosis pathway, release of cytochrome c from mitochondria to cytosol was assessed by immunoblot-ting. As shown in Fig. 7B, with HPL stimulation there was a significant decrease in mitochondrial cytochrome c, and a concomitant increase in cytoplasmic cytochrome c, indicating translocation from mitochondria to cytosol. In cells treated with MDL 72527, there was inhibition of the decrease in mitochondrial levels and the increase in cytosolic levels of cytochrome c, indicating the prevention of the translocation. These findings were confirmed by immunohistochemistry (41). Cytochrome c released from mitochondria activates caspase-9, which ultimately activates caspase-3, a final step in activation of exonucleases and apoptosis (42). After activation with HPL, macrophage caspase-3 activity increased significantly in a time-dependent manner (Fig. 7C) that paralleled that of the late apoptosis (Fig. 3D). As shown in Fig. 7D, MDL 72527 significantly reduced caspase-3 activity.

4.8. Proposed Mechanism of H. pylori-Induced Apoptosis by Polyamines

Figure 8 provides a schematic of interpretation of our findings that H. pylori itself or soluble products derived from the bacterium induce apoptosis caused by expression of ODC and SMO(PAO1). ODC generates putrescine that is converted into spermidine and spermine. The latter is back converted by the increased levels of SMO(PAO1) to spermidine, releasing H2O2 that causes depolarization of the mitochondrial membrane, release of cytochrome c to the cytosol, and activation of caspase-3, leading to apoptosis.

5. Additional Aspects of the Induction of Macrophage Apoptosis by Polyamines 5.1. Specificity of Response

Because macrophages exposed to stress, such as with H. pylori infection, undergo a vast array of innate responses, it is possible that the induction of apoptosis itself could be activating polyamine synthesis or oxidative catabolism. However, we have determined that when apoptosis is inhibited by catalase or by cyclosporine A (which blocks mitochondrial membrane depolarization), the induction of ODC or SMO(PAO1) by H. pylori is not prevented, and when apoptosis is induced by another bacterial stimulus, Citrobacter rodentium (which causes colitis in mice) or a chemical stimulus, stau-rosporine, ODC, and PAO1 are not induced (Y. Cheng, R. Chaturvedi, and K. T. Wilson, unpublished data). These data indicate that the response to H. pylori may be specific,

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