- Email: [email protected]
Immunohistochemical localization of intestinal mucosal inducible and constitute nitric oxide synthases in endotoxemic rats
A942
AGA ABSTRACTS
• IMMUNOHISTOCHEMICAL DETECTION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN HELICOBACTER PYLORI GASTRITIS. K.T. Wilson. R.F. Musselman, L. El Mahdi, and R.T. Jones. Depts. of Medicine and Pathology, VAMC and University of Maryland School of Medicine, Baltimore, MD. Helicobacter pylori (HP) infection results in gastritis and may have an important role in the etiology of gastric cancer. Nitric oxide (NO) is an immunomodulatory messenger molecule and a known mutagen. We have recently determined that HP induces iNOS expression and activity in macropbages in vitro. We hypothesize that HP stimulates increased gastric mucosal inducible nitric oxide synthase (iNOS) expression. AIMS: 1)to determine if iNOS expression is induced by HP in situ in human stomach, 2) to localize iNOS protein expression in HP gastritis, and 3) to compare these findings with constitutive NOS expression. METHODS: Endoscopic antral biopsies were formalin fixed, paraffin embedded, and 5 pm sections were cut. Eight patients with CLOtest positive biopsies, HP visible on hematoxylin and eosin stain, and histologically active gastritis were compared with HP negative control patients without gastritis. Sections were incubated with each of the following antibodies: mouoclonal anti-murine iNOS, monoclonal anti-human endothelial eNOS, or polycl0nal anti-human neuronal eNOS. Staining was detected with peroxidase-DAB. RESULTS: Abundance of mucosal stainin~ for NOS n-eNOS e-eNOS iNOS Control + - ÷+ + HP Gastritis + - ++ +++ ++ Staining for iNOS was absent in the mucosa of control patients. In tissue from patients with HP gastritis, a marked increase in iNOS was detected. Staining appeared to localize to the mucosal blood vessels and may have involved both the endothelinm and perivascular cells. In most biopsies, lamina propria cells were also positively stained for iNOS. Both n-eNOS and e-eNOS were localized mainly to the gastric epithelium and blood vessels, the latter more intensively stained with e=cNOS. In gastritis biopsies, there was an increase in e-eNOS, but the localization was not changed from that in controls. CONCLUSIONS: We have demonstrated for the first time that iNOS protein is expressed in the gastric mucosa of patients with active HP gastritis. The localization was mainly to endothelium and lamina propria. We Speculate that inducible NO production due to iNOS and/or over-expression of e-eNOS may play a role in HP-induced mucosal injury and inflammation. We are presently characterizing NOS expression in other forms of gastritis.
CYTOTOXICITY, ADHESION AND INVASION BY CLOSTRIDIUM SEPTIC~M. IN CULTURED HUMAN CELL LINES: RELATIONSHIP TO CELLULAR DIFFERENTIATION AND SWARM CELL FORMATION. L. M. Wilson, G. T. Macfarlane. MRC Dunn Clinical Nutrition Centre, Cambridge, U. K.
Clostridium septicum is an opportunistic human pathogen and is the primary aetiologic agent of neutropenic enterocolitis, a rapidly spreading and often fatal heemorrhagic infection of the ileocaecal region of the large bowel. Virulence in this bacterium is primarily expressed by the elaboration of a number of toxins and spreading factors (heemolysins, DNase, gelatinasesl neuraminidases, hyaluronidases). We have previously shown that production of these virulence determinants is co-ordinately linked to the bacterial swarm cell cycle in C septicum. The objectives of this investigation were to determine whether interactions between C. septicum and cultured human cell lines are also related to swarm cell differentiation. Two human cell lines were used to study cytotoxicity, invasion and adhesion of C. septicum : Caco-2 which are derived from colonic epithelial cells and Hep-'2 (laryngeal epithelial cells). In both cell lines, differentiation of normal motile vegetative C. septicum into swarming bacteria was accompanied by a marked decrease in cytotoxicity. This correlated with in vitro physiologic studies which showed that swarm cells do not secrete n-toxin (the only lethal cytotoxin produced by this bacterium). It was also observed that Caco-2 cells were more susceptible to ~-toxin than Hep-2 cells, indicating a degree of target cell specificity in its activity. C septicum adhered to Hep-2 cells, suggesting that Caco-2 cells lacked the necessary cell surface receptors for binding bacteria: Vegetative forms of C. septicum adhered in greater numbers than swarming bacteria to cell monolayers. Potential adherence mechanisms were investigated, including the ability of bacterial flagella to function as adhesins. Experimental results showed however, that removal of flagella by mechanical shearing, in both swarm and non-swarm cells was not accompanied by any significant reduction in adhesion. Electron micrographs of these bacteria did not indicate the presence of pili or other surface structures which are often implicated in bacterial adhesion. Incubation of the bacteria with various sugars (sucrose, D- and Lfucose, D-mannose, maltose) prior to their addition to cell monolayers did not inhibit adhesion, it may therefore be postulated that these sugars do not play a role as cell surface receptors for adhesion in C. septicum.Cell invasion is a survival strategy utilized by many pathogens capable of cell adhesion. We found that only vegetative forms of C. septicumwere able to invade Hep-2 cells. Invasion was never observed with Caco-2 cells, indicating that this process may be cell-type specific.
GASTROENTEROLOGY, Vol. 108, NO. 4
IMMUNOHISTOCHEMICAL LOCALIZATION OF INTESTINAL MUCOSAL INDUCIBLE AND CONSTITUTIVE NITRIC OXIDE SYNTHASES IN ENDOTOXEMIC RATS. K.T. Wilson L. E1 Mahdi, and R.T. Jones. Depts. of Medicine and Pathology, VAMC and Univ. of Maryland School of Medicine, Baltimore, MD. The relative activities of both constitutive (eNOS) and inducible (iNOS) nitric oxide (NO) synthases may modulate intestinal mucosal injury and inflammation. We have demonstrated that inducible nitric oxide syntbase (iNOS) mRNA expression is markedly increased in the intestinal mucosa of endotoxemic rats. AIMS: 1) to assess the level of intestinal iNOS regulation by correlating iNOS mRNA levels with protein expression and functional activity, 2) to immunolocalize iNOS in intestinal mueosa, and 3) to compare these findings with constitutive NOS expression. METHODS: Rats were treated with 1.5 mg/kg iv lipopolysaccharide (LPS) from Salmonella enteritidis or saline control and euthanized at 3 and 6 hr. 51.tm sections from paraformaldehyde fixed intestinal segments were incubated with the following antibodies: monoclonal anti-murine iNOS, monoclonal anti-human endothelial eNOS (e-eNOS), or polyelonal anti-human neuronal eNOS (n-eNOS). Staining was detected with peroxidase-DAB, Calcium dependent or independent NOS activity was measured in mucosal homogenates by the conversion of [14C]-L-arginine to [14C]-L-citrulline. RESULTS: Abundance of mucosal stainine for NOS Control LPS Colon Ileum Jejunum Colon Ileum Jejunum e-eNOS + + + ++ ÷++ +++ n-eNOS + + + + + iNOS +/+/+/++ +++ ++ In control tissues there was a low level of staining for e-eNOS in vessels and epithelium and for n-eNOS in epithelium only. In tissues from LPS-treated rats, there was a marked increase in iNOS staining; this localized to the crypt epithelium in each tissue. In ileum and jejunum there was also marked staining in the lamina propria, e-eNOS detection was increased in LPS tissues, with staining of vessels and epithelium in all segments. Staining was also observed in the lamina propria in jejunum and ileum. In comparison, in LPS tissues there was a 7-fold, 21-fold, and 5-fold increase in iNOS activity in colon, ileum, and jejunum, respectively (p <0.05 vs. control for all). There was no increase in eNOS activity with LPS. There was no significant difference in iNOS or eNOS staining or enzyme activities at 3 vs. 6 hr after LPS. CONCLUSIONS: Endotoxemia induces a marked increase in mucosal iNOS protein expression and activity in the intestine that parallels mRNA expression. Localization of stimulated iNOS expression to the epithelial crypts suggests that NO may play a role in acute injury in these metabolically active cells. The stimulated e-eNOS expression, primarily in ileum and jejunum, indicates that further studies of intestinal e-eNOS are warranted.
LOCAL SUPPRESSION OF INTERFERON-'/PRODUCTION BY HEPATIC GRANULOMA CELLS CORRELATES WITH TISSUESPECIFIC REPLICATION OF LEISHMANIA CHAGASI. M.E. Wilson, A.M. Blum, M .Sandor, A. Metwali, D. Elliott, R.G. Lynch, J.V~ Weinstock. Depts. Intemai Medicine, Microbiology & Pathology, University of Iowa and Veterans' Affairs Medical Center, Iowa City, IA. Introduction: BALB/c mice are susceptible to infection with Leishmania chagasL The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate this organ-specific multiplication ofL. chagasi, we compared cytokine production by splenic versus hepatic immune cells from infected mice. Results: The liver harbored many intracellular L. chagasi amastigotes that were all located in granulomas, whereas the spleen had few amastigotes. Granulomas were isolated from the liver and dispersed with collagenase for further analysis. Many granuloma, but few splenic lymphocytes expressed a memory/effector phenotype according to FACS analysis. Unfraetionated, dispersed granuloma cells cultured in vitro produced IL-10 and IL-6 constitutively, but no detectable interferon-y (IFN-y), IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-¥, IL-4, ][I,-6 and 1I,-10. Positive and negative selection experiments suggested the granuloma IL-10 and IL-6 were ofnon-T cell origin. Paradoxically, FACS-purified Thy-1 + granuloma lymphocytes produced IFN-y when cultured in the absence of other granuloma cells, suggesting that IFN-~/production was inhibited by a granuloma-associated non-T cell element(s). Co-culture ofgranuloma cells with splenocytes inhibited the usual splenocyte production of IFN-y and IL-4, but did not affect IL-10 release supporting this premise. Conclusion: Leishmania granulomas have an immunoregulatory mechanism that inhibits lyrnphokine secretion accounting for the absence of IFN-y in hepatic granuloma cultures. There is no such suppression in the spleen, which allows splenocytes to make IFN-y that, in turn, inhibits local parasite growth. Grant support: VAMC, Merck, AI30126, A132135, AM38327, DK07663, AHA, CCFA
AGA ABSTRACTS
• IMMUNOHISTOCHEMICAL DETECTION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN HELICOBACTER PYLORI GASTRITIS. K.T. Wilson. R.F. Musselman, L. El Mahdi, and R.T. Jones. Depts. of Medicine and Pathology, VAMC and University of Maryland School of Medicine, Baltimore, MD. Helicobacter pylori (HP) infection results in gastritis and may have an important role in the etiology of gastric cancer. Nitric oxide (NO) is an immunomodulatory messenger molecule and a known mutagen. We have recently determined that HP induces iNOS expression and activity in macropbages in vitro. We hypothesize that HP stimulates increased gastric mucosal inducible nitric oxide synthase (iNOS) expression. AIMS: 1)to determine if iNOS expression is induced by HP in situ in human stomach, 2) to localize iNOS protein expression in HP gastritis, and 3) to compare these findings with constitutive NOS expression. METHODS: Endoscopic antral biopsies were formalin fixed, paraffin embedded, and 5 pm sections were cut. Eight patients with CLOtest positive biopsies, HP visible on hematoxylin and eosin stain, and histologically active gastritis were compared with HP negative control patients without gastritis. Sections were incubated with each of the following antibodies: mouoclonal anti-murine iNOS, monoclonal anti-human endothelial eNOS, or polycl0nal anti-human neuronal eNOS. Staining was detected with peroxidase-DAB. RESULTS: Abundance of mucosal stainin~ for NOS n-eNOS e-eNOS iNOS Control + - ÷+ + HP Gastritis + - ++ +++ ++ Staining for iNOS was absent in the mucosa of control patients. In tissue from patients with HP gastritis, a marked increase in iNOS was detected. Staining appeared to localize to the mucosal blood vessels and may have involved both the endothelinm and perivascular cells. In most biopsies, lamina propria cells were also positively stained for iNOS. Both n-eNOS and e-eNOS were localized mainly to the gastric epithelium and blood vessels, the latter more intensively stained with e=cNOS. In gastritis biopsies, there was an increase in e-eNOS, but the localization was not changed from that in controls. CONCLUSIONS: We have demonstrated for the first time that iNOS protein is expressed in the gastric mucosa of patients with active HP gastritis. The localization was mainly to endothelium and lamina propria. We Speculate that inducible NO production due to iNOS and/or over-expression of e-eNOS may play a role in HP-induced mucosal injury and inflammation. We are presently characterizing NOS expression in other forms of gastritis.
CYTOTOXICITY, ADHESION AND INVASION BY CLOSTRIDIUM SEPTIC~M. IN CULTURED HUMAN CELL LINES: RELATIONSHIP TO CELLULAR DIFFERENTIATION AND SWARM CELL FORMATION. L. M. Wilson, G. T. Macfarlane. MRC Dunn Clinical Nutrition Centre, Cambridge, U. K.
Clostridium septicum is an opportunistic human pathogen and is the primary aetiologic agent of neutropenic enterocolitis, a rapidly spreading and often fatal heemorrhagic infection of the ileocaecal region of the large bowel. Virulence in this bacterium is primarily expressed by the elaboration of a number of toxins and spreading factors (heemolysins, DNase, gelatinasesl neuraminidases, hyaluronidases). We have previously shown that production of these virulence determinants is co-ordinately linked to the bacterial swarm cell cycle in C septicum. The objectives of this investigation were to determine whether interactions between C. septicum and cultured human cell lines are also related to swarm cell differentiation. Two human cell lines were used to study cytotoxicity, invasion and adhesion of C. septicum : Caco-2 which are derived from colonic epithelial cells and Hep-'2 (laryngeal epithelial cells). In both cell lines, differentiation of normal motile vegetative C. septicum into swarming bacteria was accompanied by a marked decrease in cytotoxicity. This correlated with in vitro physiologic studies which showed that swarm cells do not secrete n-toxin (the only lethal cytotoxin produced by this bacterium). It was also observed that Caco-2 cells were more susceptible to ~-toxin than Hep-2 cells, indicating a degree of target cell specificity in its activity. C septicum adhered to Hep-2 cells, suggesting that Caco-2 cells lacked the necessary cell surface receptors for binding bacteria: Vegetative forms of C. septicum adhered in greater numbers than swarming bacteria to cell monolayers. Potential adherence mechanisms were investigated, including the ability of bacterial flagella to function as adhesins. Experimental results showed however, that removal of flagella by mechanical shearing, in both swarm and non-swarm cells was not accompanied by any significant reduction in adhesion. Electron micrographs of these bacteria did not indicate the presence of pili or other surface structures which are often implicated in bacterial adhesion. Incubation of the bacteria with various sugars (sucrose, D- and Lfucose, D-mannose, maltose) prior to their addition to cell monolayers did not inhibit adhesion, it may therefore be postulated that these sugars do not play a role as cell surface receptors for adhesion in C. septicum.Cell invasion is a survival strategy utilized by many pathogens capable of cell adhesion. We found that only vegetative forms of C. septicumwere able to invade Hep-2 cells. Invasion was never observed with Caco-2 cells, indicating that this process may be cell-type specific.
GASTROENTEROLOGY, Vol. 108, NO. 4
IMMUNOHISTOCHEMICAL LOCALIZATION OF INTESTINAL MUCOSAL INDUCIBLE AND CONSTITUTIVE NITRIC OXIDE SYNTHASES IN ENDOTOXEMIC RATS. K.T. Wilson L. E1 Mahdi, and R.T. Jones. Depts. of Medicine and Pathology, VAMC and Univ. of Maryland School of Medicine, Baltimore, MD. The relative activities of both constitutive (eNOS) and inducible (iNOS) nitric oxide (NO) synthases may modulate intestinal mucosal injury and inflammation. We have demonstrated that inducible nitric oxide syntbase (iNOS) mRNA expression is markedly increased in the intestinal mucosa of endotoxemic rats. AIMS: 1) to assess the level of intestinal iNOS regulation by correlating iNOS mRNA levels with protein expression and functional activity, 2) to immunolocalize iNOS in intestinal mueosa, and 3) to compare these findings with constitutive NOS expression. METHODS: Rats were treated with 1.5 mg/kg iv lipopolysaccharide (LPS) from Salmonella enteritidis or saline control and euthanized at 3 and 6 hr. 51.tm sections from paraformaldehyde fixed intestinal segments were incubated with the following antibodies: monoclonal anti-murine iNOS, monoclonal anti-human endothelial eNOS (e-eNOS), or polyelonal anti-human neuronal eNOS (n-eNOS). Staining was detected with peroxidase-DAB, Calcium dependent or independent NOS activity was measured in mucosal homogenates by the conversion of [14C]-L-arginine to [14C]-L-citrulline. RESULTS: Abundance of mucosal stainine for NOS Control LPS Colon Ileum Jejunum Colon Ileum Jejunum e-eNOS + + + ++ ÷++ +++ n-eNOS + + + + + iNOS +/+/+/++ +++ ++ In control tissues there was a low level of staining for e-eNOS in vessels and epithelium and for n-eNOS in epithelium only. In tissues from LPS-treated rats, there was a marked increase in iNOS staining; this localized to the crypt epithelium in each tissue. In ileum and jejunum there was also marked staining in the lamina propria, e-eNOS detection was increased in LPS tissues, with staining of vessels and epithelium in all segments. Staining was also observed in the lamina propria in jejunum and ileum. In comparison, in LPS tissues there was a 7-fold, 21-fold, and 5-fold increase in iNOS activity in colon, ileum, and jejunum, respectively (p <0.05 vs. control for all). There was no increase in eNOS activity with LPS. There was no significant difference in iNOS or eNOS staining or enzyme activities at 3 vs. 6 hr after LPS. CONCLUSIONS: Endotoxemia induces a marked increase in mucosal iNOS protein expression and activity in the intestine that parallels mRNA expression. Localization of stimulated iNOS expression to the epithelial crypts suggests that NO may play a role in acute injury in these metabolically active cells. The stimulated e-eNOS expression, primarily in ileum and jejunum, indicates that further studies of intestinal e-eNOS are warranted.
LOCAL SUPPRESSION OF INTERFERON-'/PRODUCTION BY HEPATIC GRANULOMA CELLS CORRELATES WITH TISSUESPECIFIC REPLICATION OF LEISHMANIA CHAGASI. M.E. Wilson, A.M. Blum, M .Sandor, A. Metwali, D. Elliott, R.G. Lynch, J.V~ Weinstock. Depts. Intemai Medicine, Microbiology & Pathology, University of Iowa and Veterans' Affairs Medical Center, Iowa City, IA. Introduction: BALB/c mice are susceptible to infection with Leishmania chagasL The parasite load initially rises in the liver and spontaneously subsides, whereas parasite multiplication begins later and remains lower in the spleen. To investigate this organ-specific multiplication ofL. chagasi, we compared cytokine production by splenic versus hepatic immune cells from infected mice. Results: The liver harbored many intracellular L. chagasi amastigotes that were all located in granulomas, whereas the spleen had few amastigotes. Granulomas were isolated from the liver and dispersed with collagenase for further analysis. Many granuloma, but few splenic lymphocytes expressed a memory/effector phenotype according to FACS analysis. Unfraetionated, dispersed granuloma cells cultured in vitro produced IL-10 and IL-6 constitutively, but no detectable interferon-y (IFN-y), IL-4 or IL-5. In contrast, splenocytes from the same animals secreted IFN-¥, IL-4, ][I,-6 and 1I,-10. Positive and negative selection experiments suggested the granuloma IL-10 and IL-6 were ofnon-T cell origin. Paradoxically, FACS-purified Thy-1 + granuloma lymphocytes produced IFN-y when cultured in the absence of other granuloma cells, suggesting that IFN-~/production was inhibited by a granuloma-associated non-T cell element(s). Co-culture ofgranuloma cells with splenocytes inhibited the usual splenocyte production of IFN-y and IL-4, but did not affect IL-10 release supporting this premise. Conclusion: Leishmania granulomas have an immunoregulatory mechanism that inhibits lyrnphokine secretion accounting for the absence of IFN-y in hepatic granuloma cultures. There is no such suppression in the spleen, which allows splenocytes to make IFN-y that, in turn, inhibits local parasite growth. Grant support: VAMC, Merck, AI30126, A132135, AM38327, DK07663, AHA, CCFA