Antibodies against monocytes in inflammatory bowel and autoimmune liver diseases
1
April 1998 for this intestinal differentiation of macrophages was investigated in the present study. Methods- Spheroids of HT-29 cells, which show enterocytic differentiation and J82 (prostate cancer cell line) as a control were cultured in agarose coated 96 well plates with 10% serum. After 5 days elutriated monocytes from healthy donors were added and the spheroids fixed after 24 hours, 3 days or 7 days. Immunohistochemistry was performed by the APAAP (alkaline- phosphatase-anti-alkaline-phosphatase method) for CD68, CD14, CD16, CD44, CD1 lb, myeloperoxidase and EP4 Results: After 24 hours monocytes/macrophages were found to be attached to the surface of the spheroids of HT-29 and J82 ceils and showed expression of CD14, CD16 and CDllb. After 3 days macrophages had invaded the spheroids. In HT-29 cells the expression on CD14, CD16 and CDllb was decreased but still detectable. After 7 days of coculture macrophages could be found mainly in the centers of the spheroids as shown by CD68 and CD44 staining. In spheroids of J82 cells (control) macrophages still expressed CD14, CD16 and CD1 lb. In the spheroids of the intestinal epithelial cell line HT-29 no expression of CD14, CD16 or CDllb could be detected in the CD68 positive macrophages. Conclusion: Intestinal epithelial ceils induce the specific intestinal differentiation of macrophages and a downregulation of CD14, CD16 and CD1 lb in spheroid coculture models. • G4384 THE INDUCTION OF THE SECRETION OF IL-8 IN PRIMARY HUMAN COLONIC INTESTINAL EPITHELIAL CELLS IS MEDIATED BY NF-KB. G. Rooler, D. Vogl, W. Falk, V. Gross, J. Sch~51merich, T.Andus. Dept. of Intemal Medicine I, University of Regensburg, Germany. Baekeround: Recently we established a method for the primary culture of human intestinal epithelial cells (IEC) (Gastroenterology 110: A891, 1996). Whereas primary cultures from normal mucosa secreted only low amounts of IL-8, cells from inflamed mucosa secreted significantly more. In immunohistochemical studies we found an activation of NF-~:B in IEC from inflamed mucosa (Gastroenterology 112: A1077, 1997). Methods: Primary cultures of colonic epithelial cells were established from the mucosa of seven control patients and stimulated with 10 ng/ml IL-II3 for 24 hours. 10 or 100 laM ALLN (N-acetyl-leucinyl-leucinyl-norleucinal) were used for the inhibition of NF-KB activation. The determination of IL-8 and IL-1Ra from the culture media was done by ELISA. Results: Incubation of unstimulated IEC with the NF-~:B-inhibitor ALLN (101aM or 100 ~aM) was followed by significant reduction of IL-8 secretion to 54 ± 32% (10 9M) or 20 + 16% (1001aM) of controls. IL-1[~ induced IL-8 secretion in the IEC (142 ± 62% of controls). The Coincubation of IEC with 10ng/ml IL-113 and 10 or 100~tM ALLN was followed by a reduction of IL-8 secretion to 60,8 +9,1% (10~M) or 18,1 ± 6,0% (100~M) of the IL-113 inducible secretion and to 83,4 ± 44,5% or 27,7 -+ 21,7% of the unstimulated controls. The effect of ALLN was not due to unspecific toxicity because IL-1Ra levels remained unchanged. Conclusion: Isolation of primary human colonic epithelial cells is followed by an induction of IL-8 secretion, which is at least in part mediated by NF- ~:B, because it can be inhibited by ALLN. A further induction of IL-8 can be induced by IL-113 which is also mostly mediated by NF-KB. These data demonstrate the important role of NF-KB during the activation of colonic epithelial cells. • G4385 DIFFERENTIAL EXPRESSION OF NEUROTROPHINS AND THEIR TRK TYROSINE KINASE RECEPTORS IN THE INFLAMED COLON OF IL-2 KNOCKOUT MICE. H. Rohm. I. Geerling, J. Lakshmanan, G. Adler, M. Reinshagen, Dep. of Medicine I, University of Ulm, Germany, Div. of Gastroenterology, Harbor-UCLA Medical Center, Torrance, CA. Introduction: We have shown that immunoneutralization of NGF and NT-3 significantly worsened the chronic TNB-colitis of the rat, suggesting an important protective role for neurotrophins in chronic inflammation of the colon (Gastroenterology I10, Allll, 1996). Therefore we wanted to investigate the protein and mRNA expression of neurotrophins and their receptors in the IL-2 k.o. mouse model of colitis, which is a defined immunological model. Up to 3 weeks after birth these mice show no signs of inflammation, whereas after that time the animals develop signs of autoimmune disease without colitis (prediseased) and then after about 11 weeks develop chronic ulcerative colitis (diseased) when reared in a conventional environment. Methods: Colonic extracts from IL-2 k.o. mice of different age (3 w. = no inflammation, 7 w. = prediseased and 11 w. = diseased) were used for western blot analysis using polyclonal I3-NGF and BDNF antibodies and a NT-3 prohormone specific antibody. Furthermore the protein expression of the Trk receptors was analysed using polyclonal TrkA and TrkB specific antibodies. Animals of the +/+ and +/- genotype for IL-2 were used as controls. Additionally total RNA was isolated from the colonic tissue and RT-PCR was performed using specific primers for NGF, BDNF, NT-3, TrkA, TrkB and TrkC. Results: In western blot analysis we found a strong upregulation of the 53 and 70 kD NGF prnhormones in the colon of IL-2 k.o. mice beginning already after 7 w., when there is no obvious sign of mucosal inflammation. BDNF, however is downregulated in the colon of diseased k.o. mice. An 80 kD NT-3 Prohormon is present in the colonic tissue of all mice, Immunology, Microbiology, and Inflammatory Disorders A1071 control as well as IL-2 k.o., at about the same level. The analysis of the Trk receptors indicates that TrkA is abundantly expressed in the +/+ control mice and the expression is upregulated in the +/- as well as the -/- mice. TrkB in contrast is downregulated in the prediseased and diseased IL-2 -/- mice. Except for TrkA, all the neurotrophins and their receptors were expressed on the mRNA level in the gut. There was no significant regulation of the mRNA expression during the development of colonic inflammation in IL-2 k.o. mice. Discussion: We could show that the neurotrophins NGF and BDNF and their receptors TrkA and TrkB are differentially expressed in the inflamed colon of IL-2 k.o. mice compared to the colon of control mice. Since NGF and BDNF as well as their Trk receptors are expressed by immune cells, such as T- and B-lymphocytes, we suggest that these factors play an important role regulating the inflammatory response and might even be involved in the pathophysiology of IBD. G4386 ANTIBODIES AGAINST MONOCYTES IN INFLAMMATORY BOWEL AND AUTOIMMUNE LIVER DISEASES. C. Roozendaal, E.J. Hummel, M.A. de Jong, J.H. Kleibeuker, A.P. Van den Berg, P.C. Limburg, and C.G.M. Kallenberg; Depts. of Clinical Immunology and Gastroenterology and Hepatology, University Hospital Grnningen, The Netherlands. Autnantibodies against cytoplasmic constituents of neutrophil granulocytes (ANCA) have been described in systemic vasculitides and in several other autoimmune-mediated disorders, e.g. inflammatory bowel and autoimmune liver diseases. Some of the target antigens of ANCA are also present in monocytes. However, until now, it is unknown whether autoantibodies specifically directed against monocytes exist. We studied the presence of antibodies against monocytes in samples from patients with ulcerative colitis (UC), Crohn's disease (CD), primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH). Monocytes were purified by elutriation, allowed to adhere to glass slides, and fixed with ethanol. The presence of antibodies against these cells was determined by indirect immunofluorescence (IIF). In addition, the presence of ANCA was tested by IIF on ethanol-fixed granulocytes, and the presence of anti-nuclear antibodies (ANA) was tested by IIF on ethanol-fixed HEp2 cells. Antibodies reacting with monocytes were detected in 15/50 (30%) UC samples, in 10/79 (13%) CD samples, in 9/26 (35%) PSC samples, in 2/8 (25%) PBC samples, and in 17/30 (57%) AIH samples. Besides a nuclear staining pattern, that in many cases correlated with positivity in the ANA test, we found a very remarkable staining of the indentation of the nuclear membrane in 24% of UC samples, 4% of CD samples, 19% of PSC samples, and 40% of AIH samples. All samples revealing this staining pattern were p-ANCA positive, and part of them reacted with various defined ANCA antigens, which suggests that the antigen(s) responsible for this staining pattem were not specific for monocytes, but were also present in granulocytes. In conclusion, by using IIF, we found that a substantial proportion of samples from patients with inflammatory bowel or autoimmune liver diseases contained antibodies against monocyte constituents. However, these samples were also positive for either ANCA or ANA. We were not able to detect any antibodies that were solely directed against monocytes in samples from patients with inflammatory bowel or autoimmune liver diseases. G4387 CATALASE AND ALPHA-ENOLASE: TWO NOVEL GRANULOCYTE AUTOANTIGENS IN INFLAMMATORY BOWEL DISEASES. C. Roozendaal. l M.H. Zhao,5 G. Horst, 1 C.M. Lockwood, 5 J.H. Kleibeuker, 2 P.C. Limburg, 3 G.F. Nelis,4 and C.G.M. Kallenberg. 1; 1Department of Internal Medicine, University Hospital, Groningen, The Netherlands; 4Department of Gastroenterology, Sophia Hospital, Zwolle, The Netherlands; 5Department of Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom. In the inflammatory bowel diseases, the target antigens of antineutrophil cytoplasm autoantibodies (ANCA) have not been fully identified, which limits the analysis of the diagnostic significance as well as of the possible pathophysiological role of these antibodies. Aim of this study was to identify the target antigens of ANCA in large groups of patients with ulcerative colitis (UC, n=96) and Crohn's disease (CD, n=112). Detection of ANCA was performed by Western blotting followed by immunodetection, using a crude extract of isolated granulocytes as the source of antigens. We detected autoantibodies against the well-defined ANCA antigens lactoferrin (26% of UC samples and 11% of CD samples) and bactericidal/permeability-increasing protein (2% of UC samples and 6% of CD samples), as well as antibodies against two novel granulocyte antigens: antibodies against a 57/56 kD doublet were found in 38% of UC samples and in 26% of CD samples, whereas antibodies against a 47 kD protein were found in 10% of UC samples and in 18% of CD samples. By successive cation and anion exchange chromatography of the crude granulocyte extract, we obtained a fraction that was highly enriched for the 57/56 kD and the 47 kD antigens. Internal amino acid sequence analysis identified the 57 kD protein as catalase and the 47 kD protein as alpha-enolase. This study is the first to report catalase and alpha-enolase as granulocyte antigens for autoantibodies in inflammatory bowel diseases. The patho- physiological significanceof these novel autoantibodies remains to be elucidated.
Transcript of Antibodies against monocytes in inflammatory bowel and autoimmune liver diseases
April 1998
for this intestinal differentiation of macrophages was investigated in the present study. Methods- Spheroids of HT-29 cells, which show enterocytic differentiation and J82 (prostate cancer cell line) as a control were cultured in agarose coated 96 well plates with 10% serum. After 5 days elutriated monocytes from healthy donors were added and the spheroids fixed after 24 hours, 3 days or 7 days. Immunohistochemistry was performed by the APAAP (alkaline- phosphatase-anti-alkaline-phosphatase method) for CD68, CD14, CD16, CD44, CD1 lb, myeloperoxidase and EP4 Results: After 24 hours monocytes/macrophages were found to be attached to the surface of the spheroids of HT-29 and J82 ceils and showed expression of CD14, CD16 and CDllb. After 3 days macrophages had invaded the spheroids. In HT-29 cells the expression on CD14, CD16 and CDllb was decreased but still detectable. After 7 days of coculture macrophages could be found mainly in the centers of the spheroids as shown by CD68 and CD44 staining. In spheroids of J82 cells (control) macrophages still expressed CD14, CD16 and CD1 lb. In the spheroids of the intestinal epithelial cell line HT-29 no expression of CD14, CD16 or CDllb could be detected in the CD68 positive macrophages. Conclusion: Intestinal epithelial ceils induce the specific intestinal differentiation of macrophages and a downregulation of CD14, CD16 and CD1 lb in spheroid coculture models.
• G4384
THE INDUCTION OF THE SECRETION OF IL-8 IN PRIMARY HUMAN COLONIC INTESTINAL EPITHELIAL CELLS IS MEDIATED BY NF-KB. G. Rooler, D. Vogl, W. Falk, V. Gross, J. Sch~51merich, T.Andus. Dept. of Intemal Medicine I, University of Regensburg, Germany.
Baekeround: Recently we established a method for the primary culture of human intestinal epithelial cells (IEC) (Gastroenterology 110: A891, 1996). Whereas primary cultures from normal mucosa secreted only low amounts of IL-8, cells from inflamed mucosa secreted significantly more. In immunohistochemical studies we found an activation of NF-~:B in IEC from inflamed mucosa (Gastroenterology 112: A1077, 1997). Methods: Primary cultures of colonic epithelial cells were established from the mucosa of seven control patients and stimulated with 10 ng/ml IL-II3 for 24 hours. 10 or 100 laM ALLN (N-acetyl-leucinyl-leucinyl-norleucinal) were used for the inhibition of NF-KB activation. The determination of IL-8 and IL-1Ra from the culture media was done by ELISA. Results: Incubation of unstimulated IEC with the NF-~:B-inhibitor ALLN (101aM or 100 ~aM) was followed by significant reduction of IL-8 secretion to 54 ± 32% (10 9M) or 20 + 16% (1001aM) of controls. IL-1[~ induced IL-8 secretion in the IEC (142 ± 62% of controls). The Coincubation of IEC with 10ng/ml IL-113 and 10 or 100~tM ALLN was followed by a reduction of IL-8 secretion to 60,8 +9,1% (10~M) or 18,1 ± 6,0% (100~M) of the IL-113 inducible secretion and to 83,4 ± 44,5% or 27,7 -+ 21,7% of the unstimulated controls. The effect of ALLN was not due to unspecific toxicity because IL-1Ra levels remained unchanged. Conclusion: Isolation of primary human colonic epithelial cells is followed by an induction of IL-8 secretion, which is at least in part mediated by NF- ~:B, because it can be inhibited by ALLN. A further induction of IL-8 can be induced by IL-113 which is also mostly mediated by NF-KB. These data demonstrate the important role of NF-KB during the activation of colonic epithelial cells.
• G4385
DIFFERENTIAL EXPRESSION OF NEUROTROPHINS AND THEIR TRK TYROSINE KINASE RECEPTORS IN THE INFLAMED COLON OF IL-2 KNOCKOUT MICE. H. Rohm. I. Geerling, J. Lakshmanan, G. Adler, M. Reinshagen, Dep. of Medicine I, University of Ulm, Germany, Div. of Gastroenterology, Harbor-UCLA Medical Center, Torrance, CA.
Introduction: We have shown that immunoneutralization of NGF and NT-3 significantly worsened the chronic TNB-colitis of the rat, suggesting an important protective role for neurotrophins in chronic inflammation of the colon (Gastroenterology I10, A l l l l , 1996). Therefore we wanted to investigate the protein and mRNA expression of neurotrophins and their receptors in the IL-2 k.o. mouse model of colitis, which is a defined immunological model. Up to 3 weeks after birth these mice show no signs of inflammation, whereas after that time the animals develop signs of autoimmune disease without colitis (prediseased) and then after about 11 weeks develop chronic ulcerative colitis (diseased) when reared in a conventional environment. Methods: Colonic extracts from IL-2 k.o. mice of different age (3 w. = no inflammation, 7 w. = prediseased and 11 w. = diseased) were used for western blot analysis using polyclonal I3-NGF and BDNF antibodies and a NT-3 prohormone specific antibody. Furthermore the protein expression of the Trk receptors was analysed using polyclonal TrkA and TrkB specific antibodies. Animals of the +/+ and +/- genotype for IL-2 were used as controls. Additionally total RNA was isolated from the colonic tissue and RT-PCR was performed using specific primers for NGF, BDNF, NT-3, TrkA, TrkB and TrkC. Results: In western blot analysis we found a strong upregulation of the 53 and 70 kD NGF prnhormones in the colon of IL-2 k.o. mice beginning already after 7 w., when there is no obvious sign of mucosal inflammation. BDNF, however is downregulated in the colon of diseased k.o. mice. An 80 kD NT-3 Prohormon is present in the colonic tissue of all mice,
Immunology, Microbiology, and Inflammatory Disorders A1071
control as well as IL-2 k.o., at about the same level. The analysis of the Trk receptors indicates that TrkA is abundantly expressed in the +/+ control mice and the expression is upregulated in the +/- as well as the -/- mice. TrkB in contrast is downregulated in the prediseased and diseased IL-2 -/- mice. Except for TrkA, all the neurotrophins and their receptors were expressed on the mRNA level in the gut. There was no significant regulation of the mRNA expression during the development of colonic inflammation in IL-2 k.o. mice. Discussion: We could show that the neurotrophins NGF and BDNF and their receptors TrkA and TrkB are differentially expressed in the inflamed colon of IL-2 k.o. mice compared to the colon of control mice. Since NGF and BDNF as well as their Trk receptors are expressed by immune cells, such as T- and B-lymphocytes, we suggest that these factors play an important role regulating the inflammatory response and might even be involved in the pathophysiology of IBD.
G4386
ANTIBODIES AGAINST MONOCYTES IN INFLAMMATORY BOWEL AND AUTOIMMUNE LIVER DISEASES. C. Roozendaal, E.J. Hummel, M.A. de Jong, J.H. Kleibeuker, A.P. Van den Berg, P.C. Limburg, and C.G.M. Kallenberg; Depts. of Clinical Immunology and Gastroenterology and Hepatology, University Hospital Grnningen, The Netherlands.
Autnantibodies against cytoplasmic constituents of neutrophil granulocytes (ANCA) have been described in systemic vasculitides and in several other autoimmune-mediated disorders, e.g. inflammatory bowel and autoimmune liver diseases. Some of the target antigens of ANCA are also present in monocytes. However, until now, it is unknown whether autoantibodies specifically directed against monocytes exist. We studied the presence of antibodies against monocytes in samples from patients with ulcerative colitis (UC), Crohn's disease (CD), primary sclerosing cholangitis (PSC), primary biliary cirrhosis (PBC), and autoimmune hepatitis (AIH). Monocytes were purified by elutriation, allowed to adhere to glass slides, and fixed with ethanol. The presence of antibodies against these cells was determined by indirect immunofluorescence (IIF). In addition, the presence of ANCA was tested by IIF on ethanol-fixed granulocytes, and the presence of anti-nuclear antibodies (ANA) was tested by IIF on ethanol-fixed HEp2 cells. Antibodies reacting with monocytes were detected in 15/50 (30%) UC samples, in 10/79 (13%) CD samples, in 9/26 (35%) PSC samples, in 2/8 (25%) PBC samples, and in 17/30 (57%) AIH samples. Besides a nuclear staining pattern, that in many cases correlated with positivity in the ANA test, we found a very remarkable staining of the indentation of the nuclear membrane in 24% of UC samples, 4% of CD samples, 19% of PSC samples, and 40% of AIH samples. All samples revealing this staining pattern were p-ANCA positive, and part of them reacted with various defined ANCA antigens, which suggests that the antigen(s) responsible for this staining pattem were not specific for monocytes, but were also present in granulocytes. In conclusion, by using IIF, we found that a substantial proportion of samples from patients with inflammatory bowel or autoimmune liver diseases contained antibodies against monocyte constituents. However, these samples were also positive for either ANCA or ANA. We were not able to detect any antibodies that were solely directed against monocytes in samples from patients with inflammatory bowel or autoimmune liver diseases.
G4387
CATALASE AND ALPHA-ENOLASE: TWO NOVEL GRANULOCYTE AUTOANTIGENS IN INFLAMMATORY BOWEL DISEASES. C. Roozendaal. l M.H. Zhao, 5 G. Horst, 1 C.M. Lockwood, 5 J.H. Kleibeuker, 2 P.C. Limburg, 3 G.F. Nelis, 4 and C.G.M. Kallenberg. 1; 1Department of Internal Medicine, University Hospital, Groningen, The Netherlands; 4Department of Gastroenterology, Sophia Hospital, Zwolle, The Netherlands; 5Department of Medicine, Addenbrooke's Hospital, Cambridge, United Kingdom.
In the inflammatory bowel diseases, the target antigens of antineutrophil cytoplasm autoantibodies (ANCA) have not been fully identified, which limits the analysis of the diagnostic significance as well as of the possible pathophysiological role of these antibodies. Aim of this study was to identify the target antigens of ANCA in large groups of patients with ulcerative colitis (UC, n=96) and Crohn's disease (CD, n=112). Detection of ANCA was performed by Western blotting followed by immunodetection, using a crude extract of isolated granulocytes as the source of antigens. We detected autoantibodies against the well-defined ANCA antigens lactoferrin (26% of UC samples and 11% of CD samples) and bactericidal/permeability-increasing protein (2% of UC samples and 6% of CD samples), as well as antibodies against two novel granulocyte antigens: antibodies against a 57/56 kD doublet were found in 38% of UC samples and in 26% of CD samples, whereas antibodies against a 47 kD protein were found in 10% of UC samples and in 18% of CD samples. By successive cation and anion exchange chromatography of the crude granulocyte extract, we obtained a fraction that was highly enriched for the 57/56 kD and the 47 kD antigens. Internal amino acid sequence analysis identified the 57 kD protein as catalase and the 47 kD protein as alpha-enolase. This study is the first to report catalase and alpha-enolase as granulocyte antigens for autoantibodies in inflammatory bowel diseases. The patho- physiological significance of these novel autoantibodies remains to be elucidated.
