- Email: [email protected]
Intestinal mucosal CCR5 expression is down-regulated in HIV infection
or sterile-filtered supematants recovered from EcN culture broth. Cell cycle kinetics and apoptosis were assessed by cyclin B1, DNA content, and caspase 3 substrate expression measured by flow cytometry. Protein levels of cyclin A, caspase 3 and 8, bax, bcl-2, and cytochrome c were determined by immunobloning. RESULTS: When PBT were activated and cultured in the presence of stenle-filtered EcN supematants, a significantly lower number of cells entered the cell cycle and traversed the S/G2/M phase compared to PBT cultured without EcN supernatants (p<0.01). No effects were observed in the presence of heat inactivated EcN. EcN supematants decreased cyclin D3, B1, and Rb expression, resulting in a reduced generation of daughter cell populations as determined by CFDA staining. In PBT, neither heat inactivated EcN nor sterile-filtered EcN supematants altered apoptosis rates. In contrast, when LPT were stimulated and cultured in the presence of EcN supernatants, apoptosis rate and caspase-3 and -8 activity increased significantly (p<0.05), whereas bax, bcI-2, and cytochrome c expression remained unchanged compared to LPT cultured in the absence of EcN supernatants. Heat-inactivated EcN did not alter LPT apoptosis rate and LPT cell cycling was not altered by EcN, regardless the co-culture method used. CONCLUSIONS Supernatants from prohiotic EcN cultures, but not heat-inactivated EcN, modulate cell cycle progression and apoptosis of T cells, indicating that soluble mediators and not cell fragments of EcN exert biological functions in T cells. In PBT, EcN supematants signihcantly inhibit cell cycle progression, resulting in a limited capacity to expand. In contrast to the effects on PBT, these bacteria markedly increased LPT apoptosis but failed to affect LPT ceil cycle progression, indicating a differential modulation of T cell function by probiotic EcN and suggesting a mechanism by which EcN limits intestinal inflammation.
Sl105 Chronic Psychological Stress Induces Intestinal Sensitization to Luminal Antigens in the Rat Pingchang Yang, Jennifer Jury, Mary H. Perdue Background: The prevalence of food allergies associated with gastrointestinal hypersensinvity is rising. The etiology of such conditions is not fully understood. Recent evidence suggests a relationship between stress and gastrointestinal disorders including food allergy, IBS and IBD. Our previous studies revealed that chronic psychological stress increases intestinal epithelial permeability allowing uptake of macromolecular antigens from the lumen. The aim of this study was to examine the role of stress in sensitization o[ rat intestinal tissues to luminal antigens. Methods: WKY rats were subjected to water avoid stress (WAS, rat on platform surrounded by water) 1 h daily for 10 consecutive days, whereas control rats were exposed to sham stress (SS, no water). Horseradish peroxidase (HRP, a model antigen) was delivered by garage (1 mg/rat) on day 5, along with appropriate adjuvants. To determine the role of corticotropin-releasing hormone (CRH) in the stress induced effects, additional rats were injected ip with the CRH antagonist, a-helical-CRH (aCRH, 25 rag), 30 rain before each WAS session. Following the final session, blood was obtained and jejunal tissues were removed for histology and Ussing chamber studies. Sensitization was indicated by specific IgE (by passive cutaneous anaphylaxas), inflammatory cells in jejunal mucosa (mast cells, MC; eosinophils, EOS; mononuclear cells, MNC), and the intestinal response to HRP antigen added to the chambers: increase in short-circuit current (lse) and permeability (HRP flux). Results: All rats subjected to WAS became sensitized as indicated by: anti-HRP IgE titer; significant increases in numbers of MC, EOS and MNC in intestinal mucosa; and responses to HRP challenge in Ussing chambers. None of the SS or aCRH treated WAS rats developed any positive indications of sensitization. Conclusion: The present study demonstrates that the concurrence of chronic psychological stress and antigen presence in the gastrointestinal tract can initiate intestinal sensitization that can be prevented by antagonism of peripheral CRH receptors.
Sl108 Different Migration Pattern of Dendritic Cells from Intestine to Mesenteric Lymph Nodes Between Intestinal Lymph and Spleen-Derived Cells Hisashi Kohayashi, Souichirou Miura, Hiroshi Nagata, Hiromasa Ishii Background: Dendritic cells (DC) in the small intestine are strong antigen presenting ceils, and can efficiently activate resting T cells. A number of DCs are continuously transported from the intestine to mesenteric lymph nodes, but the factors affecting its migration has not been clearly demonstrated in vivo. The objective of this study was to deternane the migration kinetics of DCs into mesenteric lymph nodes via intestinal lymph and to investigate the difference in the migration pattern between intestinal lymph and spleen-derived DCs. Methods: DCs were obtained from thoracic duct lymph of mesenteric lymphadenectomizated rats or spleen. Intestinal lymph DCs and splenic DCs were enriched by centrifigation over metrizamide, positively selected by OX62, and labeled with fluorochrome carboxyflurescein diacetate succinimidyl ester. Labeled DCs were locally injected into the subserosa of mesenteric boder of smallintestine near the cecum. The time course of DC migration into mesenteric lymph nodes were determined in cryostat sections of resected specimensas. Expression of cell surface molecules on DCs was determined by FACSean. In some experiments, DC maturation was stimulated with TNF-a, IL-4 and GM-CSF, then DC migration was compared. Results and Conclusion: DCs from intestinal lymph express ICAM-1, CDllb/c, CD80/86 and MHC class II, but these cells also have ability to phagocyte latex particles, suggesting the presence of immature DCs. They were time-dependently accumulated in mesenteric lymph nodes, with peak accumulation of 48 hours, and were significantly decreased at 72 hours. Maturation of DCs from/ntestinal lymph enhanced the DC transport to mesenteric lymph nodes with peak at 24 hours. On the other hand splenic D~s showed more maturation phenotypes and showed faster migration to mesenteric lymph nodes, as shown in cytokinestimulated intestinal lymph DC. These results suggest that migration ability of DCs from intestinal lymph is significantly different from that of DC from spleen.
Tal~ 1 Oroup SS WAS W~RH
~c~JVcn~
~ HRP ) Flux
0
0
5,8 2,6
24.7 7.3
24.T~9,7
24,0~7.1
'1:64
17.1~2,0
15.6~2,5"
35.9~9.2"
38.1qZ1 ~
64.7,21.7'
0
0
6.151.4
22.6~6.4
23.4q4.7
24.3q0.3
~
uc~)
, EOS(M~d)
"NC~)
Sl106 Intestinal Mucosal CCR5 Expression is Down-Regulated in HIV Infection lan McGowan, Jufie Elliott, Philip Taing, Marie Fuerst, John Boscardin, Peter Anton Background: CCR5 is a key receptor for HIV infection and has been identified on CD4 + lymphocytes in both blood and intestinal mucosa. In seronegative controls, CCR5 expression is increased in mucosa compared to in blood. Following HIV infection, the predominant viral tropism is for the CCR5 receptor and CCR5 expression in blood is increased. The purpose of this study was to determine whether HIV infection results in altered CCR5 coreceptor expression in intestinal mucosa. Methods: Mononuclear cells from blood and intestinal mucosa were obtained from HIV-infected (n = 20) and control subjects (n = 8) and were assessed by flow cytometry for (i) Percentage CD4 + / C D 4 5 +, CD8 +/CD45 + and CD4+/CCR5+ phenotypes and (ii) Percentage and intensity of CCR5 expression on CD4 + cells. Data for cell pheuotype are expressed as percentages and the CCR5 receptor intensity. Data for CCR5 median fluorescent intensity (MFI) were log transformed for statistical analysis and reflect the number of CCR5 antibody molecules bound per cell. Results: Of the 20 subjects with HIV infection the mean plasma viral load was 33,766 copies/ ml plasma (range: <:50-293,864), mean CD4 count was 318 (range: 153-612) and all subjects were receiving comb/nation antiretroviral therapy. CD4 lymphopenia was seen in both blood and mucosal samples. An increase in CD8 + cells was seen in both compartments. As previously reported, an increase in CD4+/CCRS+ cells was seen in blood from HIVinfected subjects. However, a szgnificant decrease in CCR5 frequency and MFI was seen in intestinal mucosal samples from the HIV group. Conclusions: In contrast to blood, the frequency and intensity of CCR5 expression on mncosal CD4 + lymphocytes is reduced in HIV-inlected subjects. This may occur as a response to the increased mucosal levels of RANTES (the natural ligand of CCRS), which is increased in this population. Alternatively, infection of CCR5 + ceils with HIV may lead to a selective loss of this cell population. Whatever the mechanistic basis of this observation, sufficient CCR5 + cells remain to facilitate further cycles of mucosal infection. Cell phenotype
Blood Control HN
CD4+tCD4,~ CD~/CD4~ CD4+/CCRS+ CCR5 MFI
483% 22.0% 20.0% 54.9% 21.5% 39.8% 81.1 470.2
P value: HN vs Control < 0.0001 < 0.0001 0.005 NS
Mucosa ConVol HN 29.3% 28.0% 56.2% t64.1
12.3% 45.7% 30.8% 63.5
$1109 The 1L-10/IL-12 Axis and Regulation of DC-SIGN Expression in HIV-infected Human Gut Mucosa Kevin Gurney, Julie Elfiott, Hoorig Nassanian, fan McGowan, Carol Song, Benhur Lee, Peter Anton Background: DC-SIGN is highly expressed on dendritic cells in the lamina propria of the gut mucosa, and on dendritic cells in the subepithehal dome of Peyer's patches. Since DCSIGN has been postulated to play a role in the binding and transmission of HIV from the mucosa to secondary lymphoid organs, we undertook a study to examine the regulation of DC-SIGN in the gut mucosa in the context of HIV infection. Methods: Rectosigmoid biopsy tissue sections from HIV+ vs HIV- patients were examined for DC-SIGN expression using computer-assisted quantitative morphometry. Real-time RT-PCR was used to measure the mRNA levels of various pro- (Thl) and anti-inflammatory (Th2) cytokines. To re-capitulate our in situ findings, peripheral blood monocyte derived dendritic cells (MDDCs) were cultured with various cytokine combinations and DC-SIGN expression levels were measured by flow cytometric analysis. Results: Firstly, we found that there was great variability in the number of DC-SIGN + cells amongst individuals, but the variance amongst HIV + samples was greater than that between HIV- healthy controls (HIV+ 129.4 to 154.6] vs HIV- [22 to 63]). Secondly, HIV+ subjects, as a whole, had significantly higher numbers of DCSIGN + cells per standard area when compared to HIV- individuals (80.2 +/- 30.5 (HIV + ) vs 46.2 +/- 15.8 (HIV-), mean +/- S.D., p<:0.0002). There was no significant correlation with tissue viral loads. Thirdly, to assess the immune status in situ, we measured the levels of various cytokines in the respective tissue biopsies. We found a significant positive correlation between DC-SIGN expression and the IL-10/IL-12 ratio (r= 0.877, p<0.0001, n = 16) amongst patients with undetectable tissue viral loads (<10 copies/ug of tissue). In in vitro cultures of MDDC, the Th2 cytokines 1L-4 and 1L-13, but not IL-10, added independently with GM-CSF were critical for inducing high-level DC-SIGN expression. However, supplementing IL-10 to IL-4 + GM-CSF at the initiation of culture of the freshly isolated moncytes rather than at seven days of culture significantly enhanced expression of DCSIGN. This suggests signals received early on largely shape the outcome of the developing dendritic cell. Conclusions: Since the IL-10/IL-12 ratio is considered to be reflective of the Thl/Th2 cytokine milieu, our data suggest that a gut Th2 environment in situ favors the expression of DC-SIGN. Our findings contribute to our understanding of DC-SIGN regulation in the mucosal environment and HIV pathogenesis in the gut.
P value: HN va Control 0.00t 0.04 0.0t 0.0003
Sl107 Dual Function Of Probiotic E. Coli Nissle 1917 (EcN): Inhibition Of Cell Cycle Progression For Peripheral T Cells and Pro-Apoptotic For Mucosal T Cells Andreas Sturm, Klaus Rilling, Hanna Harder D'hcurense, Bertram Wiedenmann, Axel Dignass BACKGROUND: The crucial role of the intestinal microflora in the preservation of the mucosal immune-homeostasis is indisputable, however, the underlying mechanism are insufficiently characterized. We therefore aimed to characterize the modulatory effects of probiotic EcN on cell cycle progression and apoptosis of peripheral (PBT) and lamina propria (LPT) T cells. METHODS: PBT and LPT from donors without intestinal inflammation were stimulated with anti-CD3 mAb and cultured in the absence or presence of heat inactivated EcN
A-155
AGA Abstracts
Sl105 Chronic Psychological Stress Induces Intestinal Sensitization to Luminal Antigens in the Rat Pingchang Yang, Jennifer Jury, Mary H. Perdue Background: The prevalence of food allergies associated with gastrointestinal hypersensinvity is rising. The etiology of such conditions is not fully understood. Recent evidence suggests a relationship between stress and gastrointestinal disorders including food allergy, IBS and IBD. Our previous studies revealed that chronic psychological stress increases intestinal epithelial permeability allowing uptake of macromolecular antigens from the lumen. The aim of this study was to examine the role of stress in sensitization o[ rat intestinal tissues to luminal antigens. Methods: WKY rats were subjected to water avoid stress (WAS, rat on platform surrounded by water) 1 h daily for 10 consecutive days, whereas control rats were exposed to sham stress (SS, no water). Horseradish peroxidase (HRP, a model antigen) was delivered by garage (1 mg/rat) on day 5, along with appropriate adjuvants. To determine the role of corticotropin-releasing hormone (CRH) in the stress induced effects, additional rats were injected ip with the CRH antagonist, a-helical-CRH (aCRH, 25 rag), 30 rain before each WAS session. Following the final session, blood was obtained and jejunal tissues were removed for histology and Ussing chamber studies. Sensitization was indicated by specific IgE (by passive cutaneous anaphylaxas), inflammatory cells in jejunal mucosa (mast cells, MC; eosinophils, EOS; mononuclear cells, MNC), and the intestinal response to HRP antigen added to the chambers: increase in short-circuit current (lse) and permeability (HRP flux). Results: All rats subjected to WAS became sensitized as indicated by: anti-HRP IgE titer; significant increases in numbers of MC, EOS and MNC in intestinal mucosa; and responses to HRP challenge in Ussing chambers. None of the SS or aCRH treated WAS rats developed any positive indications of sensitization. Conclusion: The present study demonstrates that the concurrence of chronic psychological stress and antigen presence in the gastrointestinal tract can initiate intestinal sensitization that can be prevented by antagonism of peripheral CRH receptors.
Sl108 Different Migration Pattern of Dendritic Cells from Intestine to Mesenteric Lymph Nodes Between Intestinal Lymph and Spleen-Derived Cells Hisashi Kohayashi, Souichirou Miura, Hiroshi Nagata, Hiromasa Ishii Background: Dendritic cells (DC) in the small intestine are strong antigen presenting ceils, and can efficiently activate resting T cells. A number of DCs are continuously transported from the intestine to mesenteric lymph nodes, but the factors affecting its migration has not been clearly demonstrated in vivo. The objective of this study was to deternane the migration kinetics of DCs into mesenteric lymph nodes via intestinal lymph and to investigate the difference in the migration pattern between intestinal lymph and spleen-derived DCs. Methods: DCs were obtained from thoracic duct lymph of mesenteric lymphadenectomizated rats or spleen. Intestinal lymph DCs and splenic DCs were enriched by centrifigation over metrizamide, positively selected by OX62, and labeled with fluorochrome carboxyflurescein diacetate succinimidyl ester. Labeled DCs were locally injected into the subserosa of mesenteric boder of smallintestine near the cecum. The time course of DC migration into mesenteric lymph nodes were determined in cryostat sections of resected specimensas. Expression of cell surface molecules on DCs was determined by FACSean. In some experiments, DC maturation was stimulated with TNF-a, IL-4 and GM-CSF, then DC migration was compared. Results and Conclusion: DCs from intestinal lymph express ICAM-1, CDllb/c, CD80/86 and MHC class II, but these cells also have ability to phagocyte latex particles, suggesting the presence of immature DCs. They were time-dependently accumulated in mesenteric lymph nodes, with peak accumulation of 48 hours, and were significantly decreased at 72 hours. Maturation of DCs from/ntestinal lymph enhanced the DC transport to mesenteric lymph nodes with peak at 24 hours. On the other hand splenic D~s showed more maturation phenotypes and showed faster migration to mesenteric lymph nodes, as shown in cytokinestimulated intestinal lymph DC. These results suggest that migration ability of DCs from intestinal lymph is significantly different from that of DC from spleen.
Tal~ 1 Oroup SS WAS W~RH
~c~JVcn~
~ HRP ) Flux
0
0
5,8 2,6
24.7 7.3
24.T~9,7
24,0~7.1
'1:64
17.1~2,0
15.6~2,5"
35.9~9.2"
38.1qZ1 ~
64.7,21.7'
0
0
6.151.4
22.6~6.4
23.4q4.7
24.3q0.3
~
uc~)
, EOS(M~d)
"NC~)
Sl106 Intestinal Mucosal CCR5 Expression is Down-Regulated in HIV Infection lan McGowan, Jufie Elliott, Philip Taing, Marie Fuerst, John Boscardin, Peter Anton Background: CCR5 is a key receptor for HIV infection and has been identified on CD4 + lymphocytes in both blood and intestinal mucosa. In seronegative controls, CCR5 expression is increased in mucosa compared to in blood. Following HIV infection, the predominant viral tropism is for the CCR5 receptor and CCR5 expression in blood is increased. The purpose of this study was to determine whether HIV infection results in altered CCR5 coreceptor expression in intestinal mucosa. Methods: Mononuclear cells from blood and intestinal mucosa were obtained from HIV-infected (n = 20) and control subjects (n = 8) and were assessed by flow cytometry for (i) Percentage CD4 + / C D 4 5 +, CD8 +/CD45 + and CD4+/CCR5+ phenotypes and (ii) Percentage and intensity of CCR5 expression on CD4 + cells. Data for cell pheuotype are expressed as percentages and the CCR5 receptor intensity. Data for CCR5 median fluorescent intensity (MFI) were log transformed for statistical analysis and reflect the number of CCR5 antibody molecules bound per cell. Results: Of the 20 subjects with HIV infection the mean plasma viral load was 33,766 copies/ ml plasma (range: <:50-293,864), mean CD4 count was 318 (range: 153-612) and all subjects were receiving comb/nation antiretroviral therapy. CD4 lymphopenia was seen in both blood and mucosal samples. An increase in CD8 + cells was seen in both compartments. As previously reported, an increase in CD4+/CCRS+ cells was seen in blood from HIVinfected subjects. However, a szgnificant decrease in CCR5 frequency and MFI was seen in intestinal mucosal samples from the HIV group. Conclusions: In contrast to blood, the frequency and intensity of CCR5 expression on mncosal CD4 + lymphocytes is reduced in HIV-inlected subjects. This may occur as a response to the increased mucosal levels of RANTES (the natural ligand of CCRS), which is increased in this population. Alternatively, infection of CCR5 + ceils with HIV may lead to a selective loss of this cell population. Whatever the mechanistic basis of this observation, sufficient CCR5 + cells remain to facilitate further cycles of mucosal infection. Cell phenotype
Blood Control HN
CD4+tCD4,~ CD~/CD4~ CD4+/CCRS+ CCR5 MFI
483% 22.0% 20.0% 54.9% 21.5% 39.8% 81.1 470.2
P value: HN vs Control < 0.0001 < 0.0001 0.005 NS
Mucosa ConVol HN 29.3% 28.0% 56.2% t64.1
12.3% 45.7% 30.8% 63.5
$1109 The 1L-10/IL-12 Axis and Regulation of DC-SIGN Expression in HIV-infected Human Gut Mucosa Kevin Gurney, Julie Elfiott, Hoorig Nassanian, fan McGowan, Carol Song, Benhur Lee, Peter Anton Background: DC-SIGN is highly expressed on dendritic cells in the lamina propria of the gut mucosa, and on dendritic cells in the subepithehal dome of Peyer's patches. Since DCSIGN has been postulated to play a role in the binding and transmission of HIV from the mucosa to secondary lymphoid organs, we undertook a study to examine the regulation of DC-SIGN in the gut mucosa in the context of HIV infection. Methods: Rectosigmoid biopsy tissue sections from HIV+ vs HIV- patients were examined for DC-SIGN expression using computer-assisted quantitative morphometry. Real-time RT-PCR was used to measure the mRNA levels of various pro- (Thl) and anti-inflammatory (Th2) cytokines. To re-capitulate our in situ findings, peripheral blood monocyte derived dendritic cells (MDDCs) were cultured with various cytokine combinations and DC-SIGN expression levels were measured by flow cytometric analysis. Results: Firstly, we found that there was great variability in the number of DC-SIGN + cells amongst individuals, but the variance amongst HIV + samples was greater than that between HIV- healthy controls (HIV+ 129.4 to 154.6] vs HIV- [22 to 63]). Secondly, HIV+ subjects, as a whole, had significantly higher numbers of DCSIGN + cells per standard area when compared to HIV- individuals (80.2 +/- 30.5 (HIV + ) vs 46.2 +/- 15.8 (HIV-), mean +/- S.D., p<:0.0002). There was no significant correlation with tissue viral loads. Thirdly, to assess the immune status in situ, we measured the levels of various cytokines in the respective tissue biopsies. We found a significant positive correlation between DC-SIGN expression and the IL-10/IL-12 ratio (r= 0.877, p<0.0001, n = 16) amongst patients with undetectable tissue viral loads (<10 copies/ug of tissue). In in vitro cultures of MDDC, the Th2 cytokines 1L-4 and 1L-13, but not IL-10, added independently with GM-CSF were critical for inducing high-level DC-SIGN expression. However, supplementing IL-10 to IL-4 + GM-CSF at the initiation of culture of the freshly isolated moncytes rather than at seven days of culture significantly enhanced expression of DCSIGN. This suggests signals received early on largely shape the outcome of the developing dendritic cell. Conclusions: Since the IL-10/IL-12 ratio is considered to be reflective of the Thl/Th2 cytokine milieu, our data suggest that a gut Th2 environment in situ favors the expression of DC-SIGN. Our findings contribute to our understanding of DC-SIGN regulation in the mucosal environment and HIV pathogenesis in the gut.
P value: HN va Control 0.00t 0.04 0.0t 0.0003
Sl107 Dual Function Of Probiotic E. Coli Nissle 1917 (EcN): Inhibition Of Cell Cycle Progression For Peripheral T Cells and Pro-Apoptotic For Mucosal T Cells Andreas Sturm, Klaus Rilling, Hanna Harder D'hcurense, Bertram Wiedenmann, Axel Dignass BACKGROUND: The crucial role of the intestinal microflora in the preservation of the mucosal immune-homeostasis is indisputable, however, the underlying mechanism are insufficiently characterized. We therefore aimed to characterize the modulatory effects of probiotic EcN on cell cycle progression and apoptosis of peripheral (PBT) and lamina propria (LPT) T cells. METHODS: PBT and LPT from donors without intestinal inflammation were stimulated with anti-CD3 mAb and cultured in the absence or presence of heat inactivated EcN
A-155
AGA Abstracts