Respiratory Burst Activity of Intestinal Macrophages in Normal and Inflammatory Bowel Disease

Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

Gut, 1989, 30, 1362-1370 Respiratory burst activity of intestinal macrophages in normal and inflammatory bowel disease

Y R MAHIDA, K C WU, AND D P JEWELL From the Gastroenterology Unit, Radcliffe Infirmary, Oxford

SUMMARY Macrophages isolated from normal mucosa (greater than 5 cm from tumour) and inflamed mucosa (from patients with inflammatory bowel disease) of colon and ileum were studied for their ability to undergo a respiratory burst as assessed by reduction of nitroblue tetrazolium to formazan. Using phorbol myristate acetate (PMA) and opsonised zymosan as triggers, only a minority (median: 8% for zymosan and 9% for PMA) of macrophages isolated from normal colonic mucosa demonstrated release of oxygen radicals. In contrast, a significantly greater (median: 17% for zymosan and 45% for PMA) proportion of macrophages isolated from inflamed colonic mucosa were able to undergo respiratory burst. Studies with normal and inflamed ileum showed similar results. Stimulation of macrophages isolated from normal colon with interferon-y produced only a small increase in the proportion of cells showing release of oxygen radicals. We conclude that the respiratory burst capacity of majority of macrophages isolated from normal colon and ileum is downregulated and a greater proportion of macrophages isolated from inflamed colon and ileum are able to undergo a respiratory burst. http://gut.bmj.com/ An activated macrophage is capable of mediating macrophages are increased in number in the mucosa" antimicrobial and cytotoxic effects.' The activation of both being especially prominent in granulomata of process is accompanied by a number of physiological Crohn's disease. There is increased monocyte turn- and biochemical changes. These include enhanced over'2 and activation'3 14 in active disease and these secretion of neutral proteases,2 acid hydrolases,3 cells are likely to be the main source of the intestinal enhanced expression of HLA class II molecules,4 macrophages. Currently, there is very little informa- down regulation of receptors for mannose terminated tion on the function of macrophages in normal and on October 2, 2021 by guest. Protected copyright. glycoconjugates5` and an enhanced or upregulated inflamed intestine. respiratory burst.7 During a respiratory burst, oxygen In this study, macrophages isolated from histo- is taken up by the cell and enzymatically converted by logically normal colon or ileum and from the colon membrane associated NADPH oxidase to super- and ileum of patients with active inflammatory bowel oxide anion (02), hydrogen peroxide (H202) and disease (IBD) were studied for their ability to under- hydroxyl radical (OH). A respiratory burst can be go a respiratory burst. Opsonised zymosan, phorbol triggered by phagocytosis ofparticles such as zymosan myristate acetate (PMA) and N-formylmethionyl- or by soluble surface active agents such as phorbol leucyl-phenylalanine (FMLP) were used as triggers. myristate acetate (PMA).8 Some of the reactive Respiratory burst activity was assessed by reduction metabolites of oxygen (RMO) can be released out- of nitroblue tetrazolium to formazan."1 6 In some side the cell where damage to surrounding cells can studies, the effect of interferon-gamma (IFN-y) and occur.9"' lipopolysaccharide (LPS) on respiratory burst Ulcerative colitis and Crohn's disease are chronic capacity of isolated macrophages was studied. inflammatory disorders of unknown aetiology, and Methods Address for correspondence: Dr D P Jewell. Gastroenterology U!nit, Radcliffe Infirmary. Oxford OX2 6HE. STUDY POPULATION Accepted for publication 17 February 1989. Patients (10 men, 14 women, age 23-83 years) 1362 Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

Respiratory burst activity ofintestinal macrophages in normal and inflammatory bowel disease 1363

undergoing colonic resection for carcinoma (16), TRIGGERS familial polyposis (two), idiopathic constipation (one) Phorbol 12-myristate 13-acetate (PMA; Behring or right hemicolectomy for caecal carcinoma (five) Diagnostics) was kept in small aliquots at -70°C were studied. in a stock solution of 1 mg/mI in dimethylsulphoxide Thirty two patients (18 men, age 14-70 years) with (Grade 1, Sigma). An aliquot was thawed inflammatory bowel disease involving the colon and immediately before use. 12 patients (seven men, age 16-71 years) with ileal Opsonised zymosan was prepared as previously Crohn's disease were studied. All patients under- described. Zymosan (Sigma, type A) was boiled in going intestinal resection for inflammatory bowel water. After washing it was resuspended in HBSS at disease had active disease. All except two (with 20 mg/mI and stored at 4°C. Opsonised zymosan was colonic Crohn's disease) were receiving intravenous prepared by incubating the stock zymosan with fresh hydrocortisone. human serum for 30 min at 37°C. It was then centrifuged and resuspended at 1 mg/ml in HBSS. CELL ISOLATION N-formylmethionyl-leucyl-phenylalanine (FMLP) Normal mucosa from patients without inflammatory (Sigma) was stored in phosphate buffered saline bowel disease was obtained at least 5 cm from tumour (pH7) at a concentration of 2x 10 M at 4°C. and was macroscopically as well as histologically normal. Inflamed, and in some cases non or minimally DETECTION OF RESPIRATORY BURST ACTIVITY inflamed mucosa was studied from ulcerative colitis Release of RMO by macrophages in response to the or Crohn's disease colon or ileum. Mononuclear cells triggers was assessed by reduction of nitroblue tetra- were isolated by a modification of the technique zolium (NBT; Sigma) to a deep blue-black formazan described by Bull and Bookman.'7 reaction product. Nitroblue tetrazolium was dissolved The mucosa was transported in cold (4°C) RPMI in RPMI at a concentration of 1 mg/mi and filtered 1640 (Gibco) and extensively washed with Hanks' with a 0-2 [t filter just before use. Balanced Salt Solution without calcium and 1-5 x 106 intestinal mononuclear cells in 4 ml culture magnesium (CMF HBSS; Flow laboratories). 5-10 medium were incubated at 37°C with 1 ml NBT mm strips of mucosa were dissected and incubated solution (1 mg) and either PMA at a final concentra- with 1 mmol dithiothreitol (Sigma) for 20 min at tion of 200 ng/ml, 75kg opsonised zymosan (final room temperature (with shaking every five minutes). concentration, 15 Fg/ml) or FMLP at a final concen- Epithelial cells were removed by shaking the mucosa tration of 10` M. Cells stimulated with PMA and http://gut.bmj.com/ with 5 mM EDTA (BDH Chemicals, Poole, England) FMLP were incubated for 30 minutes and those with at 37°C followed by washing with CMF-HBSS. This opsonised zymosan for 60 minutes. Controls included was repeated twice and after the last EDTA step, incubating the cells with culture medium and NBT washing was performed with Hanks Balanced Salt only or with dimethylsulphoxide, NBT and culture Solution with calcium and magnesium (HBSS; medium. All the cell cultures were shaken at intervals Flow laboratories). The mucosa was then cut into of 15 minutes. Control experiments and those with 2 mm pieces and digested with collagenase (from triggers were performed concomitantly. on October 2, 2021 by guest. Protected copyright. Clostridium histolyticum; Boehringer Mannheim) at At the end of the incubation period, cytocentrifuge a concentration of 100 mg/100 ml culture medium preparations (Cytospin 2; Shandon) were made, air (10% fetal calf serum in RPMI 1640; Gibco) with dried and fixed in acetone. They were then stored at shaking for three hours at 37°C. After digestion, the -20°C before staining. cells were filtered through a 100 [ nylon mesh and Cytocentrifuge preparations of mononuclear cells extensively washed with HBSS before centrifugation incubated with PMA or FMLP were stained with on Ficoll-Paque (Pharmacia) to obtain mononuclear monoclonal antibody YI/82A which is specific for cells. After further washing, the mononuclear cells monocytes and macrophages' ` (kindly donated by were resuspended in culture medium. To determine Dr D Y Mason, John Radcliffe Hospital, Oxford). the proportion of macrophages present, cytocentri- Preparations of mononuclear cells incubated with fuge preparations of the mononuclear cells were zymosan were stained with an anti-HLA-D mono- made and stained with macrophage specific monoclonal antibody 20152122 (kindly donated by Dr S clonal antibody Y1/82A'X `9 using the peroxidase Fuggle, John Radcliffe Hospital). technique.` Staining of the cytocentrifuge preparations The respiratory burst activity was assayed within was performed using the peroxidase technique.` two hours of isolation (except when the mononuclear Endogenous peroxidase activity was blocked with cells were stimulated with IFN-y and LPS). All the methanol and hydrogen peroxide. Controls included media contained 100 U/mI penicillin, 100 Fg/ml omitting the primary antibody or using an irrelevant streptomycin and 5 Fg/ml gentamycin. monoclonal antibody. Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

1364 YR Mahida, K C Wu, and D PJewell

In cytocentrifuge preparations of cells incubated analyses were two-tailed. As data were not normally with opsonised zymosan, macrophages were distributed, results are expressed as a median and considered to be those cells that were HLA-D range. positive and had phagocytosed the zymosan. For cells incubated with PMA or FMLP, macrophages were Results considered to be Y1/82A stained cells. Of these populations of macrophages, the percentage (in each The viability of isolated intestinal and peripheral cytocentrifuge preparation) showing release of RMO blood mononuclear cells was always 90% or more (as shown by blue-black- formazan staining) was assessed by trypan blue exclusion. determined by counting at least 200 macrophages per Monocytes from three normal, healthy individuals preparation. All the cytocentrifuge preparations were were used to study the effect ofcollagenase treatment coded and analyses were performed blind by one on respiratory burst activity. There was no significant investigator (YRM). Results of many of these were difference in the proportion of collagenase treated or confirmed by other investigators (variation less than untreated adherent cells that were able to undergo 10%). respiratory burst (untreated - mean 82%; collagenase treated - mean 81%). IFN-y AND LPS STIMULATION In cytospin preparations of isolated intestinal In some experiments, mononuclear cells isolated mononuclear cells from normal and inflamed ileum from normal or inflamed mucosa were stimulated and colon, there was no significant difference in the with IFN-y and LPS. The source of IFN-y was proportion of macrophages (identified with mono- supernatant from Chinese hamster ovary cell line clonal antibody Y1/82A) present; normal colon: (donated by Dr Scott, Wellcome biotech). Its activity median 13x5% (range 7-22%); IBD colon median was determined by immunoradiometric assay. It was 12% (range 7-21%); normal ileum 12% (range 10- used in a final concentration of 100-1000 U/ml. LPS 18%); ileal Crohn's disease median 14% (range was from E coli 055:B5 (Sigma) and was used at a 1(-19%). concentration of 10 [ig/ml. The mononuclear cells (in suspension) were incu- NITROBLUE TETRAZOLIUM REDUCTION BY bated with these stimuli for 42 hours. Respiratory ISOLATED INTESTINAL MACROPHAGES IN THE burst activity by the macrophages, in response to ABSENCE OF A TRIGGER

PMA, was then determined as described above. Initially two controls were used. In one the cells were http://gut.bmj.com/ incubated with culture medium and NBT only for one MON OCYTES hour and in the other, they were incubated with To study the effect of collagenase treatment dimethylsulphoxide in addition to NBT and culture on respiratory burst activity, peripheral blood medium. After some experiments, however, no monocytes were studied. Venous blood was difference between these two controls were observed collected in heparinised tubes and mononuclear cells (data not shown). Therefore in subsequent experi- were isolated by centrifugation on Ficoll-Paque ments, only the control with dimethylsulphoxide was (Pharmacia). 107 mononuclear cells were shaken with used. on October 2, 2021 by guest. Protected copyright. 1 mg/ml collagenase (in culture medium) at 370C for 3 In the absence of a trigger, there was a significant hours. Monocytes adherent to glass coverslips were (p<0O05) increase in the proportion of macrophages obtained from collagenase treated and untreated isolated from inflamed colonic and ileal mucosa that mononuclear cells by incubation at 370C for 30 reduced NBT compared with those isolated from minutes. Non-adherent cells were removed by gentle mucosa of normal colon and terminal ileum (Fig. 1). washing with warm (37°C) 5% FCS/RPMI. Cover- slips with adherent monocytes were incubated with RESPIRATORY BURST ACTIVITY OF ISOLATED culture medium, NBT (1 mg/ml) and PMA (at a final INTESTINAL MACROPHAGES IN RESPONSE TO concentration of 200 ng/ml) at 37°C for 30 minutes. OPSONISED ZYMOSAN After washing, the coverslips were fixed in 0-1% Preliminary studies using varying doses and time gluteraldehyde for 15 minutes, washed and examined intervals showed that 15 [ig/ml of opsonised zymosan by phase contrast microscopy. The percentage of incubated for one hour gave optimal results for adherent cells undergoing respiratory burst (as shown mononuclear cells obtained from both normal and by blue-black formazan staining) was determined. inflamed mucosa. In cytocentrifuge preparations, 75-90% of STATISTICAL ANALYSIS HLA-D positive macrophages (as assessed by their Statistical analysis was performed using Wilcoxon's morphology) had phagocytosed zymosan. There was rank-sum test (paired or unpaired as indicated). The no difference between normal and inflamed intestine Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

Respiratory burst activity ofintestinal macrophages in normal and inflammatory bowel disease 1365

VZ 50 0 0 0 m 40 0 z C) 0 ._ 0 0 In0 0 30 0 0 0.c0 3 m, 11 0 -cCD cn 20 a 0 . CD mo- 0 0 E 0 Cl a 0 10- -.- E -5- 4_ S 0 0J 0 0 g (P N0 C,0J1 P1 dc, ~ ' .c.. 10 ~0, Fig. 3 Percentage ofisolated intestinal macrophages Fig. 1 Percentage ofmacropliages reducing NBTin thle undergoing a respiratory burst in response to opsonised absence ofany trigger. Isolated intestinal mononuclear cells zymosan. Intestinal mononuclear cells (in suspension) were were incubated with NBT, culture medium and incubated with opsonived zymosan and NB Tfor 60 minutes. dimethylsulphoxide only. Cytocentrifuge preparations were Cytocentrifuge preparations were then made and stained wit/i then made and the proportion ofmacrophages (YI/82A antibody 201521 (anti HLA-D). The proportion of positive) reducing NBT was determined. macrophages (HLA -D positive cells witli ingested zymosan) showing release of RMO was determined.

(data not given). These cells were easy to identify and greater proportion of macrophages isolated from of these, the percentage undergoing respiratory burst inflamed colonic mucosa showed evidence of release http://gut.bmj.com/ as shown by deep blue black staining was determined of RMO (median 17%; range 9-49%; p<001) (Fig. 2). (Fig. 3). Only a small proportion of macrophages isolated Similarly, a significantly greater proportion of from normal colon were able to undergo a respiratory macrophages isolated from inflamed mucosa of ileal burst (median 8%; range 2-33%). A significantly Crohn's disease showed evidence of having under- gone a respiratory burst (median 22%; range 5-49%)

compared to macrophages isolated from normal on October 2, 2021 by guest. Protected copyright. terminal ileal mucosa (median 9%; range 5-17%; p<005). There was no difference in the ability of macrophages isolated from different areas of normal colon to release RMO in response to opsonised p zymosan (data not shown). There was also no difference between ulceratative colitis and Crohn's disease in the proportion of isolated colonic macrophages able to undergo a respiratory burst. In six inflammatory bowel disease colons resected (four with ulcerative colitis and two with Crohn's disease), it was possible to study from the same colon, macrophages isolated from inflamed as well as from areas which were normal or near normal macroscopically and histologically. In five of six Fig. 2 Isolated intestinal macrophage (stained with anti- colons, fewer macrophages isolated from non or HLA-D monoclonal antibody, 201521) with a phagocytosed minimally inflamed mucosa showed evidence of opsonised zymosan particle, showing release ofoxygen release of RMO compared with those isolated from radicals as shown by the blue black reaction product severely inflamed areas. In the sixth colon, no (arro wed). Nucleus - N, peroxidase stain - P. difference was seen (Fig. 4). Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

1366 Y R Mahida, K C Wu, and D P Jewell

50- cn 100 .0 4. 0 40- X 80- .h- 4._ ax T .a) 30- .' 60- VX ~0 C CD) cm C: 20- 40- E0 0.0) CF I- 0 10- 20-0

E S O0 Inflamed IBD Non or min Inflamed IBD Non or min inflamed IBD inflamed IBD Fig. 4 Respiratory burst activity ofmacrophages isolated Fig. 6 Respiratory burst activity ofmacrophages isolated fron non or minimally inflamed areas ofsix IBD colons. from inflamed and non or minimally inflamed areas offive Opsonised zymosan was the trigger used. Proportion of IBD colons. PMA was used as a trigger. Proportion of macrophlages (HLA-D positive with ingested zymosan) macrophlages (YI/82A positive) reducing NBT, in reducing NBT, in cytocentrifuge preparations, was cytocentrifuge preparations was determined (different determined (different symbols indicate separate patients). symbols represent separate patients).

RESPIRATORY BURST ACTIVITY OF ISOLATED product) and the proportion releasing RMO (blue INTESTINAL MACROPHAGES IN RESPONSE TO black staining) was determined. Studies using PMA another macrophage specific monoclonal antibody Macrophages were easy to identify with the mono- 3C1020 gave similar results to those obtained with Y1/ clonal antibody Y1/82A (giving a brown reaction 82A (data not shown). The latter was chosen because http://gut.bmj.com/ of good and consistent staining using the peroxidase technique. In this system, contaminating polymorphs and eosinophils could be ignored as they did not stain 0 .0 with the monoclonal antibody (endogenous peroxi- 0 ° 80- dase activity having been blocked). as 0 As was found for opsonised zymosan, only a small proportion of macrophages isolated from normal

to on October 2, 2021 by guest. Protected copyright. 2c 60- colon and ileum were able to undergo a respiratory c0 ID burst (median 9%; range 1-45% and median 6%; ' range 4-11% respectively). There was a significantly 40- higher proportion of macrophages isolated from 0 0 0 mucosa of inflamed colon (p<0-01) and ileum

°n 20- (p<0-01) that showed evidence of release of RMO E . (median 45%; range 21-91% and median 43%; range 34-77% respectively) (Fig. 5). Macrophages from l V , inflamed mucosa also showed more intense blue --- c,o,, black staining suggesting a greater release of RMO 4%o per cell. In five colons resected (three with ulcerative colitis and two with Crohn's disease) macrophages isolated Fig. 5 Percentage ofisolated intestinal macrophages from severely inflamed and non or minimally undergoing a respiratory burst in response to PMA. inflamed areas of the same colon were studied Intestinal mononuclear cells (in suspension) were incubated with PMA and NBTfor30 minutes. Cytocentrifuge (Fig. 6). In all five, a greater proportion of macro- preparations were then made and stained with monoclonal phages isolated from severely inflamed mucosa antibody Y1182A. The proportion ofmacrophages (Y1182A showed evidence of release of RMO compared with positive) reducing NBT was determined. those from non or minimally inflamed mucosa. Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

Respiratory burst activity ofintestinal macrophages in normal and inflammatory bowel disease 1367

30- Table Respiratory burst activity ofisolated intestinal macropliages following incubation witli L PS an(l/or IFN-y. 0 The intestinal mononuiclear cells were incubated (in1 20- suspension) wit/i LPS, IFN-y or culture medium onl tit 37°C( for 42 Ii. PMA and NBT wvere thieni added and tfl cells incubated for a furthler 30 minutes. Cytocentrifuge pr eparations were tlien made and stained wit/i monoclonal ~0 antibody Y1182A. Pertcentage ofmacrophiages (Y1182A positive) undergoing a respiratory buurst was then deter-mined. C lo Culture, tnledXilutr LPS lkN-y lkN-y E on/ lOig/lt?l10 680 Ul/mIl +LPS 0 T NT T NT T NT T NT Normal colon 1 29) 58 2 32 29 4(0 3 15 16 21 4 6 7 12 11 IBD Fig. 7 Respiratory burst activity ofisolated colonic colon 1 39 35 42 macrophages in the presence (T) or absence (NT) or FMLP. 2 62 63 65 Isolated intestinal cells 3 74 77 8() mononuclear (in suspension) were 4 83 86 87 incubated with NBT and withl or without FMLPfor 30 50 47 53 minutes. Cytocentrifuge preparations were then made and the proportion ofmacropliages (Y1182A positive) slhowing release ofRMO was determined (different symbols represent incubation period (with IFN-y or LPS) was always separate patients). Normal colon: FMLP v no trigger- p: not significant; Normal ileum: FMLP v no trigger p: not 80% or more. significant; IBD colon: FMLP v no trigger- p

FMLP: normal ileum v ileal Crolin's- p<0)05; (unpaired (data not shown). http://gut.bmj.com/ Wilcoxon's rank-sum test) After incubation with IFN-y there was a small increase in the proportion of macrophages from normal colonic mucosa that showed evidence of release of RMO (Table). Stimulation with LPS did RESPIRATORY BURST ACTIVITY OF ISOLATED not make any significant difference. In two experi- INTESTINAL MACROPHAGES IN RESPONSE TO ments the mononuclear cells were incubated with FMLP IFN-y and LPS, however the proportion of macro- Preliminary studies showed that 10 M of FMLP was phages showing release of RMO was similar to that on October 2, 2021 by guest. Protected copyright. optimal for triggering the respiratory burst (data not after incubation with IFNy alone. Incubating mono- shown). N-formylmethionyl-leucyl-phenylalanine nuclear cells isolated from inflamed mucosa with was not able to trigger macrophages isolated from either IFN-y or LPS did not produce any significant normal colon and ileum to undergo a respiratory change in the proportion of macrophages undergoing burst (Fig. 7). For macrophages isolated from a respiratory burst (Table). inflamed ileal and colonic mucosa, FMLP was much less potent at triggering respiratory burst activity Discussion than opsonised zymosan or PMA. This was shown by a smaller proportion of cells isolated from inflamed In the human ileum and colon macrophages are mucosa demonstrating release of RMO as well as found in the lamina propria and are most prominent much less intense blue black staining per cell. close to the basement membrane of the epithelium.` Studies in our unit have shown morphological and RESPIRATORY BURST ACTIVITY OF ISOLATED INTESTINAL immunohistochemical heterogeneity of these cells in MACROPHAGES AFTER STIMULATION WITH IFN-y OR LPS normal and inflamed ileum and colon" as well as in In preliminary studies, IFN-y at a concentration of the granulomata of Crohn's disease.3 There is little 680 U/mI was optimal for stimulating intestinal information on the functions of these intestinal macrophages to undergo a respiratory burst. The cells.se viability of the mononuclear cells at the end of the This study investigated the capacity of isolated Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

1368 YR Mahida, K C Wu, and D PJewell intestinal macrophages to undergo a respiratory to present antigen to T Cells but are not activated as burst as shown by the reduction of NBT to a deep assessed by other functional tests. blue black formazan reaction product in response to It has been postulated that as monocytes migrate various triggers. This test gives a semiquantitative and mature into tissue macrophages, their ability to measure of respiratory burst activity in individual secrete large amounts of superoxide and hydrogen cells.'" peroxide may be down regulated.' This is supported Superoxide anion, hydrogen peroxide and by in v,itro studies of superoxide anion and hydrogen hydroxyl radicals can be more accurately determined peroxide production by monocytes after isolation quantitatively.' These assays were not performed in and during differentiation into macrophages.' this study because of difficulties in consistently IFN-y has been shown to activate macrophage excluding polymorphonuclear leucocyte contamina- oxidative metabolism., After stimulation with IFN-y, tion especially from mononuclear suspensions one would have expected majority of the macro- isolated from inflamed mucosa. In preliminary phages from normal colonic mucosa to be able to studies varying degrees of polymorphonuclear leuco- undergo a respiratory burst (in the presence of cyte contamination occurred despite centrifugation PMA). Only a small proportion were able to do so, on Ficoll-Paque (performed twice) and separation of however, suggesting that the resident population of cells by adherence to fibronectin coated plates. This intestinal macrophages in the normal colon are may reflect properties of young polymorphonuclear 'desensitised' to the effect of IFN-y. The activity of cells recruited into the mucosa. Any quantitative this batch of IFN-y had previously been determined analysis of RMO in the presence of contaminating and it has also been shown to induce HLA-DR polymorphonuclear leucocytes would not accurately in an epithelial cell line.14 The possibility that the reflect production of these radicals by the macro- 'desensitisation' to IFN-y may be caused by the phages. presence of tumour in the resected specimen cannot The technique (NBT reduction) used in this study be excluded. One of the patients studied, however, allows examination of most of the macrophage popu- had intestinal resection for idiopathic constipation, lation rather than that selected - for example, by suggesting that this 'desensitisation' may be a pheno- adherence or elutriation. In addition, this technique menon of normal intestinal macrophages. also enables study of the relationship of macrophage This effect may be mediated by trace levels of phenotype (as determined by monoclonal antibody bacterial lipopolysaccharide. ' In this respect, or histochemical staining) and the functional capacity intestinal macrophages may have some similarities to http://gut.bmj.com/ of the cell to release RMO. Our own and previous murine Kupffer cells which fail to respond to IFN-y in studies25 35031 have shown that most of the human v,itro with a rise in their respiratory burst activity.1" intestinal macrophages express MHC class II mole- Both may be exposed to trace levels of LPS which cules. Our studies have also shown that most of these may cross the bowel wall into the portal circulation cells also stain with monoclonal antibody Y1/82A." under physiological conditions.31 It is also possible Using two different ways to define the macrophage that the lack of respiratory burst by these cells under and we have normal conditions, protects from oxidative injury.15 population using different triggers, on October 2, 2021 by guest. Protected copyright. shown that a greater proportion of macrophages In active inflammatory bowel disease, there is isolated from the mucosa of ulcerative colitis and infiltration by polymorphonuclear leucocytes, Crohn's disease are able to undergo a respiratory lymphocytes and macrophages." The greater propor- burst compared with those isolated from normal tion of intestinal macrophages from inflammatory terminal ileal or colonic mucosa. bowel disese mucosa that are able to undergo a During a respiratory burst, superoxide anion is respiratory burst may be either the result of an formed by one-electron reduction of oxygen, a upregulation of respiratory burst capacity of the reaction catalysed by membrane-associated NADPH resident population of cells or may reflect an elicited oxidase. In activated macrophages, this response is population of macrophages. It is generally believed enhanced.' Using this criteria, it appears that only a that tissue macrophages in inflammatory lesions are small proportion of normal intestinal macrophages predominantly derived from blood monocytes.15 Our are activated and that this proportion is increased in studies with IFN-y suggest that it is probably the inflamed mucosa. As majority of human intestinal elicited population of macrophages (derived from macrophages express HLA class 1I molecules," circulating monocytes) that contribute to the however it has been suggested that they are all increased proportion able to release RMO. activated. ' These differences may reflect hetero- Oxygen radicals produced by activated macro- geneity in the activation of the macrophages. It is also phages (as well as polymorphonuclear leucocytes) possible that the intestinal macrophages express may be involved in mediating damage in the inflamed HLA-DR because they may be frequently required mucosa.9 The oxygen metabolites are likely to be Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

Respiratory burst activity ofintestinal macrophages in normal and inflammatory bowel disease 1369

generated by phagocytosis of bacterial and other metabolites and cellular injury. Lab Invest 1982; 47: products penetrating into the mucosa because of a 5-18. breach in the epithelial barrier. Chemotactic peptides 11 Strober W, James SP. The immunological basis of inflammatory bowel discasc. J Clin Immunol 1986; 6: like FMLP" may also play a role. Apart from 415-32. superoxide anion (02 ), hydrogen peroxide (H202) 12 Meuret G, Bitzi A, Hammer GB. macrophagc turnover and hydroxyl radical (OH'), other oxygen meta- in Crohn's disease and ulcerative colitis. Gastro- bolites that may be involved in tissue damage are enterology 1978; 74: 501-3. hypohalides. Hypochlorite (OCI) is one of the main 13 Mee AS, Szawatakowski M, Jewell DP. Monocytes in hypohalides and is formed through the activity of the inflammatory bowel disease: phagocytosis and intra- azurophilic granule enzyme myeloperoxidase.4' cellular killing. J Clin Patliol 1980; 33: 921-25. Oxygen radicals may intereact with proteinases 14 Doe WF, Forsman B. Chronic inflammatory bowel secreted by the same phagocytic cells.' Thus local disease: increased plasminogen activator secretion by mononuclear phagocytes. Clin Exp Immunol 1982; 48: inactivation of antiproteinases by RMO4' may allow 256-60. local uncontrolled action of enzymes secreted by 15 Nathan DG, Baehner RL, Weaver DK. Failure of phagocytes. Oxygen radicals may also denature nitroblue tetrazolium reduction in the phagocytic proteins and make them susceptible to proteinases.43 vacuoles of leukocytes of chronic granulomatous disease. J Clin Invest 1969; 48: 895-904. Dr Mahida was supported by the Medical Research 16 Ezekowitz RAB, Orkin SH, Newburger PE. Recom- Council. We are grateful to Dr Siamon Gordon (Sir binant interferon gamma augments phagocyte super- William Dunn School of Pathology) for his helpful oxide production and x-chronic granulomatous disease advice and to Mr Kettlewell and Mr Mortensen for gene expression in x-linked variant chronic granulo- the operation resection specimens. matous disease. J Clin Invest 1987; 80: 1009-16. 17 Bull DM, Brookman MA. Isolation and functional References characterization of human intestinal mucosal lymphoid cells. J Clin Invest 1977; 59: 966-74. 1 Adams DO. Hamilton TA. The cell biology of macro- 18 Hogg N, Horton MA. Myeloid antigens: new and phage activation. Ann Rev Immunol 1984; 2: 283-318. previously defined clusters. In: McMichael AJ, ed. 2 Johnson WJ, Pizzo SV, Imber MJ, Adams DO. Recep- Leucoyte typing III. White cell differentiation antigens. tors for maleated proteins regulate the secretion of Oxford: Oxford University Press, 1987: 576-602. neutral proteases by murine macrophages. Science 1982; 19 Davey FR, Cordell JL, Erber WN, Pulford KAF, Gattcr 218: 574-6. KC, Mason DY. Monoclonal antibody (Yl/82A) with 3 Bonney RJ, Davies P. Possible autoregulatory functions specificity towards peripheral blood monocytes and http://gut.bmj.com/ of the secretory products of mononuclear phagocytes. tissue macrophages. J Clin Patl/ol 1988; 41: 753-8. Contemp Top Immunobiol 1984; 13: 199-223. 20 Gatter KC, Falinia B, Mason DY. The use of mono- 4 Unanue R. The regulatory role of macrophages in clonal antibodies in histopathological diagnosis. In: antigenic stimulation. Part II. Symbiotic relationship Anthony PP, MacSween NM, eds. Recent advances in between lymphocytes and macrophages. Acta Immunol histopathology. Vol 12. Edinburgh: Churchill Living- 1981;31: 1. stone, 1984: 135-67. 5 Ezekowitz RAB, Gordon S. Down-regulation of 21 Berton G, Gordon S. Superoxide release by peritoneal mannosyl receptor mediated endocytosis and antigen and bone marrow derived mouse macrophages. Modu- on October 2, 2021 by guest. Protected copyright. F4/80 in Bacillus Calmette-Guerin-activated mouse lation by adherence and cell activation. Immnunology macrophages Role of T lymphocytes and lymphokines. 1983; 49: 693-705. J Exp Med 1982; 155: 1623-37. 22 Smith KGC, Austyn JM, Harris G, Beverley PCL, 6 Mokoena T. Gordon S. Human macrophage activation. Morris MJ. T cell activation by anti-T3 antibodies: Modulation of mannosyl, fucosyl receptor activity in comparison of IgG1, and IgG2b switch variants and vitro by lymphokines, gamma and alpha interferons and direct evidence for accessory function of macrophage Fc dexamethazone. J Clin Invest 1985; 75: 415-32. reccptors. EurJ Intnunol 1986; 16: 478-86. 7 Nathan CF, Murray HW, Wiebe ME, Rubin BY. 23 Van Voorhis WC, Stcinman RM, Hair LS, et al. Specific Identification of interferon-gamma as the lympokine antimononuclear phagocyte monoclonal antibodics. that activates human macrophage oxidative metabolism Application to the purification of dendritic cells and the and antimicrobial activity. J Exp Med 1983; 158: 670-89. tissue localization of macrophages. J Exp^ Med 1983; 8 Badeway JA, Robinson JM, Karnovsky MJ, 158: 126-45. Karnovsky ML. Reduction and excitation of oxygen by 24 Donnellan WL. The structurc of the colonic mucosa. phagocytic leucocytes: biochemical and cytochemical The epithelium and subcpithelium reticulohistiocytic techniques. In: Weir DM, Herzenberg LA, Blackwell complex. Gastroeniterology 1965; 49: 496-514. C. Handbook of experimental immunology. 2. Cellular- 25 Selby WS, Poulter LW, Hobbs S, Jewell DP, Janossy G. immunology. Oxford: Blackwell, 1986: 50: 1-50.6. Heterogeneity of HLA-DR-positive histiocytes in 9 Henson PM, Johnson RB. Tissue injury in inflam- human intestinal lamina propria: a combined histo- mation. J Clin Invest 1987; 79:669-74. chemical and immunohistological analysis. J Clin Patlo/l 10 Weiss SJ, LoBuglio AF. Phagocyte-generated oxygen 1983: 36: 379-84. Gut: first published as 10.1136/gut.30.10.1362 on 1 October 1989. Downloaded from

1370 Y R Mahida, K C Wu, and D PJewell

26 Mahida YR. Gionchetti P, Vaux D, Patel S, Jewell DP. lipopolysaccharide prevent interferon-gamma or Macrophage subpopulations in lamina propria of tumour necrosis factor-alpha from enhancing mouse normal and inflamed colon and terminal ileum. Gut peritoneal macrophage respiratory burst capacity. 1989; 30: 826-34. J Immunol 1987; 139: 1971-77. 27 Mahida YR, Patel S, Wu K, Jewell DP. Macrophage 36 Lepay DA, Nathan CF, Steinman RM, Murray HW, and lymphoid subpopulations in the granulomata of Cohn ZA. Murine Kupffer cells. Mononuclear phago- Crohn's disease. [Abstract]. Gut 1987; 28: A 1388. cytes deficient in generation of reactive oxygen inter- 28 Verspaget H, Beeken W. Mononuclear phagocytes in mediates. J Exp Med 1985; 161: 1079-84. the gastrointestinal tract. Acta Chir Scand 1985; 525 37 Mathison JC, Ulevitch RJ. The clearance, tissue distri- [Suppil 113-26. bution, and cellular localization of intravenously 29 Ezckowitz RAB, Sim RB, MacPherson GG, Gordon S. injected lipopolysaccharide in rabbits. J Immunol 1979; Interaction of human monocytes, macrophages and 123:2133. polymorphonuclear leukocytes with zymosan in vitro. 38 Gordon S, Crocker PR, Morris L, Lee SH, Perry HV, Role of type 3 complement receptors and macrophage- Hume DA. Localization and function of tissue macro- derived complement. J Clin Invest 1985; 76: 2368-76. phages. In: Biochemistry of macrophages. Ciba 30 Beeken W, Miereme Ooms M, Ginsel IA, Leith PCJ, Foundation Symposium 118. Chichester: John Wiley, Verspaget H. Enrichment of macrophages in cell 1986: 54-62. suspensions of human intestinal mucosa by elutriation 39 Anonymous. Bone marrow origin of Kuppfer cells. centrifugation. J Immunol Methods 1984; 73: 189-201. [Editorial]. Lancet 1980: i: 131-2. 31 Golder JP, Doe WF. Isolation and preliminary charac- 40 Hobson CH, Butt TJ, Ferry DM, Hunter J, terization of human intestinal macrophages. Gastro- Chadwick VS, Broom MF. Enterohepatic circulation of enterology 1983; 84: 795-802. bacterial chemotactic peptide in rats with experimental 32 Mowat AM. The cellular basis of gastrointestinal colitis. Gastroenterology 1988; 94: 1006-13. immunity. In: Marsh MN, ed. Immunopatliology of tlie 41 Klebanoff SJ. Mycloperoxidase: contribution to the small intestine. Chichcster: John Wiley, 1987: 41-72. microbicidal activity of intact leukocytes. Science 1970: 33 Nakagawara A, Nathan CF, Cohn ZA. Hydrogen 169: 1095-7. peroxide metabolism in human monocytes during differ- 42 Carp H, Janoff A. In vitro suppression of serum entiation in vitro. J Clin Invest 1981; 68: 1243-52. elastase-inhibitory capacity by reactive oxygen species 34 Lowes JR, Radwan P, Priddle JD, Jewell DP. Induction generated by phagocytosing polymophonuclear leuko- of HLA-DR molecules on a colonic epithelial cell line by cytcs. J Clin Inv'est 1979; 63: 793-7. gamma-interferon [Abstract]. Clin Sci 1987; 74: [suppl 43 Fligiel SE, Lec EC, McCoy JP, Johnson KJ, Vavani J. no 18]: 31. Protein degradation following treatment with hydrogen

35 Ding AH, Nathan CF. Trace levels of bacterial peroxide. Am J Pathol 1984; 115: 418-25. http://gut.bmj.com/ on October 2, 2021 by guest. Protected copyright.