J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

J Clin Pathol 1983;36:379-384

Heterogeneity of HLA-DR-positive histiocytes in human intestinal lamina propria: a combined histochemical and immunohistological analysis

WS SELBY, LW POULTER, S HOBBS, DP JEWELL, G JANOSSY From the Department ofGastroenterology, John Radcliffe Hospital, Oxford, and Department of , Royal Free Hospital School of Medicine, London

SUMMARY HLA-DR-positive histiocytes in the lamina propria of the human intestine have been characterised using combined histochemical and immunohistological techniques. In the small intestine, 80-90% of the HLA-DR+ histiocytes had irregular surfaces with stellate processes, and exhibited strong membrane adenosine triphosphatase (ATPase) activity, but weak acid phos- phatase (ACP) and non-specific esterase (NSE) activities (HLA-DR+ ACP+'- NSA+'- ATP++; type 1 ). In contrast, in the lamina propria of the colon the majority (60-70%) of HLA-DR+ cells were large, round cells with strong ACP and NSE activities but no detectable ATPase activity (HLA-DR+ ACP++ NSE++ ATP+'-; type 2 cell). The colon also contained a population of type 1 cells (30-40%). In active inflammatory bowel disease affecting the colon a third population of HLA-DR+ histiocytes was seen. These cells were irregular in outline, with many processes, and were ACP++ NSE+ ATP+'- (type 3 cell). The type 3 cells appeared to replace type 2 cells. After treatment, the appearances returned to normal. These findings suggest that the different populations of HLA-DR+ histiocytes in the human intestine may have several functions, reflecting the different forms of present in the intestine. The alterations in inflammatory bowel disease may represent activation in response to http://jcp.bmj.com/ an invading antigen.

Within the lamina propria of the human small and be of considerable value in differentiating these two large intestine are many histiocytic-type cells which cell types, both of which are HLA-DR+, in normal exhibit strong expression of HLA-DR (Ia-like) and pathological tissues.'4 .' 2 These cells are found in the villi of the In order to characterise the HLA-DR+ histiocytes

small intestine and in the subepithelial region of the in human intestinal lamina propria we have on October 1, 2021 by guest. Protected copyright. colon. These sites correspond to those previously examined the activities of acid phosphatase (ACP), described for intestinal .34 non-specific esterase (NSE) and adenosine triphos- HLA-DR+ -like cells identified in phatase (ATPase) in frozen sections of normal and other tissues have been shown to represent a diseased intestine. Double enzyme techniques and heterogeneous population, comprised of such cells the combination of immunofluorescence and as the Langerhans cells in the skin,5 the interdigitat- cytochemistry on the same section has allowed us to ing (ID) cells of lymph nodes and ,67 cortical demonstrate that the intestinal HLA-DR-positive cells of the thymus8 and inflammatory cells in the population is indeed heterogeneous. This synovial membrane.9 Histochemistry has been of heterogeneity may have functional significance both value in defining the characteristics of several cell in normal intestine and in that of patients with gas- populations and particularly in the discrimination of trointestinal disease, such as ulcerative colitis or specific cell types of the -macrophage Crohn's disease. series.""' Indeed, the demonstration of acid phos- phatase in inflammatory macrophages and -of Material and methods adenosine triphosphatase activity in interdigitating, antigen-presenting cells,'2 13 has now been shown to SUBJECTS AND TISSUES Proximal small intestinal biopsies were obtained by Accepted for publication 15 September 1982 peroral suction from one normal subject and from 379 J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

380 Selby, Poulter, Hobbs, Jewell, Janossy three patients undergoing investigation for diar- method was used to detect ATPase. This used rhoea. Ileal or colonic tissue, or both, was taken adenosine triphosphate as a substrate and lead endoscopically or at laparotomy from seven patients nitrate as a coupler; sections were developed after with either colonic neoplasia or the irritable colon incubation in a solution of ammonium sulphide. syndrome. All specimens were histologically normal Staining for these two enzymes was done separately as shown by study of frozen or formalin-fixed sec- as well as in combination on the one section. Stain- tions. Five patients with ulcerative colitis (active in ing for ACP was also combined with three, inactive in two) and one with active Crohn's immunofluorescence for HLA-DR antigens on the disease were also studied. Four of the patients were same section. In this case, the Gomori lead method receiving corticosteroids and three were taking sul- for ACP was used'8 as the pararosaniline method phasalazine. The specimens were orientated on resulted in considerable autofluorescence. The two cork, covered with OCT compound (Ames Co) and methods for ACP gave similar results. Non-specific frozen in isopentane suspended over liquid nitrogen. esterase activity was detected using a naphthyl ace- They were stored in liquid nitrogen until used. tate as a substrate.

IMMUNOFLUORESCENCE Results HLA-DR antigens were detected by an antiserum raised in chickens'5 using a rhodamine-conjugated SMALL INTESTINE (TABLE) second layer antiserum. In several sections the HLA-DR-positive histiocytic cells occurred predo- anti-HLA-DR antiserum was used in combination minantly in the villi. Double-labelling with the anti- with antisera to human IgA, IgM or IgG or with a Ig antisera confirmed that these cells were neither monoclonal antibody to human leucocyte antigen plasma cells nor B . In the small intes- (2D1) in a double marker immunofluorescence tine approximately 80-90% of the cells were small, technique described in full elsewhere.'6 Tissue sec- stellate cells with processes extending from the cell tions were also studied using the monoclonal anti- surface (Fig. la). Cytochemical analysis demons- body NA1/34 which reacts with cortical trated that this cell type expressed strong membrane as well as Langerhans cells.'4 1' Immunofluorescence activity of ATPase, while activity of ACP was either was detected using a Standard 14 Zeiss microscope weak or negative (Fig. 2a). Activity of NSE was with IV Fl epifluorescence condenser. Selective similar to that of ACP. This type of cell (HLA-DR+ filters for rhodamine and fluorescein enabled simul- ACP`' NSE+'- ATP++) was designated type 1. A taneous detection of HLA-DR antigens and Ig sub- smaller proportion of the HLA-DR+ histiocytes http://jcp.bmj.com/ class on the same section. (approximately 10-20%) were large and round in appearance and had strong cytoplasmic ACP and HISTOCHEMISTRY NSE activities but weak or negative ATPase activ- Histochemical methods for detecting ACP, NSE and ity. This cell type (HLA-DR+ ACP++ NSE++ ATPase have been described in full elsewhere.'4 ATP+'-) was designated type 2. Of some interest Briefly, the pararosaniline method for ACP was was the observation that in some of the HLA-DR+

used. This involves incubation of the slides in a cells the distribution of the HLA-DR antigens on October 1, 2021 by guest. Protected copyright. medium containing the substrate naphthol ASBI appeared to be cytoplasmic as well as membrane- phosphate and used hexazotised pararosaniline as a associated. simultaneous coupler. The lead precipitation In the ileum, most of the villi contained type 1 cells. However, in one of the ileal specimens, from a patient with the irritable colon syndrome, there were occasional villi which were packed with type 2 Characteristics ofHLA-DR' histiocytes in the human cells. intestinal lamina propria Combined analysis of HLA-DR antigens and % Morphology ACP A7Pase 7ype ACP clearly identified the HLA-DR+ histiocytes, Small but these were only infrequently ACP+. intestine 80-90) small, stellate +/ ++ 1 10-20 large, round ++ +/ 2 LARGE INTESTINE Large intestine 6(0-70 large, round ±+ +/- 2 In the lamina propria of the colon HLA-DR+ his- 30-4() small, stellate +/- ++ 1 tiocytes were concentrated subepithelially in clusters Active IBD 50-6(0 small, irregular ++ +/ 3 (Fig. lb). They did not label with antisera to the Ig *3(4() small, stellate +/_ + + 1 subclasses. In contrast to the findings in the small 10-20 large, round ++ +/ 2 intestine, the majority of HLA-DR+ histiocytes in IBD = inflammatory bowel disease the colon (approximately 60-70%; Table) were of J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

Heterogeneity of HLA-DR-positive histiocytes in human intestinal lamina propria 381

Fig. 1 HLA-DR antigens in human intestinal mucosa. (a) Small intestine: the HLA-DR t histiocytes in the lamina propria are irregular in shape and show heterogeneity ofsize. (b) Large intestine: Many HLA-DR + cells are large and round (type 2 =large arrows). Smaller, irregular cells are also seen (type 1 =small arrows) X400 http://jcp.bmj.com/

Fig. 2 Double histochemical analysis for ACP and A TPase. (a) In the normal small intestine histiocytes with membrane A TPase activity predominate (type 1; cells with brown reaction product). There are occasional cells with strong cytoplasmic A CP activity (red reaction product). (b) In the colon most ofthe histiocytes are large, round cells with strong A CP activity (type 2 cells; red reaction product). There are also a few type I cells (brown reaction product). on October 1, 2021 by guest. Protected copyright. the large, round type, with strong ACP and NSE ulcerative colitis or Crohn's disease were studied it activities but weak or undetectable ATPase activity was found that the characteristics of the HLA-DR+ (type 2 cells). Thirty to 40% of HLA-DR+ cells histiocytes were clearly different from those seen in were type 1 cells (Fig. 2b). All of the cells with the normal large intestine. The majority of the cells strong ACP activity were also HLA-DR+ and, con- were irregular in outline with many elongated pro- versely, all large, round HLA-DR+ cells had strong cesses (Fig. 3) and, unlike the histiocytes in the small ACP activity. intestine, had strong cytoplasmic ACP activity but Caecal biopsies were examined from the patients weak or no ATPase activity (Fig. 4, Table). NSE in whom ileal tissue was studied. In each case, type 2 activity was variable. This cell type was designated cells predominated in the caecum and type 1 cells type 3. Type 2 cells appeared to be considerably predominated in the ileum (see above). reduced in number, making up some 10-20% of the Both in the small intestine and in the large HLA-DR+ population in the inflamed colon. Type 1 intestine, the HLA-DR+ cells showed weak cells were present in proportions similar to those reactivity with 2D1, but were NA1/34-. seen in the normal colon. The HLA-DR+ histiocytes in the colon of patients with inflammatory bowel INFLAMMATORY BOWEL DISEASE disease were not clustered but rather were scattered When sections of colonic biopsies from patients with diffusely throughout the subepithelial region, sepa- J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

382 Selby, Poulter, Hobbs, Jewell, Janossy

Fig. 4 Colonic biopsy from a patient with active ulcerative colitis, showing A CP and A TPase activities. Most ofthe ACP+ lamina propria histiocytes are smaller and more irregular than in normal colon, and are scattered more Fig. 3 Cotonic biopsy from a panent witn acnve Cronn s difftsely (type 3 cells). Type 2 cells are infrequent. Some colitis, showing the distribution ofHLA-DR antigens. The type I cells (brown A TPase activity) can be seen. Strong HLA-DR' histiocytes appear small and irregular. A TPase activity occurs in the vessel walls. Individual cell outlines are difficult to distinguish, and there are many processes extending throughout the lamina propria. The colonic is also HLA-DR+ X400 rophage family" are indeed heterogeneous,2425 This has recently been demonstrated in a variety of situa- tions using a combined analysis similar to that used rated from each other by oedema and by the in this study.9 14 26 This heterogeneity of HLA-DR+ inflammatory cell infiltrate. histiocyte populations, in particular the presence of After treatment, the HLA-DR-positive histiocyte either strong ACP activity or strong ATPase activ- http://jcp.bmj.com/ populations returned to normal. ity, may have important functional significance. For example, the antigen-presenting interdigitating cells Discussion of the paracortex are stellate or dendri- tic in appearance, exhibit strong expression of The combination of immunofluorescent and his- HLA-DR antigens and have strong ATPase activity tochemical analysis of tissue sections had indicated with weak or negative ACP activity.'47257 These

that there are at least two populations of HLA-DR+ cells are thought to be important in the ingestion of on October 1, 2021 by guest. Protected copyright. histiocytic-type cells within the normal lamina prop- soluble antigens and presentation of these antigens ria. In the small intestine the majority of these cells to T lymphocytes.7 Similar features and role have are ATPase++ ACP+'- NSE+'- and are relatively been shown for Langerhans cells of the skin28"" small with a stellate appearance (type 1). In the although these cells are NA1/34+.'4 The predomin- colon, large, round HLA-DR+ cells predominate. ant HLA-DR+ histiocyte in the small intestine, the They are ACP++ NSE++ ATPase+'- (type 2). type 1 cell, has similar morphology and enzyme Several workers have examined the histochemical activity to the interdigitating cell. The small intesti- characteristics of the human small intestine4 19-21 nal mucosa is exposed to a large variety of soluble and large intestine.22 These studies confirmed our food antigens, but has only a scanty bacterial popu- findings with respect to ACP activity, although lation.3" This may explain the predominance of results for other enzymes were inconsistent. In the HLA-DR+ cells of the antigen-presenting type most comprehensive study, Riecken et :23 described within the small intestine. These cells may ingest histochemical characteristics of small intestinal mac- soluble antigens which have crossed the epithelial rophages similar to those observed in the present barrier and then present these to local helper T Iym- study. However, they made no mention of mac- phocytes, which then, in turn, regulate the immune rophage morphology, nor did they examine the dis- response to the antigens. The predominance of tribution of HLA-DR antigens. helper-type T lymphocytes in the lamina propria of It is now well established that cells of the "mac- the intestine, and the close relation between these J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

Heterogeneity ofHLA-DR-positive histiocytes in human intestinal lamina propria 383 lymphocytes and the HLA-DR+ cells, would sup- 2 Selby WS, Janossy G, Goldstein G, Jewell DP. T subsets in normal human intestinal mucosa-the distribution port this hypothesis.2 and relationship to MHC-derived antigens. Clin Exp Immunol The characteristics of the predomninant HLA- 1981;44:453-8. DR+ population in the colon (type 2 cell) are those 3 Donnellan WL. The structure of the colonic mucosa. The of phagocyctic "scavenger" macrophages.'4 31 Cells epithelium and subepithelial reticulohistiocytic complex. Gas- troenterology 1965;49:496-514. of this type are responsible for the ingestion of par- 4Sawicki W, Kucharczyk K, Szymanskak K, Kujawa M. Lamina ticulate antigens.27 The colon contains an extremely propria macrophages of intestine of the guinea pig. Possible large bacterial flora.32 This could account for the role in of migrating cells. Gastroenterology presence of a high proportion of type 2 cells in the 1977;73: 1340-4. Klareskog L, Tjernlund UM, Forsum U, Peterson PA. Epider- colonic lamina propria, where they could deal with mal Langerhans cells express Ia antigens. Nature any penetrating bacterial antigens or cellular debris. 1977;268:248-50. Type 1 cells are also present and these may process 6 Steinman RM, Lustig DS, Cohn ZA. Identification of a novel cell any soluble antigens entering the mucosa. type in peripheral lymphoid organs of mice. III. Functional properties in vivo. J Exp Med 1974;139:1431-45. In patients with active inflammatory bowel dis- Lampert IA, Pizzolo G, Thomas A, Janossy G. Immunohistolog- ease there was an increase within the colon of small, ical characteristics of cells involved in dermatopathic lym- irregular HLA-DR+ ACP++ NSE+ ATPase`' phadenopathy. J Pathol 1980;131:145-55. (type 3) cells. These cells appeared at the expense of Janossy G, TLidman N, Selby WS et al. Human inducer and sup- pressor T lymphocytes occupy different microenvironments. the type 2 cells. The findings suggest that the his- Nature 1980;289:81-4. tiocytes in inflammatory bowel disease may be 9Poulter LW, Duke 0, Hobbs S, Janossy G, Panayi G. important in pathogenesis, the altered characteris- Histochemical discrimination of HLA-DR-positive-cell tics perhaps representing activation in response to populations in the normal and arthritic synovial lining. Clin Exp Immunol 1982;48:381-8. foreign antigen. An increase in the number of mac- Van-Furth R, Langevoort UL, Schaberg A. Mononuclear phago- rophages in the mucosa of patients with Crohn's dis- cytes in human pathology-proposal for an approach to ease has recently been reported, and these mac- improved classification. In: Van-Furth R, ed. Mononuclear rophages were shown to have large con- in immunity, infection and pathology. Blackwell: scattered distribution Oxford, 1975. taining dense inclusions.33 The Poulter LW, Turk JL. Studies of the effect of soluble lymphocyte of type 3 cells resulting from oedema and the products on macrophage physiology. II. Cytochemical changes inflammatory cell infiltrate may allow T cell- associated with activation. Cell Immunol 1975;20:25-32. macrophage interactions to take place throughout Muller-Hermelink HK. Characterisation of the B-cell and T-cell regions of human lymphatic tissue through enzyme histochem- the mucosa where numbers of helper T and ical analysis of the human jejunal epithelium in non-tropical suppressor-cytotoxic T cells are increased (Selby-et sprue. Gastroenterology 1961 ;40:735-65. http://jcp.bmj.com/ al, submitted). Increased turnover of circulating 3 Rowden G, Lewis MG, Sullivan AK. Ia antigen expression on in patients with inflammatory bowel dis- human epidermal Langerhans cells. Nature 1977;268:247-8. Hobbs G. ease and increased monocyte lysosomal enzyme Poulter LW, Chilosi M, Seymour GJ, S, Janossy Immunofluorescence membrane staining and cytochemistry, activity provides evidence for the recruitment of applied in combination for analysing cell interactions in situ. activated macrophages into the intestinal In: Polak J, Van Moorden S, eds. Immunocytochemistry: mucosa.34135 After treatment, the appearances practical applications in pathology and biology. Bristol: J Wright, 1982.

returned to normal, indicating that the alterations on October 1, 2021 by guest. Protected copyright. 5 Janossy G, Bollum FJ, Bradstock KF, McMichael A, Rapson N, seen in the histiocyte populations in inflammatory Greaves MF. Terminal transferase-positive human bone mar- bowel disease are not primary to the disease process. row cells exhibit the antigenic phenotype of common acute Heterogeneity of non-lymphoid, NSE-positive lymphoblastic leukaemia. J Immunol 1979;123: 1525-9. cells to mucosa Selby WS, Janossy G, Jewell DP. Immunological characterisation thought have arisen from intestinal of intraepithelial lymphocytes of the human gastrointestinal of rats has recently been shown in vitro.36 The ability tract. Gut 1981:22:169-76. to isolate and study histiocytes from human intesti- 7 McMichael AJ, Pilch JR, Galfre G, Mason DY, Fabre JW, Mils- nal lamina propria37 will allow closer analysis of the tein C. A human antigen defined by a hybrid myeloma monoclonal antibody. Eur J Immunol 1979;9:205- functions of these important cell populations. Such 10. studies, in combination with continued examination I8 Gomori G. Microscopic histochemistry. Chicago: University of the cellular microenvironment in situ, shall Press, 1952. undoubtedly increase our understanding of the 9 Padykula HA, Strauss EW, Ladman AJ, Gardner FH. A mor- phologic and histochemical analysis of the human jejunal immunological mechanisms active in the human epithelium in non-tropical sprue. Gastroenterology intestinal tract. 1961 ;40:735-65. Spiro HM, Filipe MI, Stewart JS, Booth CC, Pearse AGE. Func- References tional histochemistry of the small bowel mucosa in malabsorp- tive syndromes. Gut 1964;5: 145-54. 'Scott H, Solheim BG, Brandtzaeg P, Thorsby E; HLA-DR-like 21 Samloff IM, Davis JS, Schenk EA. A clinical and histochemical antigens in the epithelium of the human small intestine. Scand study of coeliac disease before and during a gluten-free diet. J Immunol 1980;12:77-82. Gastroenterology 1965;48:155-72. J Clin Pathol: first published as 10.1136/jcp.36.4.379 on 1 April 1983. Downloaded from

384 Selby, Poulter, Hobbs, Jewell, Janossy

22 Monis B, Mendeloff AI. Studies in ulcerative colitis: TPN-linked ecology of the upper small bowel. Am J Gastroenterol dehydrogenases and non-specific esterase in rectal biopsy 1976;65:57-62. specimens. Gastroenterology 1965;48: 173-84. 3' Poulter LW, Hobbs S, Bofill M, Janossy G. Immunohistological 23 Riecken EO, Stewart JS, Booth CC, Pearse AGE. A histochemi- analysis of delayed type hypersensitivity reactions in man using cal study on the role of lysomal enzymes in idiopathic steator- combined immunofluorescence and cytochemical staining on rhoea before and during a gluten-free diet. Gut 1966;7:317- tissue sections. Submitted for publication. 1982. 32. 32 Gorbach SL, Nahas L, Lerner PI, Weinstein L. Studies of intesti- 24 Humphrey JH. Differentiation of function among macrophages. nal microflora. I. Effects of diet, age and periodic sampling on In: Microenvironments in haemopoietic and lymphoid differ- numbers of faecal microorganisms in man. Gastoentrology entiation. Ciba Found Symp 1981;84:302-12. 1967;53:845-55. 25 Balfour BM, Drexhage HA, Kamperdijk EWA, Hoefsmit 33 Thyberg J, Graf W, Klingenstrom P. Intestinal fine structure in EChM. Antigen-presenting cells, including Langerhans cells, Crohn's disease. Lysosomal inclusions in epithelial cells and veiled cells and interdigitating cells. In: Microenvironments in macrophages. Virchows Arch (Pathol Anat) 1981;39: 141-52. haemopoietic and lymphoid differentiation. Ciba Found Symp 3 Meuret G, Bitzi A, Hammer B. Macrophage turnover in Crohn's 1981;84:281-98. disease and ulcerative colitis. Gastroenterology- 1978;74:501- 26 Seymour GR, Poulter LW, Bofill M et al. The reactivity of a 3. monoclonal antibody against cells of the monocyte/ 3S Mee AS, Jewell DP. Monocytes in inflammatory bowel disease: macrophage series in sections of normal and inflamed human monocyte and serum lysosomal enzyme activity. Clin Sci tissues. Submitted for publication. 1982. 1 980;58: 295-300. 27 Hoefsmit EChk4, Kamperdijk EWA, Hendrikes HR, Beeten 36 Steer H. Study of acute localised of the gastrointes- RHJ, Balfour BW. Lymph node macrophages. In: Carr I, tinal tract: the effluent lymph. Gut 1981;22:827-35. Daens WT, eds. The renculoendothelial system. Vol. 1. 37 Bull DM, Bookham MA. Isolation and functional characterisa- Morphology. Oxford: Blackwell Scientific Publications, 1980. tion of human intestinal mucosal lymphoid cells. J Clin Invest 28 Stingl G, Katz SI, Clement L, Green I, Shevach EM. 1977;59:966-74. Immunologic functions of Ia-bearing epidermal Langerhans cells. J Immunol 1978;121:2005-13. 29 Braathen LR, Thorsby E. Studies on human epidermal Langerhans cells. I. Allo-activating and antigen-presenting Requests for reprints to: Dr WS Selby, Department of capacity. Scand J Immunol 1980;11:401-8. Gastroenterology, John Radcliffe Hospital, Oxford OX3 3t Dickman MD, Chappelka AR, Schaedler RW. The microbial 9DU, England. http://jcp.bmj.com/ on October 1, 2021 by guest. Protected copyright.