Aggravation of Different Types of Experimental Colitis by Depletion or Adhesion Blockade of Neutrophils

Background & Aims: Neutrophils are generally thought to play an important proinflammatory role in the pathogenesis of inflammatory bowel disease. The objective of this study was to evaluate whether blocking the invasion of neutrophils by anti-L-selectin monoclonal antibodies modulates chemically induced colitis and how this modulation is accomplished. Methods: Trinitrobenzene sulfonic acid/dinitrobenzene sulfonic acid (TNBS/DNBS)-induced colitis was studied in rats on treatment with anti-L-selectin monoclonal antibodies (mAb) or antineutrophil antiserum. Different anti-L-selectin mAb, either blocking or nonblocking, as well as F(ab)2 fragments were evaluated. Additionally, leukocyte migration was examined using intravital microscopy. Furthermore, the effect of neutrophil depletion in rat TNBS-induced colitis was studied either prior to or after colitis induction as well as murine CD4+CD45RBhigh transfer colitis. Finally, bacterial translocation during DNBS-induced colitis was studied in neutrophil-depleted and control rats. Results: Anti-L-selectin mAb treatment resulted in increased mortality and bowel inflammation as well as hemorrhagic eye secretion. No clear difference was found between blocking and nonblocking mAb or F(ab)2 fragments. For all investigated antibodies/fragments, either complete blockade of leukocyte invasion or marked neutrophil depletion was found. Accordingly, neutrophil depletion by antiserum resulted in aggravation of rat DNBS-induced colitis as well as murine transfer colitis. Conclusions: Adhesion blockade or neutrophil depletion aggravates rat TNBS/DNBS-induced colitis together with extraintestinal manifestations of the eyes. Therefore, neutrophils appear to have an important role in mucosal repair processes. Importantly, adhesion blockade as a therapeutic concept can be detrimental in inflammatory bowel disease. Background & Aims: Neutrophils are generally thought to play an important proinflammatory role in the pathogenesis of inflammatory bowel disease. The objective of this study was to evaluate whether blocking the invasion of neutrophils by anti-L-selectin monoclonal antibodies modulates chemically induced colitis and how this modulation is accomplished. Methods: Trinitrobenzene sulfonic acid/dinitrobenzene sulfonic acid (TNBS/DNBS)-induced colitis was studied in rats on treatment with anti-L-selectin monoclonal antibodies (mAb) or antineutrophil antiserum. Different anti-L-selectin mAb, either blocking or nonblocking, as well as F(ab)2 fragments were evaluated. Additionally, leukocyte migration was examined using intravital microscopy. Furthermore, the effect of neutrophil depletion in rat TNBS-induced colitis was studied either prior to or after colitis induction as well as murine CD4+CD45RBhigh transfer colitis. Finally, bacterial translocation during DNBS-induced colitis was studied in neutrophil-depleted and control rats. Results: Anti-L-selectin mAb treatment resulted in increased mortality and bowel inflammation as well as hemorrhagic eye secretion. No clear difference was found between blocking and nonblocking mAb or F(ab)2 fragments. For all investigated antibodies/fragments, either complete blockade of leukocyte invasion or marked neutrophil depletion was found. Accordingly, neutrophil depletion by antiserum resulted in aggravation of rat DNBS-induced colitis as well as murine transfer colitis. Conclusions: Adhesion blockade or neutrophil depletion aggravates rat TNBS/DNBS-induced colitis together with extraintestinal manifestations of the eyes. Therefore, neutrophils appear to have an important role in mucosal repair processes. Importantly, adhesion blockade as a therapeutic concept can be detrimental in inflammatory bowel disease. See editorial on page 2049.The immune system exhibits 2 forms of migration: homeostatic migration and inflammatory migration. Although homeostatic migration is part of lymphocyte recirculation, inflammatory migration is characterized by migration of peripheral leukocytes into sites of infection and inflammation.1Steeber D.A. Tedder T.F. Adhesion molecule cascades direct lymphocyte recirculation and leukocyte migration during inflammation.Immunol Res. 2000; 22: 299-317Crossref PubMed Scopus (88) Google Scholar The migratory process can be divided into several overlapping steps including rolling, sticking, and transepithelial migration. Selectins are involved in the initial adhesion process, ie, rolling. They are composed of E-selectin, L-selectin, and P-selectin sharing overlapping functions.2Jung U. Ley K. Mice lacking two or all three selectins demonstrate overlapping and distinct functions for each selectin.J Immunol. 1999; 162: 6755-6762PubMed Google Scholar, 3Ebnet K. Vestweber D. Molecular mechanisms that control leukocyte extravasation: the selectins and the chemokines.Histochem Cell Biol. 1999; 112: 1-23Crossref PubMed Scopus (209) Google Scholar L-selectin is the only constitutively expressed selectin and is found on leukocytes, particularly neutrophils.4Rainer T.H. L-selectin in health and disease.Resuscitation. 2002; 52: 127-141Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Because L-selectin plays an important role in early leukocyte-endothelial interactions, its modification or blockade may have dramatic effects on the progression of inflammatory responses and clinical outcomes.4Rainer T.H. L-selectin in health and disease.Resuscitation. 2002; 52: 127-141Abstract Full Text Full Text PDF PubMed Scopus (67) Google ScholarL-selectin blockade and deficiency have been found to be beneficial in various disease models such as allograft rejection,5Tang M.L. Hale L.P. 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Attenuation of experimental autoimmune neuritis in the Lewis rat by treatment with an antibody to L-selectin.Neurosci Lett. 1997; 235: 9-12Crossref PubMed Scopus (16) Google Scholar and encephalomyelitis.9Archelos J.J. Jung S. Rinner W. et al.Role of the leukocyte-adhesion molecule L-selectin in experimental autoimmune encephalomyelitis.J Neurol Sci. 1998; 159: 127-134Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar In inflammatory bowel disease (IBD), adhesion blockade by a blocking anti-α4 integrin monoclonal antibody (mAb) (natalizumab) was described to be effective in cotton top tamarins10Podolsky D.K. Lobb R. King N. et al.Attenuation of colitis in the cotton-top tamarin by anti-α 4 integrin monoclonal antibody.J Clin Invest. 1993; 92: 372-380Crossref PubMed Scopus (316) Google Scholar and in a phase II trial in Crohn’s disease.11Ghosh S. Goldin E. Gordon F.H. et al.Natalizumab for active Crohn’s disease.N Engl J Med. 2003; 348: 24-32Crossref PubMed Scopus (767) Google Scholar However, in a recent phase III trial, efficacy of natalizumab in active Crohn’s disease was not confirmed.12Sandborn W.J. Colombel J.F. Enns R. et al.Natalizumab induction and maintenance therapy for Crohn’s disease.N Engl J Med. 2005; 353: 1912-1925Crossref PubMed Scopus (809) Google Scholar In addition, adhesion blockade was detrimental in some animal models of chronic inflammation, eg, mice lacking intercellular adhesion molecule 1 in experimental autoimmune encephalomyelitis,13Samoilova E.B. Horton J.L. Chen Y. Experimental autoimmune encephalomyelitis in intercellular adhesion molecule-1-deficient mice.Cell Immunol. 1998; 190: 83-89Crossref PubMed Scopus (29) Google Scholar and more severe experimental glomerulonephritis in P-selectin knockout mice.14Rosenkranz A.R. Mendrick D.L. Cotran R.S. et al.P-selectin deficiency exacerbates experimental glomerulonephritis: a protective role for endothelial P-selectin in inflammation.J Clin Invest. 1999; 103: 649-659Crossref PubMed Scopus (103) Google Scholar In active IBD, an abundance of monocytes, neutrophils, and lymphocytes infiltrate the mucosa, and neutrophils so far appeared to promote inflammatory progression in IBD.15Brannigan A.E. O’Connell P.R. Hurley H. et al.Neutrophil apoptosis is delayed in patients with inflammatory bowel disease.Shock. 2000; 13: 361-366Crossref PubMed Scopus (165) Google Scholar, 16Carter L. Wallace J.L. Alterations in rat peripheral blood neutrophil function as a consequence of colitis.Dig Dis Sci. 1995; 40: 192-197Crossref PubMed Scopus (12) Google Scholar, 17Palmen M.J. Dieleman L.A. van der Ende M.B. et al.Non-lymphoid and lymphoid cells in acute, chronic and relapsing experimental colitis.Clin Exp Immunol. 1995; 99: 226-232Crossref PubMed Scopus (41) Google Scholar However, in chemically induced IBD animal models, no pathogenic role of neutrophils could be found.18Buell M.G. Berin M.C. Neutrophil-independence of the initiation of colonic injury Comparison of results from three models of experimental colitis in the rat.Dig Dis Sci. 1994; 39: 2575-2588Crossref PubMed Scopus (75) Google Scholar, 19Yamada T. Zimmerman B.J. Specian R.D. et al.Role of neutrophils in acetic acid-induced colitis in rats.Inflammation. 1991; 15: 399-411Crossref PubMed Scopus (55) Google ScholarBecause controversial data exist regarding the role of neutrophils in IBD, this study was performed to evaluate the role of neutrophil adhesion in trinitrobenzene sulfonic acid/dinitrobenzene sulfonic acid TNBS/DNBS-induced colitis in rats. TNBS/DNBS-induced colitis is associated with increased infiltration of granulocytes and macrophages as well as increased mucosal permeability because of epithelial necrosis. The animals exhibit severe diarrhea, weight loss, and bowel wall thickening. After colitis induction, the injured tissue heals continuously, and no relapses occur. Therefore, it reflects important repair processes in IBD including the role of leukocytes in this process.20Hoffmann J.C. Pawlowski N.N. Kuhl A.A. et al.Animal models of inflammatory bowel disease: an overview.Pathobiology. 2002; 70: 121-130Crossref PubMed Scopus (93) Google Scholar We will show that blocking neutrophil invasion or depletion of neutrophils aggravate TNBS/DNBS-induced colitis in rats and transfer colitis in mice, indicating an important role for neutrophils in mucosal repair processes. Furthermore, the severe barrier defect in neutrophil-depleted rats led to translocation of gut bacteria into other organs, probably resulting in increased mortality and extraintestinal manifestations.Materials and MethodsAnimalsInbred female Lewis rats (8–12 weeks of age) were obtained from Charles River (Sulzfeld, Germany) or Harlan (Borchen, Germany). Animals were housed under conventional conditions at the Research Institute for Experimental Medicine (Berlin, Germany) with exception of the first experiment, which was performed at the University Hospital of Saarland Animals Facilities (Homburg, Germany). Animals for intravital microscopy (IVM) were kept under conventional conditions at the animal facility at the Department of Surgery at the University of Regensburg (Regensburg, Germany).Donor wild-type mice (C57BL/6) were obtained from I. Förster (Munich, Germany), and recombination activating gene 1-deficient mice (C57BL/6J-Rag1tm1Mom) were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6 and RAG-1-deficient (RAG-1 ko) mice were bred under specific pathogen-free conditions at the Research Institute for Experimental Medicine. During the experiments of transfer colitis, mice were housed under conventional conditions at the Research Institute for Experimental Medicine.All animals (rats and mice) were kept in polycarbonate cages and had free access to sterile standard chow and water. All experiments were performed in accordance with the German legislation on the protection of animals.AntibodiesThe following mouse anti-rat mAb were used in vivo: HRL3 (IgG1, blocking),9Archelos J.J. Jung S. Rinner W. et al.Role of the leukocyte-adhesion molecule L-selectin in experimental autoimmune encephalomyelitis.J Neurol Sci. 1998; 159: 127-134Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar OX85 (IgG1, blocking),21Nicholson M.W. Barclay A.N. Singer M.S. et al.Affinity and kinetic analysis of L-selectin (CD62L) binding to glycosylation-dependent cell-adhesion molecule-1.J Biol Chem. 1998; 273: 763-770Crossref PubMed Scopus (172) Google Scholar and HRL4 (IgG1, nonblocking)8Archelos J.J. Fortwangler T. Hartung H.P. Attenuation of experimental autoimmune neuritis in the Lewis rat by treatment with an antibody to L-selectin.Neurosci Lett. 1997; 235: 9-12Crossref PubMed Scopus (16) Google Scholar, 9Archelos J.J. Jung S. Rinner W. et al.Role of the leukocyte-adhesion molecule L-selectin in experimental autoimmune encephalomyelitis.J Neurol Sci. 1998; 159: 127-134Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 22Tamatani T. Kitamura F. Kuida K. et al.Characterization of rat LECAM-1 (L-selectin) by the use of monoclonal antibodies and evidence for the presence of soluble LECAM-1 in rat sera.Eur J Immunol. 1993; 23: 2181-2188Crossref PubMed Scopus (89) Google Scholar directed at rat L-selectin (CD62L). TS2/9 (IgG1; American Type Culture Collection)23Dengler T.J. Hoffmann J.C. Knolle P. et al.Structural and functional epitopes of the human adhesion receptor CD58 (LFA-3).Eur J Immunol. 1992; 22: 2809-2817Crossref PubMed Scopus (39) Google Scholar directed at human CD58 served as control mAb. Hybridoma cell lines of HRL3 and HRL4 were kindly provided by M. Miyasaka (Osaka Universtity Graduate School of Medicine, Japan), and the mAb OX85 was a gift from A. N. Barclay (University of Oxford, United Kingdom). For depletion of peripheral neutrophils in rats, polyclonal antiserum (rabbit anti-rat PMN; Accurate, New York, NY) and rabbit anti-rat IgG (DPC Biermann, Bad Nauheim, Germany) as control antibody were used. All mAb were purified from supernatants employing Protein G Sepharose (Amersham Pharmacia Biotech, Freiburg, Germany), diluted in phosphate-buffered saline (PBS), filtered (0.2 μm), and administered intraperitoneally (IP). F(ab)2 fragments of HRL3 and HRL4 were obtained by incubation of 2.0 μg pepsin per milligram IgG for 2 hours at 37°C, pH 3.5. The digestate was then dialyzed to neutral pH and passed over Protein A Sepharose (Amersham Pharmacia Biotech). Gel electrophoresis for characterization of the fragments demonstrated pure F(ab)2 fragments.The following rat anti-mouse mAb were used in vivo: RB6-8C5 (directed at mouse peripheral neutrophils)24Tepper R.I. Coffman R.L. Leder P. An eosinophil-dependent mechanism for the antitumor effect of interleukin-4.Science. 1992; 257: 548-551Crossref PubMed Scopus (479) Google Scholar, 25Schon M. Denzer D. Kubitza R.C. et al.Critical role of neutrophils for the generation of psoriasiform skin lesions in flaky skin mice.J Invest Dermatol. 2000; 114: 976-983Crossref PubMed Scopus (93) Google Scholar and rat IgG (Chemicon, Hampshire, United Kingdom) as control. The hybridoma cell line RB6-8C5 was kindly provided by M. Schön (Heinrich-Heine University, Düsseldorf, Germany) with permission by R. L. Coffman (Dynavax Technologies, Berkley, CA). The following antibodies were used in vitro: Annexin V-FITC (BD Biosciences, Heidelberg, Germany) and anti-CD95 (clone IPO-4; Abcam, Cambridge, United Kingdom).Induction of Rat TNBS-Induced ColitisRats were randomized into treatment or control groups in a blinded fashion. Trinitrobenzene sulfonic acid (TNBS; Sigma, Taufenkirchen, Germany) colitis in rats was induced as described previously.26Hoffmann J.C. Peters K. Henschke S. et al.Role of T lymphocytes in rat 2,4,6-trinitrobenzene sulphonic acid (TNBS) induced colitis: increased mortality after γδ T-cell depletion and no effect of γβ T-cell depletion.Gut. 2001; 48: 489-495Crossref PubMed Scopus (61) Google Scholar Because TNBS in concentrations sufficient for colitis induction in rats became unavailable during the experiments described herein, some experiments were conducted using dinitrobenzene sulfonic acid (DNBS; ICN, Eschwege, Germany), which is a reasonable substitute for TNBS.27Wallace J.L. Le T. Carter L. et al.Hapten-induced chronic colitis in the rat: alternatives to trinitrobenzene sulfonic acid.J Pharmacol Toxicol Methods. 1995; 33: 237-239Crossref PubMed Scopus (77) Google Scholar DNBS was given at a dose of 30 mg in 250 μL 50% wt/vol ethanol (mAb treatment prior to colitis induction) and 40 mg in 500 μL 50% wt/vol ethanol (mAb treatment after colitis induction), respectively. Twenty-four hours prior to colitis induction, the animals were deprived of food.Neutrophil Depletion in VivoFor neutrophil depletion, rats were injected IP with 3 mg rabbit polyclonal antiserum against polymorphonuclear neutrophils (anti-rat PMN) and mice with 200 μg of the mAb RB6-8C5. Successful depletion of segmented neutrophils was confirmed by differential counts of blood smears prepared from animals prior to antibody treatment and at the end of the experiment. Blood smears were prepared from 2 μL blood collected from tail vein, air-dried, and stained with Giemsa Wright stain (Sigma, Taufenkirchen, Germany). Two hundred cells per slide were counted to determine the percentage of neutrophils.Induction of Mouse Transfer ColitisTransfer colitis was induced in 30 RAG-1 ko mice, 6–8 weeks of age, by injection of 4 × 105 CD4+CD45RBhigh T cells IP. CD4+CD45RBhigh T cells were isolated from spleen cells from C57BL/6 mice employing magnetic cell separation technology. Spleen cells were isolated as described elsewhere,28Kuhl A.A. Pawlowski N.N. Grollich K. et al.Aggravation of intestinal inflammation by depletion/deficiency of {γ}{δ} T cells in different types of IBD animal models.J Leukoc Biol. 2007; 81: 168-175Crossref PubMed Scopus (70) Google Scholar followed by enrichment of CD4+ T cells employing the FITC-labeled anti-CD4 mAb RM4-5 (BD Biosciences) in optimal dilution and the anti-FITC MultiSort Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Subsequently, CD4+ T cells were labeled with highly diluted (1:400) anti-CD45RB-PE (BD Biosciences) and anti-PE MicroBeads (Miltenyi Biotec) following manufacturers’ instruction. Viability of CD4+CD45RBhigh T cells was confirmed by trypan blue dye exclusion and purity by flow cytometry (FACSCalibur; Beckton Dickinson); only CD4+CD45RBhigh T cells of >90% viability and >97% purity were transferred into RAG-1 ko mice.Treatment ProtocolFemale Lewis rats were treated with various anti-L-selectin mAb and their F(ab)2 fragments prior to colitis induction. The clinical colitis progression was examined in 3 sets of experiments. In the first experiment, rats were treated with either 500 μg blocking anti-L-selectin mAb OX85 (n = 20), blocking anti-L-selectin mAb HRL3 (n = 10), or 500 μg control mAb TS2/9 (n = 20). In the second experiment, rats received either 500 μg nonblocking anti-L-selectin mAb HRL4 (n = 10) or 500 μg TS2/9 (n = 10) as control. In the third experiment, rats received F(ab)2 fragments of HRL3 (n = 5) and HRL4 (n = 5) or whole HRL3 mAb (n = 3) as positive control and TS2/9 (n = 3) as negative control. In all experiments, mAb and F(ab)2 fragments (500 μg each) were applied IP on days −1, 0, 2, 4, and 6. TNBS colitis was induced on day 0, and animals were killed on day 10. The clinical course was assessed daily in all experiments, and animals were electively killed upon signs of severe colitis (eg, weight loss >20%, lethargy, or shagginess). The rolling behavior of leukocytes after treatment with 500 μg anti-L-selectin mAb OX85 (n = 4) and 500 μg control antibody TS2/9 (n = 4) in TNBS colitic rats was studied by IVM (see below). Further IVM studies were conducted in DNBS colitic rats treated with 500 μg HRL3 (n = 3), HRL4 (n = 3), and TS2/9 (n = 3).For neutrophil depletion, rats received either 3 mg polyclonal anti-neutrophil antiserum (anti-rat PMN, n = 12) or 3 mg control antibody (anti-rat IgG, n = 8) on days −1, 0, 2, 4, and 6. DNBS colitis was induced on day 0 in all control animals and in 8 depleted animals. Four anti-rat PMN-treated rats served as a second control group (sham group) and received no DNBS. Rats with established colitis received 3 mg anti-rat PMN (n = 5) and 3 mg control antibody (n = 4) on days 2, 4, and 6. The clinical courses in both experiments were assessed daily, and animals were killed on day 8 or upon signs of severe colitis (see above). For bacterial translocation studies, rats received either 3 mg polyclonal antiserum (anti-rat PMN, n = 10) or 3 mg control antibody (anti-rat IgG, n = 10) on days −1, 1, 3, and 5. DNBS colitis was induced on day 0 in all animals, which were killed on day 7 and examined for the bacterial load in extraintestinal organs to assess bacterial translocation.Mice received for neutrophil depletion either 200 μg mAb RB6-8C5 (n = 15) or 200 μg control antibody (rat IgG, n = 15) every third day. Antibody treatment started with transfer colitis induction (n = 10 each) or with first signs of colitis (eg, 5% weight loss, diarrhea, occult blood).Assessment of Inflammatory ChangesAt the end of the study, animals were killed by carbon dioxide anesthesia, the large intestine was excised, and the colon length was measured. The macroscopic appearance of the colon was scored in a blinded fashion as follows: 0, no evidence of inflammation; 1, erythema only; 2, erythema, slight edema, and small erosions; 3, 2 or more bleeding ulcers and/or inflammation and/or moderate adhesions; 4, severe ulceration and/or stenosis with prestenotic dilations and/or severe adhesions. Samples of the sigmoid colon were fixed in 4% formaldehyde/PBS and embedded in paraffin. Sections (5 μm) were stained with H&E, and the degree of inflammation was scored by a blinded pathologist as described previously29Kuhl A.A. Loddenkemper C. Westermann J. et al.Role of γδ T cells in inflammatory bowel disease.Pathobiology. 2002; 70: 150-155Crossref PubMed Scopus (23) Google Scholar: briefly, 0, no signs of inflammation; 1, low level of leukocyte infiltration (10–30 leukocytes per high-power field [hpf]); 2, moderate level of leukocyte infiltration (31–70 leukocytes per hpf); 3, high level of leukocyte infiltration (>71 leukocytes per hpf), high vascular density, thickening of the bowel wall; 4, transmural infiltrations, loss of goblet cells, high vascular density, strong bowel wall thickening, ulcerations, cryptic abcesses.Blood CountBlood samples were taken at the beginning (tail vein) and end (heart blood) of the experiments for blood count using an automatic cytometer (Beckmann Coulter, Germany).IVMMicroscopic observations of adhesion processes in vivo were made employing IVM as previously described.30Farkas S. Herfarth H. Rossle M. et al.Quantification of mucosal leucocyte endothelial cell interaction by in vivo fluorescence microscopy in experimental colitis in mice.Clin Exp Immunol. 2001; 126: 250-258Crossref PubMed Scopus (27) Google Scholar IVM was performed in colitic rats (on day 2) that had received 500 μg anti-L-selectin mAb OX85 (n = 4), HRL3 (n = 3), HRL4 (n = 3), and control mAb TS2/9 (n = 7) 1 day prior to and simultaneously with colitis induction.Sampling Procedures and Cultural AnalysisTo assess bacterial translocation, rats were killed with carbon dioxide. Cardiac blood, tissue samples from spleen, mesenteric lymph nodes, and proximal colon were removed under sterile conditions.Luminal contents from colon were resuspended in PBS and weighed, and 100-μL aliquots of serial dilutions plated onto solid medium (Oxoid, Wesel, Germany). Bacteria were grown at 37°C for 2 days under aerobic or for 5 days under anaerobic conditions, and total numbers were determined by colony counting on Columbia blood agar. Bile esculin, McConkey medium, Rogosa medium, and Columbia blood agar supplemented with hemin, kanamycin, and vancomycin (Merck, Darmstadt, Germany) were used for quantitative identification of enterococci, enterobacteria, lactic acid bacteria (mainly lactobacilli), and Bacteroides/Prevotella species, respectively. The amounts of gram-negative and gram-positive bacteria were determined by counting of distinct colony morphotypes on Columbia blood agar. Bacteria were subcultured and further investigated by Gram’s staining and by biochemical analysis with the API20E, API50CH, and API Rapid ID 32A systems (BioMérieux, Nuertingen, Germany). Results were expressed as colony-forming unit per gram luminal colon content.Bacterial translocation into mesenteric lymph nodes, spleen, and cardiac blood was determined by culture of respective samples in brain heart infusion and in thioglycolate broths (Oxoid, Wesel, Germany) for at least 1 week at 37°C, and bacterial growth was monitored by turbidity assessment. Aliquots from turbid broths were cultured on solid media under aerobic and anaerobic conditions, and the bacteria were identified microbiologically and biochemically as described above.Induction and Analysis of Apoptosis in Rat Lymphocytes and Neutrophils In VitroLymphocytes and neutrophils were isolated from heparinized heart blood of female Lewis rats (n = 10). After sedimentation of erythrocytes through Plasmasteril (Fresenius, Bad Homburg, Germany), lymphocytes and neutrophils were separated by gradient centrifugation via Ficoll-Paque (Biochrom KG, Berlin, Germany). Lymphocytes were isolated from the interphase and the neutrophils from the sediment. Remaining erythrocytes in the sediment were removed by rapid lysis in ice-cold distilled water. Lymphocytes and neutrophils were suspended in complete media at a final concentration of 1 × 106 cells/mL. The cells were incubated in triplicate (200 μL/well) for 18 hours in 96-well, round-bottom plates (NUNC, Wiesbaden, Germany) in the presence of PBS, 25 μg/mL HRL3, HRL4, OX85 or anti-CD95 at 37°C in 5% CO2 humidified air. Cells were harvested, washed twice in cold PBS and resuspended at 5 × 105 cells/100 μL 1X Annexin V binding buffer (BD Biosciences) and labeled for Annexin V and propidiumiodide (Sigma) according to the manufacturers’ instructions. Annexin V labels early and late apoptotic cells, whereas propidiumiodide only labels necrotic cells. The labeled cells were analyzed by flow cytometry using a FACS Vantage cell sorter (BD Biosciences) and the CellQuest software (BD Biosciences).Statistical AnalysisStatistical analysis was carried out using SPSS (SPSS Inc, Chicago, IL) for Windows (Microsoft Corp, Redmond, WA). Survival was analyzed using Kaplan–Meier analysis. For other comparisons, the Mann–Whitney U test was used. Values were expressed as mean (95% confidence intervals [CI]) and standard error of the mean (SEM). Differences were considered statistically significant for P < .05.ResultsBlocking Anti-L-Selectin mAb Aggravate Rat TNBS-Induced ColitisApplication of anti-L-selectin mAb starting prior to colitis induction led to more severe TNBS-induced colitis. Rats treated with either anti-L-selectin mAb OX85 or HRL3 displayed hemorrhagic secretion around the eyes and nose (Figure 1), increased mortality (Figure 2), and elevated macroscopic and histologic scores (Table 1, group A) compared with controls. Plasmatic and cellular coagulation as well as platelet counts were found to be normal (international normalized ratio, partial thromboplastin time, thrombin time, fibrinogen, and thrombocyte aggregation; data not shown). At autopsy, all OX85- and HRL3-treated animals displayed patchy colon necroses (Figure 3), which sometimes led to fecal peritonitis because of bowel perforation (data not shown). Importantly, the effects of OX85 and HRL3 were reproduced at another animal facility (data not shown).Figure 2Increased mortality of rats with TNBS-induced colitis because of mAb treatment with OX85 (dashed line, n = 20) and HRL3 (dotted line, n = 10) compared with TS2/9-treated controls (solid line, n = 20). Shown is the Kaplan–Meier analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Inflammatory Damage in TNBS-Induced Colitis Upon Treatment With Blocking, Group A, and Nonblocking Anti-L-Selectin mAb, Group BGroup AMacroscopic scoreMicroscopic scoreGroup BMacroscopic scoreMicroscopic scoreControl (TS2/9)2.0 ± 0.21.9 ± 0.2Control (TS2/9)2.4 ± 0.32.1 ± 0.3OX853.6 ± 0.1aP < .01 vs control.2.8 ± 0.2aP < .01 vs control.HRL43.8 ± 0.1aP < .01 vs control.3.3 ± 0.2bP < .05 vs control.HRL33.5 ± 0.2aP < .01 vs control.3.3 ± 0.3aP < .01 vs control.a P < .01 vs control.b P < .05 vs control. Open table in a new tab Figure 3Severe colitis with necrosis in rats with TNBS-induced colitis because of anti-L-selectin mAb treatment. Representative picture of patchy necrosis (arrow) upon OX85 treatment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because adhesion blockade by OX85 and HRL3 was thought to be the most likely explanation for the occurrence of fulminant colitis, a nonblocking anti-L-selectin mAb, HRL4, was studied next. Surprisingly, HRL4 treatment resulted also in increased mortality