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Macrophage Subpopulations in Lamina Propria of Normal and Inflamed Colon and Terminal Ileum

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Macrophage Subpopulations in Lamina Propria of Normal and Inflamed Colon and Terminal Ileum Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from Gut, 1989, 30, 826-834 Macrophage subpopulations in lamina propria of normal and inflamed colon and terminal ileum Y R MAHIDA, S PATEL, P GIONCHETTI, D VAUX, AND D P JEWELL From the Gastroenterology Unit, Radcliffe Infirmary, Oxford SUMMARY The aim of this study was to characterise human intestinal macrophages in normal and inflamed ileum and colon. Immunoperoxidase staining with a panel of monoclonal antibodies and histochemical staining for acid phosphatase and non-specific esterase was performed. In the superficial lamina propria, normal colonic macrophages were larger and more strongly positive for acid phosphatase and non-specific esterase than those in normal terminal ileum. There were more macrophages staining with monoclonal antibody RFD1 in the superficial lamina propria of the latter. Studies in inflammatory bowel disease tissue showed the presence of macrophages staining with monoclonal antibodies RFD9 and 3G8 which were rarely present in normal tissue and represented a different pattern from that seen in infectious colitis. Studies on isolated intestinal macrophages confirmed the findings in tissue sections. Subpopulations ofintestinal macrophages are likely to have different functional roles. Phenotypic changes during inflammation may be induced by mediators of inflammation or may represent a recently recruited population of cells. http://gut.bmj.com/ Macrophages arise from the bone marrow where the In active inflammatory bowel disease, there is an monoblast differentiates into promonocytes and then increase in the mucosal macrophage population. into monocytes which are released into the blood.' Morphological and histochemical heterogeneity of From the blood, monocytes migrate to tissues where intestinal macrophages has previously been demon- they become macrophages. During an inflammatory strated with a distinct population being prominent in reaction, large numbers of monocytes are recruited active inflammatory bowel disease.6 We have studied into the lesion. The functionally diverse subpopula- this heterogeneity further, in normal and inflamed on September 29, 2021 by guest. Protected copyright. tions of these cells as well as the resident population small and large intestine, using a panel of monoclonal of macrophages are not easy to identify just on antibodies and histochemical staining. Tissue morphology and therefore new techniques are sections and cells isolated from normal and inflamed required to identify them.2 Monoclonal antibodies mucosa were studied. have provided a powerful means to identify and study unique subsets of lymphocytes. By using similar Methods- antibodies it should be possible to study the hetero- geneity of intestinal macrophages. PATIENTS Human intestinal macrophages are found in the Normal colonic and ileal mucosa was obtained lamina propria, frequently close to the basal mem- from 23 patients (16 women, median age 65 years brane of the epithelium.3 They appear to be closely (range 38-83)) undergoing intestinal resection for associated with lymphocytes, plasma cells, and carcinoma. The mucosa was obtained at least 5 cm epithelial cells and are likely to have a number of from tumour. specialised functions. These include antigen presen- Mucosa involved with active inflammatory bowel tation;4 microbicidal and tumoricidal activity; disease was obtained from ileum and colon resected secretion of mediators that regulate other cells and at operation or from rectal biopsy specimens from secretion of a variety of enzymes.' patients with active ulcerative colitis or Crohn's colitis rectal Address for correspondence: Dr D P Jewell, Gastroenterology Unit, Radcliffe (with involvement). Thirty two patients Infirmary, Oxford. were studied (20 women, median age 40 years (range Accepted for publication 31 October 1988. 17-77)). Ten of the 32 patients were on intravenous 826 Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from Macrophage slibpopulations in lamina propria ofnormal and inflamed colon and terminal ileum 827 steroids, 13 on steroid enemas only an eighteen were Table 1 Monoclonal antibodies used on sulphasalazine. Rectal biopsy specimens from six patients with ulcerative colitis in remission (all on Antibody Cells labelled Source sulphasalazine only) and from three patients with RFDI (IgG)' '6 Dendritic' cells Dr Poulter Clostridium difficile colitis were also studied. Royal Free Hospital London TISSUE SECTIONS RFD7 (lgG)"'` I Mature macrophages Dr Poulter Royal Free Hospital Tissue was placed on a small piece of cork and London covered with OCT mounting medium (Ames) and RFD9 (IgG)'`' Epitheliod cells and Dr Poulter frozen in isopentane in liquid nitrogen. Cryostat tingible body Royal Free Hospital sections 4 p.m thick were cut from the blocks at macrophages London -25°C, fixed in acetone for 10 and stored at EBM1 1 (lgG)' Tissue macrophages ProfessorJ 0 D McGee minutes, John Radcliffe Hosp. -20°C until used. Oxford 3C10 (lgG)` Monocytes and Dr Ralph Steinman ISOLATION OF INTESTINAL MONONUCLEAR macrophages The Rockefeller Univ. CELLS New York 3G8 (IgG) Fcy receptor on poly- Dr H B Fleit Cells isolated from normal and inflamed colonic morphonuclear State Univ of New mucosa were studied. Macroscopically normal leucocytes natural York, New York mucosa, greater than 5 cm from tumour and inflamed killer cells and mucosa was obtained from fresh operation resection some macrophages Y1/82A (IgG)` Monocytes and Dr D Y Mason specimens. macrophages John Radcliffe Hosp. Mononuclear cells were obtained by a modified Oxford EDTA-collagenase technique of Bull and 201521 (IgG) Anti pan HLA-D Dr S Fuggel Bookman. Specimens were washed with calcium and John Radcliffe Hosp. Oxford magnesium free Hanks Balanced Solution (HBSS; RDFRI (IgM)` Anti HLA-DR Dr Poulter Flow Laboratories). Mucosa was dissected from Royal Free Hosp. muscularis mucosa and fragments incubated with London 1 mM dithiothreitol at for 15 minutes. (Sigma) 20°C http://gut.bmj.com/ After washing with HBSS, epithelial cells were removed by 3x30 minute incubations in 5 mM incubated with peroxidase-conjugated rabbit anti- EDTA (BDH chemicals) at 37°C with shaking. mouse antibody (Dako; diluted in TBS/normal Mucosa was subsequently cut into small pieces human serum) for 30 minutes, washed with TBS for and digested with collagenase (from Clostridium five minutes and incubated with peroxidase conju- histolyticum; Boehringer Mannheim) at a concentra- gated swine antirabbit antibody (Dako; diluted in tion of 100 mg/100 ml culture medium (containing TBS/normal human serum) for 30 minutes. After 10% FCS/RPMI; Gibco) for three hours or 20 mgIlO0 further washing, peroxidase activity was developed on September 29, 2021 by guest. Protected copyright. ml culture medium for 12 hours. After washing, with diaminobenzidine and hydrogen peroxide. The mononuclear cells were obtained by centrifugation preparations were counterstained in Harris' haema- over Ficoll-Paque (Pharmacia). All the media used toxylin, dehydrated, and mounted in DPX. Two contained 100 U/ml penicillin, 5 p.g/ml gentamicin controls were used, omitting the first antibody and and 100 p.g/ml streptomycin. secondly, using OX7 (mouse anti-rat Thy 1.1 mono- Cytospin preparations with about 60000 mono- clonal antibody kindly donated by Dr A Williams, nuclear cells per slide were made, air dried and fixed MRC Cellular Immunology Unit, Oxford) as the with acetone. They were stored at -20°C until used. primary antibody (at similar concentrations to those used for the macrophage monoclonal antibodies). MONOCLONAL ANTIBODIES Tissue sections were also stained for acid phos- Table 1 lists the monoclonal antibodies used. phatase (ACP) using naphthol AS TR phosphoric acid sodium salt as substrate9 and for non-specific STAINING esterase (NSE) using naphthyl acetate as substrate.9 Tissue sections and cytospin preparations were In some studies acid phosphatase activity was stained with the monoclonal antibodies using the detected in combination with immunoperoxidase peroxidase technique.' Endogenous peroxidase staining' using monoclonal antibodies as described. activity was blocked with methanol and hydrogen For double immunofluorescence, monoclonal anti- peroxide. The preparations were incubated with the bodies of different class (IgG and IgM) were used and monoclonal antibody for 60 minutes and washed in labelled with fluorescein-conjugated goat anti-mouse Tris buffered saline (TBS; pH 7). They were then IgG and TRITC-conjugated goat antimouse IgM Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from 828 Mahida, Patel, Gionchetti, Vaux, andJewell (Nordic).` Sections were examined under a standard Table 2 Macrophages in tissue sections stainedfor ACP 14 Zeiss microscope equipped with a x40 phase oil and NSE and with monoclonal antibodies RFDI, RFD9, objective and epifluorescence condenser containing and 3G8 selective filters for FITC and TRITC. ACP and NSE RFDI RFD9 and 3G8 CELL COUNTS Normal Large, strongly Positive cells Rarely any For each monoclonal antibody and acid phosphatase colon positive cells in predominantly positive cell in macrophages as a percentage of the superficial in superficial lamina propria staining, positive lamina propria lamina propria total number of mononuclear cells in the superficial Normal Smaller, less Positive cells Rarely any lamina propria was determined by using a graticule, terminal strongly throughout positive cell in using the same magnification
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