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Presence of the purinergic receptor P2X7 in colonic macrophages and T cells from normal and IBD mucosa
A348 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
1871
1873
IGA SERUM ANTIBODY REACTIVITY WITH 12, A NOVEL CROHN'S DISEASE MARKER: ANTI-12 LEVEL IS INDEPENDENT FROM ASCA AND ANCA LEVELS, EVEN THOUGH IGA ASCA POSITIVITY IS CORRELATED WITH ANTI-12 POSITIVITY. Carol J. Landers, Huiying Yang, Ying-Chao Lin, Eric A. Vasiliauskas, Jonathan Braun, Stephan R. Targan, Cedars-Sinai Med Ctr, Los Angeles, CA; UCLA, Los Angeles, CA.
PRESENCE OF THE PURINERGIC RECEPTOR P2X 7 IN COLONIC MACROPHAGES AND T CELLS FROM NORMAL AND IBDMUCOSA. Chris K. Li, Keith Bowers, Shri Pathmakanthan, Mandy Lawson, MelBinder Fagura, Chris J. Hawkey, Acad Med Ctr, Nottingham, United Kingdom; AstraZeneca R&D Chamwood, Loughborough, United Kingdom.
We have isolated a novel mucosal Crohn's disease (CD) associated bacterial antigen (12)using representational display analysis and demonstrated varying levels of specific IgA serum reactivity to this antigen in -50% of CD patients (Gastro 116:03030, April 1999). Previously ASCA and pANCA serological markers were found in 55% and 15% of CD patients respectively. Aim: Determination of any relationship in positivity or level between these three markers in a cohort of 153 consecutively ascertained CD patients. Methods: ASCA and pANCA were analyzed by Prometheus Laboratories, San Diego, CA. Serum 12 reactivity was measured by direct ELISA with IgA specific detection of antibody bound to 12 glutathione S-transferase (OST) fusion protein versus antibody bound to OST alone. Anti-I2 positive was defined as greater than the mean plus 2 standard deviations of anti-I2 binding of sera from 20 normal subjects. Nonparametric analyses were used for correlation and comparisons between groups since 12 levels were not normally distributed. A combined ASCA variable (com-ASCA) was created by taking the larger value of the two (IgA and IgO) standardized deviate from their respective means. Results: Overall,anti-I2 was present in the serum of 51% of the cohort with 63% of ASCA", 35% of ASCA-, 44% of pANCA ". and 33% of ASCA-/pANCA patients testing positive. 12 positivity was highly associated with ASCA positivity (p=O.ool) but not ANCA positivity (p=0.5). The level of 12 serum reactivity was also significantly correlated with com-ASCA level (r=0.27, p=0.OOO7). The correlation between 12 and IgA ASCA levels (r=0.32, p=O.OOOI) is much greater than that between 12 and IgO ASCA levels (r=0.16, p=0.053). In multiple regression, both IgA and IgO ASCA levels are associated with anti-I2 levels, however IgA contributed only 10.4% (p=O.Oool) and IgO only 2.9% (p=0.03) of the total variance. Conclusions: I. Serum antibodies to 12 are expressed in many ASCA-I ANCA- CD patients (33%). 2. The correlation between 12 IgA level and IgA ASCA level suggests some common immune responses to bacterial antigens in a subgroup of CD patients. 3. The large proportion of anti-I2 level variance not explained by ASCA levels suggests that there is some independence of 12 from ASCA. 4. Therefore, the relationship between anti-I2 and ASCA is likely complex and may represent interactions between immune alterations in CD.
1872 ULCERATIVE COLITIS (UC) AND CROHN'S DISEASE (CD) TISSUE GENE EXPRESSION PROFILING BY DNA MICROARRAYS: DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS. Ian C. Lawrance, Claudio Fiocchi, Shukti Chakravarti, Case Western Reserve Univ, Cleveland, OH. Background & aim. To elucidate global changes in gene expression in IBD pathogenesis, UC and CD tissue transcripts were profiled using highdensity oligonucleotide microarrays. Methods. RNA from whole colonic tissue typical of UC and CD, and control (n=6/group) was used to prepare biotinylated cRNA for hybridization to oligonucleotide HuOene FI sets (OeneChips, Affymetrix). OeneChips were analyzed (Fluidics Station, OeneChip3.1 Analysis Suite Affymetrix) and changes >3 fold over control were evaluated. Results. UC and CD display remarkably different gene expression profiles. Differentially regulated genes (3-100 fold) were assigned to 8 categories (Table). 82 genes in UC and 36 in CD were overexpressed, while 65 in UC and 29 in CD were downregulated. Both novel and 'lBO-related' genes, including those for HLA class II antigens, defensins, metalloproteinases, phospholipase A2 and collagen I were differentially regulated. Unlike CD, UC appears as an acute inflammatory disease with major changes in inflammatory-mediator, cytokine/growth factor and cancer-related categories. Conclusion. Microarray expression profiling demonstrates, for the first time, broad and fundamental differences in pathogenic mechanisms of UC versus CD. These studies will aid in developing specific targeted therapies for IBD and identify candidate genes for genome-wide susceptibility gene searches.
I II III IV V VI VII VIII
Functional categories
UC
CD
Cytokines &growth factors Inflammation mediators Transcription factors &cell proliferation.mediators Cancer-related HLA & immune function Antimicrobial Mucins, ECM & Remodeling enzymes Miscellaneous
13 12 21
4 1 16 1 6 2 14 21
5
24 2 22 48
Background: Extracellular ATP is increasingly recognized as an important signalling molecule in imrnunoregulation via purinergic receptor P2X7 . This is an ATP-gated ion channel which can form large membrane pores and has been implicated in the apoptosis and cytokine release of macrophages and T cells. We therefore investigated the expression and activation of P2X7 in lamina propria macrophages and T cells from normal and IBD mucosa. Materials and Methods: Lamina propria mononuclear cells migrating from normal and IBD mucosa were purified by magnetic bead sorting. Expression of P2X7 mRNA in isolated cells was studied by RT-PCR. Agonist concentration-effect curves were constructed to 2'& 3'-0-(4-benzyolbenzyoI) adenosine 5' -triphosphate (BzATP) and adenosine 5'-triphosphate (ATP) for two functional assays measured using flow cytometry. Pore formation, which is characteristic for P2X7 receptor stimulation, was studied by measuring the influx of the fluorescent label ethidium bromide. Early apoptotsis was measured by annexin V (AV)staining. The release of IL-I f3 in LPS-primed macrophages in the response to BzATP stimulation was measured by sandwich ELISA. Results: P2X7 mRNA was present in colonic macrophages and T cells from both normal and IBD mucosa. In cells from normal tissue BzATP stimulated the influx of ethidium bromide in a time- and concentration-dependent manner; macrophages with an ECso of 2.l7:t 1.58 /-LM (n=6) and T cells with an ECso of3.74:t 1.81 /-LM (n=6). Moreover, BzATP and ATP stimulated AV staining in macrophages with ECso's of 2.59:t0.72 /-LM (n=8) and 5.67:t 1.5/-LM (n=4) respectively and T cells with ECso's of 3.89:t 1.43 /-LM (n=8) and 6.66:t2.3/-LM (n=4) respectively. The maximal responses to ATP for macrophages and T cells was reduced to 58:t20% (n=4) and 44:t21 % (n=4) of that of BzATP respectively. Moreover, in LPS-primed macrophages, BzATP induced the release of mature IL-l f3 in a concentration-dependent manner. Using diseased tissue we found the proportion of T cells recovered from UC (36:t20%, n=5) and CD (41:t22%, n=4) tissue that were AV positive in the absence of BzATP were significantly higher (p<0.02) than compared to normal tissue (l6:t 14%, n=8). Further addition of 100 /-LM BzATP to T cells and macrophages from UC and CD tissue did not cause further AV staining. Conclusions: Expression and functional data are consistent with the presence of P2X7 receptor on macrophages and T cells from both normal and IBD colon. The role of this receptor in the pathology of IBD deserves further investigation.
1874 EFFECT OF SODIUM BUTYRATE ON REACTIVE OXYGEN GENERATION BY HUMAN NEUTROPHILS. Qiang Liu, Tadashi Shimoyama, Katsuhiko Suzuki, Kazuma Danjo, Shigeyuki Nakaji, Kazuo Sugawara, Hirosaki Univ, Hirosaki, Japan. Introduction: Short chain fatty acids (SCFA) enema is based on the hypothesis that a lack of luminal SCFA, in particular butyrate, would lead to a Agmetabolically starvedah coloncytes that would be one etiologic factor in ulcerative colitis (UC). However, other roles of butyrate in this therapy have not been well characterized. In the present study, we explored how butyrate effects on reactive oxygen species (ROS) generation by neutrophils that has been suggested to be responsible for the tissue damage that occurs during the active phase of UC. Methods: Sodium butyrate (SB) solution was keeping pH 7.4 and isotonic. Blood samples from healthy volunteers were at first incubated with SB for 10 min and further incubated with stimulus for 30 min. ROS generation by neutrophils was differentiated by different assays. Hydroethidine (HE) oxidation measures largely superoxide anion. Dichlorofluorescin (DCFH) oxidation measures largely hydrogen peroxide. Luminol-dependent chemiluminescence (LmCL) measures MPO-mediated ROS. Opsonized zymosan (OZ) and phorbol 12myristate 13-acetate (PMA) were employed as stimuli. Results: SB (up to 50 mM final concentration) alone did not elevate HE oxidation and DCFH oxidation in the absence of stimuli. SB also did not alter HE oxidation upon the stimulation of OZ or PMA. However, SB elevated DCFH oxidation in a dose-dependent manner in the presence of stimuli. DCFH oxidation was significantly increased to 125A}8 % (p=0.028) of control with OZ and to 191A}30% (p=0.0016) with PMA by 25 mM SB. Contrary to these results; SB inhibited markedly LmCL responses in a dose-dependent manner. The inhibition by 50 mM SB was 61A}6% with OZ and 7IA}9% with PMA, respectively. Conclusions: The present study demonstrates that SB alone does not cause ROS generation. In the presence of OZ or PMA, SB up-regulates hydrogen peroxide generation but down-regulates MPOmediated ROS generation, the latter is more potent in killing microbe and tissue damage. These results might be attributed to an inhibitory effect of SB on MPO activity and hence attenuate the neutrophils' destructive activities in UC.
GASTROENTEROLOGY Vol. 118, No.4
1871
1873
IGA SERUM ANTIBODY REACTIVITY WITH 12, A NOVEL CROHN'S DISEASE MARKER: ANTI-12 LEVEL IS INDEPENDENT FROM ASCA AND ANCA LEVELS, EVEN THOUGH IGA ASCA POSITIVITY IS CORRELATED WITH ANTI-12 POSITIVITY. Carol J. Landers, Huiying Yang, Ying-Chao Lin, Eric A. Vasiliauskas, Jonathan Braun, Stephan R. Targan, Cedars-Sinai Med Ctr, Los Angeles, CA; UCLA, Los Angeles, CA.
PRESENCE OF THE PURINERGIC RECEPTOR P2X 7 IN COLONIC MACROPHAGES AND T CELLS FROM NORMAL AND IBDMUCOSA. Chris K. Li, Keith Bowers, Shri Pathmakanthan, Mandy Lawson, MelBinder Fagura, Chris J. Hawkey, Acad Med Ctr, Nottingham, United Kingdom; AstraZeneca R&D Chamwood, Loughborough, United Kingdom.
We have isolated a novel mucosal Crohn's disease (CD) associated bacterial antigen (12)using representational display analysis and demonstrated varying levels of specific IgA serum reactivity to this antigen in -50% of CD patients (Gastro 116:03030, April 1999). Previously ASCA and pANCA serological markers were found in 55% and 15% of CD patients respectively. Aim: Determination of any relationship in positivity or level between these three markers in a cohort of 153 consecutively ascertained CD patients. Methods: ASCA and pANCA were analyzed by Prometheus Laboratories, San Diego, CA. Serum 12 reactivity was measured by direct ELISA with IgA specific detection of antibody bound to 12 glutathione S-transferase (OST) fusion protein versus antibody bound to OST alone. Anti-I2 positive was defined as greater than the mean plus 2 standard deviations of anti-I2 binding of sera from 20 normal subjects. Nonparametric analyses were used for correlation and comparisons between groups since 12 levels were not normally distributed. A combined ASCA variable (com-ASCA) was created by taking the larger value of the two (IgA and IgO) standardized deviate from their respective means. Results: Overall,anti-I2 was present in the serum of 51% of the cohort with 63% of ASCA", 35% of ASCA-, 44% of pANCA ". and 33% of ASCA-/pANCA patients testing positive. 12 positivity was highly associated with ASCA positivity (p=O.ool) but not ANCA positivity (p=0.5). The level of 12 serum reactivity was also significantly correlated with com-ASCA level (r=0.27, p=0.OOO7). The correlation between 12 and IgA ASCA levels (r=0.32, p=O.OOOI) is much greater than that between 12 and IgO ASCA levels (r=0.16, p=0.053). In multiple regression, both IgA and IgO ASCA levels are associated with anti-I2 levels, however IgA contributed only 10.4% (p=O.Oool) and IgO only 2.9% (p=0.03) of the total variance. Conclusions: I. Serum antibodies to 12 are expressed in many ASCA-I ANCA- CD patients (33%). 2. The correlation between 12 IgA level and IgA ASCA level suggests some common immune responses to bacterial antigens in a subgroup of CD patients. 3. The large proportion of anti-I2 level variance not explained by ASCA levels suggests that there is some independence of 12 from ASCA. 4. Therefore, the relationship between anti-I2 and ASCA is likely complex and may represent interactions between immune alterations in CD.
1872 ULCERATIVE COLITIS (UC) AND CROHN'S DISEASE (CD) TISSUE GENE EXPRESSION PROFILING BY DNA MICROARRAYS: DIAGNOSTIC AND THERAPEUTIC IMPLICATIONS. Ian C. Lawrance, Claudio Fiocchi, Shukti Chakravarti, Case Western Reserve Univ, Cleveland, OH. Background & aim. To elucidate global changes in gene expression in IBD pathogenesis, UC and CD tissue transcripts were profiled using highdensity oligonucleotide microarrays. Methods. RNA from whole colonic tissue typical of UC and CD, and control (n=6/group) was used to prepare biotinylated cRNA for hybridization to oligonucleotide HuOene FI sets (OeneChips, Affymetrix). OeneChips were analyzed (Fluidics Station, OeneChip3.1 Analysis Suite Affymetrix) and changes >3 fold over control were evaluated. Results. UC and CD display remarkably different gene expression profiles. Differentially regulated genes (3-100 fold) were assigned to 8 categories (Table). 82 genes in UC and 36 in CD were overexpressed, while 65 in UC and 29 in CD were downregulated. Both novel and 'lBO-related' genes, including those for HLA class II antigens, defensins, metalloproteinases, phospholipase A2 and collagen I were differentially regulated. Unlike CD, UC appears as an acute inflammatory disease with major changes in inflammatory-mediator, cytokine/growth factor and cancer-related categories. Conclusion. Microarray expression profiling demonstrates, for the first time, broad and fundamental differences in pathogenic mechanisms of UC versus CD. These studies will aid in developing specific targeted therapies for IBD and identify candidate genes for genome-wide susceptibility gene searches.
I II III IV V VI VII VIII
Functional categories
UC
CD
Cytokines &growth factors Inflammation mediators Transcription factors &cell proliferation.mediators Cancer-related HLA & immune function Antimicrobial Mucins, ECM & Remodeling enzymes Miscellaneous
13 12 21
4 1 16 1 6 2 14 21
5
24 2 22 48
Background: Extracellular ATP is increasingly recognized as an important signalling molecule in imrnunoregulation via purinergic receptor P2X7 . This is an ATP-gated ion channel which can form large membrane pores and has been implicated in the apoptosis and cytokine release of macrophages and T cells. We therefore investigated the expression and activation of P2X7 in lamina propria macrophages and T cells from normal and IBD mucosa. Materials and Methods: Lamina propria mononuclear cells migrating from normal and IBD mucosa were purified by magnetic bead sorting. Expression of P2X7 mRNA in isolated cells was studied by RT-PCR. Agonist concentration-effect curves were constructed to 2'& 3'-0-(4-benzyolbenzyoI) adenosine 5' -triphosphate (BzATP) and adenosine 5'-triphosphate (ATP) for two functional assays measured using flow cytometry. Pore formation, which is characteristic for P2X7 receptor stimulation, was studied by measuring the influx of the fluorescent label ethidium bromide. Early apoptotsis was measured by annexin V (AV)staining. The release of IL-I f3 in LPS-primed macrophages in the response to BzATP stimulation was measured by sandwich ELISA. Results: P2X7 mRNA was present in colonic macrophages and T cells from both normal and IBD mucosa. In cells from normal tissue BzATP stimulated the influx of ethidium bromide in a time- and concentration-dependent manner; macrophages with an ECso of 2.l7:t 1.58 /-LM (n=6) and T cells with an ECso of3.74:t 1.81 /-LM (n=6). Moreover, BzATP and ATP stimulated AV staining in macrophages with ECso's of 2.59:t0.72 /-LM (n=8) and 5.67:t 1.5/-LM (n=4) respectively and T cells with ECso's of 3.89:t 1.43 /-LM (n=8) and 6.66:t2.3/-LM (n=4) respectively. The maximal responses to ATP for macrophages and T cells was reduced to 58:t20% (n=4) and 44:t21 % (n=4) of that of BzATP respectively. Moreover, in LPS-primed macrophages, BzATP induced the release of mature IL-l f3 in a concentration-dependent manner. Using diseased tissue we found the proportion of T cells recovered from UC (36:t20%, n=5) and CD (41:t22%, n=4) tissue that were AV positive in the absence of BzATP were significantly higher (p<0.02) than compared to normal tissue (l6:t 14%, n=8). Further addition of 100 /-LM BzATP to T cells and macrophages from UC and CD tissue did not cause further AV staining. Conclusions: Expression and functional data are consistent with the presence of P2X7 receptor on macrophages and T cells from both normal and IBD colon. The role of this receptor in the pathology of IBD deserves further investigation.
1874 EFFECT OF SODIUM BUTYRATE ON REACTIVE OXYGEN GENERATION BY HUMAN NEUTROPHILS. Qiang Liu, Tadashi Shimoyama, Katsuhiko Suzuki, Kazuma Danjo, Shigeyuki Nakaji, Kazuo Sugawara, Hirosaki Univ, Hirosaki, Japan. Introduction: Short chain fatty acids (SCFA) enema is based on the hypothesis that a lack of luminal SCFA, in particular butyrate, would lead to a Agmetabolically starvedah coloncytes that would be one etiologic factor in ulcerative colitis (UC). However, other roles of butyrate in this therapy have not been well characterized. In the present study, we explored how butyrate effects on reactive oxygen species (ROS) generation by neutrophils that has been suggested to be responsible for the tissue damage that occurs during the active phase of UC. Methods: Sodium butyrate (SB) solution was keeping pH 7.4 and isotonic. Blood samples from healthy volunteers were at first incubated with SB for 10 min and further incubated with stimulus for 30 min. ROS generation by neutrophils was differentiated by different assays. Hydroethidine (HE) oxidation measures largely superoxide anion. Dichlorofluorescin (DCFH) oxidation measures largely hydrogen peroxide. Luminol-dependent chemiluminescence (LmCL) measures MPO-mediated ROS. Opsonized zymosan (OZ) and phorbol 12myristate 13-acetate (PMA) were employed as stimuli. Results: SB (up to 50 mM final concentration) alone did not elevate HE oxidation and DCFH oxidation in the absence of stimuli. SB also did not alter HE oxidation upon the stimulation of OZ or PMA. However, SB elevated DCFH oxidation in a dose-dependent manner in the presence of stimuli. DCFH oxidation was significantly increased to 125A}8 % (p=0.028) of control with OZ and to 191A}30% (p=0.0016) with PMA by 25 mM SB. Contrary to these results; SB inhibited markedly LmCL responses in a dose-dependent manner. The inhibition by 50 mM SB was 61A}6% with OZ and 7IA}9% with PMA, respectively. Conclusions: The present study demonstrates that SB alone does not cause ROS generation. In the presence of OZ or PMA, SB up-regulates hydrogen peroxide generation but down-regulates MPOmediated ROS generation, the latter is more potent in killing microbe and tissue damage. These results might be attributed to an inhibitory effect of SB on MPO activity and hence attenuate the neutrophils' destructive activities in UC.