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 heterogeneity 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 monoclonal 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 objective, and counting ileum positive cells in lamina propria lamina propria in three different areas of each section. superficial superficial> lamina propria deep Cells in and around Peyer's patches were not UC and Strongly positive Positive cells Positive cells in considered, they are the subject of a separate investi- colonic cells throughout lamina propria gation. For all sections, the same length of superficial Crohn's throughout lamina propria in all sections lamina propria was used. In the cytospin prepara- mucosa lleal Strongly positive Positive cells Positive cells in tions, the percentage of positive macrophages was Crohn's cells throughout lamina propria determined by counting at least 200 mononuclear throughout lamina propria of most cells per preparation. All the counts were performed mucosa and but fewer in sections but many were confirmed submucosa superficial by one investigator (YRM) lamina propria independently by other investigators. compared to normal ileum STATISTICAL ANALYSIS Analysis between median percentages of positive macrophages (staining with monoclonal antibodies or for ACP or NSE in tissue sections or cytospin positive macrophages were also ACP positive. For preparations) was performed using Wilcoxon's rank- antibodies RFD7 and 3C10, 70-95% of cells in the

sum tests for unpaired samples. Results are superficial lamina propria were also ACP positive http://gut.bmj.com/ expressed as a median and range. whereas in the deep lamina propria, only 5-20% of cells were ACP positive. More than 90% of the ACP Results positive cells were HLA-D (201521) positive. There were no differences between proximal and distal In control tissue sections or cytospin preparations colon in the distribution of macrophages. (after either omitting the primary antibody or using an irrelevant monoclonal antibody, OX7), no NORMAL TERMINAL ILEUM peroxidase staining was seen. Macroscopically normal terminal ileum and ascend- on September 29, 2021 by guest. Protected copyright. Table 2 summarises the different staining patterns ing colon (at least 5 cm from tumour) from six right of macrophages in tissue sections stained for ACP hemicolectomy specimens resected for caecal and NSE and with monoclonal antibodies RFD1, carcinoma were studied. RFD9, and 3G8. Compared with normal colonic mucosa, macrophages in the superficial lamina propria of the NORMAL COLON terminal ileum were smaller and less strongly ACP In tissue sections, macrophages were most frequent and NSE positive (Fig. 2). In the deeper lamina in the superficial lamina propria just below the propria, the macrophages became smaller and epithelium. Here they were large, round and strongly weakly ACP and NSE positive or ACP and NSE ACP and NSE positive (Fig. 1). In the deeper lamina negative. In most sections, all the monoclonal anti- propria they became smaller, more irregular and bodies stained macrophages in the superficial as well were only weakly ACP and NSE positive or fre- as deep lamina propria. quently ACP and NSE negative. Cell counts performed using a graticule (Table 3) Monoclonal antibodies RFD1 and EBM11 stained showed that there was a significantly higher propor- macrophages predominantly in the superficial lamina tion of RFD1 positive cells in the villi of the terminal propria whereas the other antibodies (RFD7, 3C10, ileum compared with the superficial lamina propria Y1/82A, and 201521) stained macrophages both in of the colon. the superficial and deep lamina propria. Combined Combined immunoperoxidase and acid phos- staining with monoclonal antibodies and for acid phatase staining gave similar results to those phosphatase activity showed that 30-45% of RFD1 obtained for normal colon. Thus, in the superficial Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from

Macrophage subpopiulations in lamina propria ofnormal and iniflamed colon and terminal ileum 829

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Fig. 1 Sectionof normal colonic mucosa stained for acid phosp1hatase sliowing strongly positis'e mac.rolphages ins5uperfic-ial lamina propria. http://gut.bmj.com/ lamina propia 20-40% of RFD1 positive macro- positive mononuclear cells in ulcerative colitis and phages were also ACP positive whereas 80-90% of Crohn's colitis (Table 4). These findings were.con- cells that stained with RFD7 and 3C10 also showed firmed by counts on cytospin preparations of isolated ACP activity. In the deeper lamina propia, only mononuclear cells stained with the same monoclonal about 5-25% of cells that stained with RFD1, RFD7, antibodies (Table 5). Of the 13 normal colons, there and 3C10 demonstrated ACP activity. More than were no RFD9 positive cells in the lamina propria in 90% of the ACP positive cells were HLA-D (201521) nine and no 3G8 positive mononuclear cells in 10. In positive. contrast, all the ulcerative colitis and Crohn's colitis on September 29, 2021 by guest. Protected copyright. sections showed the presence of RFD9 and 3G8 (Fig. INFLAMED COLONIC MUCOSA 4) positive mononuclear cells. Staining of sequential In active inflammatory bowel disease mucosa, sections suggested that RFD9 and 3G8 positive although the overall pattern of large round macro- mononuclear cells were largely distinct populations phages in the superficial lamina propria and smaller of cells. Combined staining demonstrated that 8(- more irregular cells in the deeper regions was main- 90% of both RFD9 and 3G8 positive macrophages tained, there was a general increase in the number of demonstrated ACP activity. macrophages which were irregular in shape. Acid Many of the RFD9 positive cells were largely phosphatase and NSE positive macrophages were aggregated in seven of 14 ulcerative colitis and all the seen throughout the lamina propria and submucosa Crohn's colitis sections (Fig. 5). The unaggregated (the latter, especially with Crohn's colitis) (Fig. 3). cells tended to be less intensely stained than the Cells staining with the antibodies RFD1 and EBM 1 1 aggregated cells. were also similarly distributed. Combined staining Examination of rectal biopsy specimens from six showed that 7(-90% of RFD1, RFD7, and 3C10 patients with ulcerative colitis in remission (con- positive macrophages throughout the lamina propria firmed by routine histology) showed occasional (as well as the submucosa) showed ACP activity. RFD9 positive cells in two but no 3G8 positive cells in Most (more than 90%) of ACP positive cells were any section. HLA-D (201521) positive. Also studied were rectal biopsies from three Cell counts using a graticule showed that there was patients with Clostridiuim difficile colitis (diag- a significant increase in RFD9 positive and 3G8 nosed by the presence of the toxin and compatible Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from

830 Mahida, Patel, Gionchetti, Vaux, andJewell

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Fig. 2 Section ofnormal terminal ileal mucosa stainedfor Fig. 3 Section ofcolonic Cr4ohn 's disease stainedfor acid acid phoslplhatase showing weak actit'ity in tle macrophages phosphatase. Strongly positiv,,e cells are seen in the mucosa as in the supetficial lamina propria. well as the submucosa. on September 29, 2021 by guest. Protected copyright.

Table 3 Median (range) % ofpositive macrophages in supetficial lamina propria in tissue sections from normal Table 4 Median (range) % positive macrophages in terminal ileum and ascending colon (ofsix intestinal superficial lamina propria in tissue sections ofnormal and resections for carcinoma ofcaecum) inflammatory bowel disease colon

Nortnal terminal ileium Normal colon Monoclonal Nornmal colon UC Crohn's colitis olonoclonal aiitibli' (i -=6) (n=6) antibody (n= 13) (n = 14) (n =8) RFDl1 32 (28-35) 20-5 (13-24)* RFDl 23 (13-25) 19.5 (10-27) 20.5 (9-28) RFD7 22(14-26) 19-5 (14-24) RFD7 20 (10-28) 19.5 (12-29) 23 (13-37) RFD9 0 (0-2) 0 (0-3) RFD9 0 (0-5) 12.5 (5-27)* 7.5 (2-15)* EBII 26 (16-34) 22.5 (18 -34) EBMII 22 (18-34) 22-5 (9-31) 21-5 (13-29) 3C10 18.5 (15-24) 22 (13-43) 3C10 29 (13-43) 24 (15-31) 21-5 (14-33) 3G8 (0 1) 0(0- 1) 3G8 0(0-5) 9.5(1-15)* 11.5 (2-20)* Y1/82A 31.S5 (30-36) 31 (23-34) Y1/82A 31 (23-32) 29(17-36) 29(15-43) ACP 19 (16-29) 23 (20-29) ACP 20 (16-29) 21 (12-26) 22.5 (18-28)

*p<0.01. Macrophages staining with each monoclonal antibody or *p<0(l (normal v, inflamed). Macrophages staining with each ACP as a percentage of the total mononuclear cell population in the monoclonal antibody or for ACP as a percentage of the total superficial lamina propria was determined by using a graticule. mononuclear cell population in the superficial lamina propria was Three different areas of superficial lamina propria of each section determined using a graticule. Three different areas of superficial were examined. lamina propria of each section were examined. Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from

Macrophage subpopulations in lamina propria ofnormal and inflamed colon and terminal ileim83 831

Table 5 Median (range) % positive macroplhages in Cell counts using a graticule revealed an increase in cytospin preparations ofisolated colonic mucosal the proportion of RFD9 and 3G8 positive mono- mononuclear cells nuclear cells (Table 6). The RFD9 positive cells were largely aggregated in seven of 10 ileal Crohn's Monoclonial Normal coloni IBD colon As in bowel disease antibody (n =10) (ni = 16) sections. the active inflammatory colon, the unaggregated cells tended to be less RFD1 9.5 (5-19) 9.0 (4-16) intensely stained than the aggregated cells. RFD7 5 (1-12) 7.5 (2-9) There was a significant decrease in the proportion RFD9 0.5 (0-2) 4 (1-7)* EBMII 11(9-24) 12 (8-23) of RFDI positive cells in the superficial lamina 3C10 14 (6-20) 12 (5-20) propria of the inflamed ileum compared with normal 3G8 1 (0-2) 4 (2-11)* ileal mucosa. Combined staining showed that 70- Y1/82A 17.5 (8-24) 12 (7-21 ) 100% of macrophages staining with the antibodies RFDI, RFG7, 3C10, RFD9, and 3G8 were also *p<0.0l. Macrophages staining with each monoclonal antibody or positive for ACP. As for the colon, more than 90% of for ACP as a percentage of the isolated mononuclear cell population was determined by counting at least 200 cells per preparation. ACP positive cells were stained with antibody 201521 (HLA-D). Cytospin preparations of mononuclear cells isolated from nine specimens of ileal Crohn's showed histology). There were no RFD9 positive cells in any, RFD9 positive cells in all (median 3%; range 1-7%) but 3G8 positive mononuclear cells were present in and 3G8 positive macrophages in preparations of all three. eight out of nine specimens (median 2%; range 1-4%). In contrast no RFD9 positive or 3G8 positive ILEAL CROHN S DISEASE cells were seen in mononuclear cells isolated from Acid phosphatase and NSE positive cells were thee specimens of normal terminal ileum. present throughout the mucosa and also the submucosa. In many sections there was a general DOUBLE IMMUNOFLUORESCENT1 STAINING increase in the intensity of staining for ACP and NSE Double immunofluorescent staining of normal and in the lamina propria compared with normal ileal inflamed colonic and ileal tissue showed that tissue. There was also an increase in the number of more than 90% of macrophages staining with the http://gut.bmj.com/ irregular macrophages especially in the superficial monoclonal antibodies listed in Table 1 were HLA- lamina propria. DR positive.

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Fig. 4 Macrophages (arrowed) stained with monoclonal antibody 3G8 in a sec.tion of olonic C olins disease. Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from

832 Mahida, Patel, Gionchetti, Vaux, andJewell

Fig. 5 Aggregate of RFD9 positive cells in section ofcolonic Crohn's disease.

Discussion lamina propria they became smaller, more irregular and showed weak activity for ACP and NSE. Macro-

We have studied heterogeneity of macrophages of phages in normal terminal ileum of the same patients http://gut.bmj.com/ normal and inflamed terminal ileal and colonic showed a similar distribution but generally the cells mucosa using a panel of monoclonal antibodies and tended to be smaller and less strongly positive for ACP and NSE staining. Monoclonal antibodies used ACP and NSE. included many which have been shown to have high It has been suggested that macrophages with high specificity for macrophages.27-" lysosomal enzyme content (strongly ACP positive) In the normal colonic mucosa, macrophages below are likely to be 'scavenger' cells.'` The normal colonic the epithelium are predominantly large, round and lumen has a large bacterial flora;'7 in contrast, the strongly positive for ACP and NSE. In the deeper small intestinAl lumen has a very much smaller on September 29, 2021 by guest. Protected copyright. bacterial population.'` This may explain the presence Table 6 Median (range) % ofpositive macrophages in of macrophages with strong ACP activity in the suipeificial lamina preopria in tissue sections ofnormal and superficial lamina propria of the colon. Croln 's terminal ileum Studies using combined staining for ACP and Class II molecules have shown that there are distinct Monoclonal Normal terminal Crohn 's terminal populations of cells with high expression of Class II atitibodV,i ileium (nt =6) ileum (n = 10) molecules with low ACP activity - for example, RFDI 32 (28-35) 19.5 (1()29)* interdigitating cells of T cell zone in lymph nodes and RFD7 22 (14-26) 23.5 (15-32) Langerhans cells of skin - and of cells with weak or no RFD9 0 ((12) 5.5 (1-14)* expression of Class II antigens but strong ACP EMBII 26 (16-34) 29 (24-35) activity (tissue macrophages) (reviewed in'6 `v). 3C10 18 (15-24) 23.5 (16-37) 3G ()1((-) 55(0-18)t Development of monoclonal antibodies RFD1 and Y 1/82A 315 (3(-36) 315 (21-40) RFD7, has allowed further studies on these two ACP 19 (16-29) 23 (11-34) subpopulations of cells. RFD1 labels a product of the HLA-D locus that is preferentially located on *p

Macrophagesubpopulations in lamina propria ofnormal and inflamed colon and terminal ileum 833 positive) - for example, macrophages in the marginal staining of red pulp macrophages and Kupffer cells sinus of lymph nodes, but not interdigitating cells of by this antibody; white pulp of spleen and lymph T zones and thymic medulla." 16 nodes were negative.28 Our study shows that normal In the villi of the normal terminal ileum, a higher intestinal macrophages are virtually negative in their proportion of macrophages staining with the anti- expression of the 3G8 antigen but in inflamed ileal body RFD1 were present compared with the super- and colonic tissue, some macrophages showed ficial lamina propria of the colon. There was no sig- reactivity with this antibody. Macrophage expression nificant difference in the proportion of macrophages of this antigen occurred in inflammation as a result of staining with the other monoclonal antibodies. active inflammatory bowel disease or infection (in In the superficial lamina propria ofboth the normal contrast with reactivity with the RFD9 antibody). terminal ileum and colon, the majority of RFD7 and The significance of this is uncertain. It has been 3C10 positive macrophages were ACP positive com- suggested, however, that low-affinity FcyRs may be pared with about 30% of RFD1 positive cells. important in the clearance of immune complexes. The normal small intestinal mucosa comes in This has been supported by some recent animal contact with a large variety of soluble food antigens studies.28 Thus it is conceivable that macrophage and this may account for the higher proportion of expression of this low-affinity FcyR in inflamed RFD1 positive ('antigen presenting') cells in the villi intestine may have the role of clearing immune of the terminal ileum. complexes. In inflammatory bowel disease, there was an In conclusion, subpopulations of macrophages in increase in the intensity of ACP staining as well as in normal and inflamed human colon and terminal the number of ACP positive cells in deep lamina ileum in tissue and in isolated cells have been shown propria and submucosa. This may be caused by using a panel of monoclonal antibodies and histo- increased penetration of bacteria and its products chemical staining. The phenotypes of these cells are after damage to the epithelial barrier. likely to be influenced by their environment which in Monoclonal antibody staining also showed the the intestine is determined to a large extent by the presence of two new subpopulations of macrophages luminal contents and the integrity of the epithelial in the lamina propria of mucosa with active barrier. The latter is deranged in inflamed mucosa

inflammatory bowel disease. These subpopulations and the increased penetration of luminal antigens as http://gut.bmj.com/ stained with the antibodies RFD9 and 3G8. These well as the mediators released during inflammation cells were absent or only occasionally present in the could influence the phenotype of macrophages. In normal tissue or in ulcerative colitis in remission. addition, recently recruited monocytes from the The significance of the appearance of these two circulation are also likely to be phenotypically subpopulations of macrophages in the inflamed tissue different from the resident population of macro- is unknown. RFD9 is a marker for epithelioid cells phages. and 'tingible body' macrophages. 1621 Epithelioid Finally, this study also shows that antigenic cells in sarcoid granulomas are strongly positive with determinants on macrophages that are recognised by on September 29, 2021 by guest. Protected copyright. this antibody.2' We have recently shown strong monoclonal antibodies used in this study do not staining of epitheliod cells in Crohn's granulomas by appear to be affected by the isolation procedure. this antibody.22 Aggregates of these cells occur in active ulcerative colitis or Crohn's disease, especially Dr Mahida was supported by the Medical Research in the latter. It is possible that, under certain condi- Council. Miss S Patel was supported by Pharmacia tions, some of these aggregates may go on to form AB. We are grateful to Mr Kettlewell and Mr granulomas. Mortensen for the operation resection specimens and Monoclonal antibody 3G8 recognises the low to all those who supplied the monoclonal antibodies affinity FcyR on polymorphs, natural killer cells and (Table 1). some macrophages.2324 There are two types of References receptors with low affinity for monomeric IgG; one with broad electrophorectic mobility (51-73kd) is 1 Van Furth R. Origin and kinetics of mononuclear found on neurtophils,23 natural killer cells24 and phagocytes. In: Van Furth R, ed. Mononuclear phago- macrophages (identified by 3G8), and one on cytes, functional aspects. Boston: Nijhoff (Martinus), platelets25 and human monocyte lines.26 The FcyR 1980: 1-30. 2 Stewart CC. Methods for studying the ontogeny of recognised by 3G8 is absent on monocytes but is mononuclear phagocytes. In: Weir DM, Herzenberg present on 60% of alveolar macrophages. The 3G8 LA, Blackwell C, Herzenberg L, eds. Handbook of determinant appears on monocytes cultured in vitro experimental immunology 2. Cellular immunology. for seven days suggesting that it is an inducible Oxford: Blackwell Scientific Publications, 1986: protein.27 Sections of human spleen and liver show 44.1-17. Gut: first published as 10.1136/gut.30.6.826 on 1 June 1989. Downloaded from

834 Mahida, Patel, Gionchetti, Vaux, andJewell

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