Clinical, Histochemical, and Electron Microscopic Study of Colonic Histiocytosis

Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from Gut, 1966, 7, 458

Clinical, histochemical, and electron microscopic study of colonic histiocytosis

FRED E. PITTMAN,1 W. THOMAS SMITH, ABRAHAM MIZRAHI, WILLIAM A. BLANC, AND JOAN C. PITTMAN From the Departments ofMedicine andPathology, University ofBirmingham, and the Queen Elizabeth Hospital, Birmingham, and the Departments ofMedicine and Pediatrics, Columbia University College ofPhysicians and Surgeons, the Presbyterian and Babies Hospitals, New York, N. Y., U.S.A.

EDITORIAL COMMENT These studies inidcate that colonic histiocytosis is unrelated to Whipple's disease and emphasize that the presence of P.A.S.-positive histiocytes in the lamina propria of rectal mucosa cannot, by itself, be considered diagnostic of Whipple's disease.

Infiltration of the lamina propria of the mucosa of cytes have not been found in significant numbers in the small intestine with 'foamy' macrophages the lamina propria of rectal mucosa obtained from (histiocytes) which give a strongly positive cytoplas- normal individuals or from various patients with mic reaction with the periodic acid-Schiff (P.A.S.) diarrhoea not due to Whipple's disease (Caravati et stain is considered diagnostic of Whipple's disease al., 1963; Flick, Voegtlin, and Rubin, 1962; Martel (Black-Schaffer, 1949; Chears, Hargrove, Verner, et al., 1963). Rowlands and Landing (1960), however, Smith, and Ruffin, 1961; Farnan, 1958; Plummer, reported the post-mortem finding of P.A.S.-positive Russi, Harris, and Caravati, 1950; Sieracki and Fine, histiocytes in the lamina propria of colonic mucosa 1959); other features, such as clubbing and fusion of of two children; since these cells were not found in villi presence spaces in the jejunal mucosa, lymph nodes, or other tissues, the and the of lipid-filled the http://gut.bmj.com/ lamina propria, are variable in degree and occasion- this finding could have represented an early stage of ally absent. Similar infiltration with P.A.S.-positive Whipple's disease, a related metabolic disorder, or an histiocytes is frequently observed in mesenteric entirely different pathological process. These authors lymph nodes, and less commonly in extra-abdominal used the term 'colonic histiocytosis' to describe this tissues (Chears, Smith, and Ruffin, 1959; Enzinger finding. and Helwig, 1963; Eyler and Doub, 1956; Farnan, Recently we have investigated chronic diarrhoea 1958; Koudouris, Stern, and Utterback, 1963; in a 7-year-old girl and in a 37-year-old woman, both Sieracki and Fine, 1959; Sieracki, Fine, Horn, and of whom showed infiltration of the lamina propria to on October 1, 2021 by guest. Protected copyright. Bebin, 1960; Smith, French, Gottsman, Smith, and the rectal mucosa with large numbers of P.A.S.- Wakes-Miller, 1965). These cells have been found in positive histiocytes. Clinical, histochemical, and the lamina propria of the large bowel in several electron microscopic studies were carried out in an reported cases of Whipple's disease in which the effort to determine if this pathological picturere pre- diagnosis was confirmed by histological examination sented an early and localized form of Whipple's of other tissues (Caravati, Litch, Weisiger, Ragland, disease. The results of these studies are presented in and Berliner, 1963; Fleming, Yardley, and Hendrix, this report. 1962; Sieracki and Fine, 1959). The absence of P.A.S.-positive histiocytes in the lamina propria of CASE REPORTS the mucosa of the large bowel in proven cases of CASE 1 This 7-year-old Caucasian girl was readmitted to Whipple's disease has been seldom reported (Martel, the Babies Hospital for further evaluation of chronic Batsakis, and Bolt, 1963; Smith et al., 1965). There watery diarrhoea and faecal incontinence which had been have been two recent reports advocating the use of present since birth. Aside from the diarrhoea and previous urinary tract symptoms thought to be secondary to rectal biopsy to diagnose this disease (Caravati et al., cystitis, the patient had been in excellent health. Her 1963; Fleming et al., 1962). P.A.S.-positive histio- growth and development were normal. During a previous admission to elucidate the of the 'Present address: Department of Medicine, Tulane University School attempts aetiology of Medicine, 1430 Tulane Avenue, New Orleans, Louisiana 70112, diarrhoea, including barium studies of the oesophagus, U.S.A. stomach, and small and large intestinal tracts, were 458 Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from Clinical, histochemical, and electron microscopic study of colonic histiocytosis 459 Crosby-Kugler peroral intestinal biopsy capsule. The histology of the specimen was normal. There were no P.A.S.-positive histiocytes in the lamina propria. Bone marrow aspiration and peripheral lymph node biopsy were performed and the cytology and histology found to be normal. To complete the evaluation, sigmoidoscopy was performed. The rectal and sigmoid colonic mucosa was noted to have a slightly granular appearance. A rectal biopsy was performed and large numbers of P.A.S.- positive histiocytes were observed in the lamina propria (Figs. 1 and 2). Sigmoidoscopy and rectal biopsy were repeated to obtain material for histochemical and electron microscopic examination. Clinical study Because of the beneficial effects of 'broad-spectrum' antibiotics described in patients with Whipple's disease (Davis, McBee, Borland, Kurtz, and Ruffin, 1963; England, French, and Rawson, 1960; Kent, FIG. 1. Light micrograph (case 1) ofrectal mucosa: P.A.S.- Schapira, P.A.S. x 230. Layton, Clifton, and Schedl, 1963; Perez, positive histiocytes in the lamina propria. Pellegrino, Rybak, and Larrechea, 1963), it was decided to treat the patient with tetracycline. She was given tetracycline, 250 mg. four times daily by the oral route, for 21 days. There was no effect on the diarrhoea either during or after administration of the drug. Subsequently she was given prednisone, 10 mg. three times daily, for three months, also with no effect on the diarrhoea. Repeat sigmoidoscopy and rectal biopsy following treatment with the drugs revealed no change from previous examinations. The patient was then given 2,2-diphenyl-4- (4-carbethoxy-4-phenyl-l-piperidino) butyronitrile hydrochloride (diphenoxylate hydrochloride, Lomotil) with atropine sulphate, 2 5 mg. three times daily. The diarr- *xj z 1111._~~~~~~~~~~~~~~~1!_XIP'hoea and faecal incontinence ceased and stools became formed several days after the drug was begun. Attempts to stop the drug have resulted in a return of the patient's http://gut.bmj.com/ diarrhoea.

CASE 2 This 37-year-old woman was admitted to the FIG. 2. Light micrograph (case 1) ofrectal mucosa; higher Presbyterian Hospital for evaluation of chronic diarrhoea, magnification of the histiocytes. Mucicarmine x 450. decreased appetite, weight loss, and fatigue. Twelve years before admission an attack of uveitis of undetermined

origin resulted in total blindness in the right eye. The on October 1, 2021 by guest. Protected copyright. normal. At the time of readmission the watery diarrhoea symptoms which prompted this admission began insidi- and faecal incontinence were still present and were the ously four years previously, and had been evaluated one only symptoms. Physical examination revealed no year after onset at another hospital. Investigations at that abnormalities. Urine analysis was normal. The haemo- time, including barium studies of the oesophagus, globin was 14 6 g. per 100 ml; the white cell count was stomach, and small and large intestinal tracts, gave 9,800cells per mm.3 and the differential countwas normal; normal results. platelets were adequate on smear. The faeces were Physical examination revealed a thin (weight 39 kg., liquid and did not contain occult blood, fat globules, 86 lb.) Caucasian woman with total blindness in the right fatty acid crystals, ova, or parasites. Barium studies of the eye, which had a fixed pupil and an opaque lens. The oesophagus, stomach, and small and large intestinal remainder of the physical examination was unremarkable. tracts were again normal. Other laboratory investigations, Urine analysis was normal. The haemoglobin was 14 5 g. including erythrocyte sedimentation rate, serum electro- per 100 ml; the white cell count was 6,120 cells per mm.3 lyte concentrations, serum amylase activity, liver function and the differential count was normal; platelets were tests, serum protein electrophoresis and scanning, oral adequate on smear. The faeces were liquid to semi- glucose tolerance test, xylose excretion test, 1311-labelled formed and did not contain occult blood, fat globules, triolein and '311-labelled oleic acid absorption tests, assay fatty acid crystals, ova, or parasites. Barium studies of the of proteolytic activity in duodenal secretions, prothrom- oesophagus, stomach, and small and large intestinal bin time, urinary excretion of 5-hydroxy-indoleacetic acid, tracts were normal. Additional investigations, including faecal excretion of lactic acid, and assay of sodium and erythrocyte sedimentation rate, serum electrolyte con- chloride concentrations in the sweat, all yielded normal centrations, serum amylase activity, liver function tests, results. A jejunal mucosal biopsy was obtained with serum protein electrophoresis and scanning, oral glucose Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from 460 Fred E. Pittman, W. Thomas Smith, Abraham Mizrahi, William A. Blanc, and Joan C. Pittman

FIG. 3. Light micrograph (case 2) of rectal mucosa: P.A.S.-positive histiocytes in the lamina propria. P.A.S. x 200. FIG. 4. Light micrograph: Whipple's disease ofjejunal mucosa. Numerous P.A.S.-positive histiocytes are present in the lamina propria surrounding space which contained lipid. P.A. S. x 80.

tolerance test, xylose excretion test, 131I-labelled triolein from these two patients failed to demonstrate the presence and 1311-labelled oleic acid absorption tests, assay of of P.A.S.-positive histiocytes. A rectal mucosal biopsy proteolytic activity in duodenal secretions, prothrombin was obtained from the third patient nine months after time, urine excretion of 5-hydroxy-indole acetic acid and treatment with tetracycline and this showed a moderate Schilling test without intrinsic factor, were normal. infiltration with P.A.S.-positive histiocytes in the lamina Mucosal biopsies of the proximal and distal jejunum propria. were obtained with a Crosby-Kugler peroral intestinal biopsy capsule. The histological appearance of each FREQUENCY OF OCCURRENCE OF specimen was normal. No P.A.S.-positive histiocytes P.A.S.-POSITIVE HISTIOCYTES IN RECTAL MUCOSA

were seen in the lamina propria. Tissue obtained by http://gut.bmj.com/ percutaneous biopsy of the liver showed mild periportal Rectal and colonic mucosal specimens obtained from 131 fibrosis. Sigmoidoscopy was performed and the rectal and patients with and without disease of the large or small sigmoid colon mucosa appeared slightly granular. A bowel were examined for the presence of P.A.S.-positive rectal mucosal biopsy was obtained and the histology cells (Table I). Virtually all of the specimens contained showed large numbers of P.A.S.-positive histiocytes in the some P.A.S.-positive plasma cells just beneath the layer lamina propria (Fig. 3). Sigmoidoscopy and rectal mucosa of surface epithelium. An occasional P.A.S.-positive biopsy were repeated to obtain material for histo- histiocyte was found in approximately 25 % of the chemical and electron microscopic examination. specimens. Moderate infiltration with P.A.S.-positive on October 1, 2021 by guest. Protected copyright. Clinical study The patient was treated with tetra- histiocytes was found in biopsies obtained from one cycline, 250 mg. in 50 ml. distilled water given as a reten- patient with non-specific colitis and from one patient with tion enema four times daily. Following one week of therapy the watery diarrhoea became more severe and the drug was discontinued. She was treated subsequently with TABLE I diphenoxylate hydrochloride with atropine sulphate, 2-5 FREQUENCY OF P.A.S.-POSITIVE HISTIOCYTES IN RECTAL AND mg. three times daily by mouth. On this therapy the COLONIC MUCOSA diarrhoea became much less severe and stools were Clinical State Number of mostly formed. At the time of discharge from the Patients hospital the patient was urged to increase the amount of food she ate. During the 12 months following discharge No known bowel disease 32 she Normal mucosa adjacent to carcinoma of large bowel 30 gained 6-4 kg. (14 lb.) and had only occasional Normal mucosa adjacent to benign tumour of large diarrhoea. bowel 29 Non-specific colitis 18 Adult CASES 3-5 Jejunal mucosa was coeliac disease 8 obtained by peroral Normal mucosa adjacent to diverticulitis 4 biopsy from three untreated patients with Whipple's Rectal schistosomiasis 2 disease, showing characteristic histological features on Regional enteritis 2 light microscopy (Fig. 4). The clinical findings and brief Rheumatoid arthritis 2 Diarrhoea of undetermined origin 2 reference to the jejunal lesions in two of these patients Intestinal lymphangiectasia I have been given in a previous report (Smith et al., 1965). Acquired adult hypogammaglobulinaemia with diarrhoea 1 Light microscopy of colonic mucosa obtained at necropsy Total 131 Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from Clinical, histochemical, and electron microscopic study of colonic histiocytosis 461 TABLE 11 HISTOCHEMICAL ANALYSES OF CYTOPLASM OF HISTIOCYTES Stain Case I Case 2 Whipple's Disease Cases of Colonic Histiocytosis (Jejunal Afucosa) Reported by Rowlands and Landing (1960) Case 1 Case 2 Periodic acid-Schiff (P.A.S.) Strongly positive Strongly positive Strongly positive Purple-red Purple-red P.A.S.-diastase digested (salivary diastase) Strongly positive Strongly positive Strongly positive Purple-red Not reported Mucicarmine Strongly positive Strongly positive Weakly positive Not reported Not reported Sudan IV (frozen section) Negative Negative Positive in cystic Negative Negative areas

no bowel symptoms or disease. In neither of these reported by others (Chears and Ashworth, 1961; specimens was the infiltration as heavy as that found in Cohen, 1964; Kent et al., 1963; Kojecky, Malinsk', cases 1 and 2 of this report. Kodousek, and Marsalek, 1964; Kurtz, Davis, and Ruffin, 1962; Perez et al., 1963; Yardley and HISTOCHEMICAL STUDY Hendrix, 1961). The lamina propria contained large Standard methods (Pearse, 1960) were used on the rectal round spaces filled with electron-dense material biopsies from cases 1 and 2 and on jejunal biopsies from which is presumably lipid. Surrounding the round the patients with Whipple's disease. The histochemical spaces were histiocytes which contained cytoplasmic properties of the cytoplasm of the histiocytes in these inclusions, some of which appeared to be groups of specimens are given in Table 11 and are compared with the phagocytosed bacilliform bodies (Fig. 5). Encapsu- histochemical properties of the cytoplasm of the histio- lated, extracellular bacilliform bodies were present cytes in the colonic mucosa of the two cases of colonic histiocytosis reported by Rowlands and Landing (1960). in the lamina propria (Figs. 6 and 7). The cytoplasm of the histiocytes in the rectal mucosa of RECTAL MUCOSA FROM CASES 1 AND 2 Electron cases 1 and 2 gave a strongly positive reaction with the mucicarmine stain, whereas the histiocytes in the jejunal microscopic examination failed to reveal either en- mucosa of the patients with Whipple's disease gave a capsulated extracellular bacilliform bodies or histio- weakly positive reaction with this stain. cytes with inclusions similar to those found in histio-

cytes injejunal mucosa of the patients with Whipple's http://gut.bmj.com/ ELECTRON MICROSCOPIC STUDY disease. Clusters of predominantly rod-shaped electron-dense bodies intimately associated with the One half of each biopsy specimen was fixed immediately were seen in iced 10% formol-saline and a small portion of this was basal portion of occasional goblet cells frozen for staining for lipid.,The remainder of the formol- (Fig. 8); smaller numbers of spherical electron-dense saline-fixed tissue was subsequently embedded in paraffin bodies were also present in a similar location (Fig. 9). for light microscopy. The other half of each biopsy The exact position of these bodies was not clear. specimen was fixed either in 1 % phosphate buffered Some of them could have been in the cytoplasm of osmium tetroxide or in 6-25 % glutaric dialdehyde in 0- 1 adjacent cells such as capillary endothelial cells. on October 1, 2021 by guest. Protected copyright. M phosphate buffer at pH 7-2 followed by post-fixation They did not resemble the granules of Paneth or with 1 % osmium tetroxide. Fixation was followed by Kultschitzky (argentaffine) cells but showed some in dehydration increasing concentrations of ethanol and similarity to the lipochrome pigment granules des- propylene oxide. The specimens were embedded in Epon mucosa of 812, Methacrylate, or Araldite (Luft, 1961; Sabatini, cribed in endothelial cells in the jejunal Bensch, and Barrnett, 1963). Sections were cut with glass patients with Whipple's disease (Kent et al., 1963). knives on a Huxley-Cambridge ultramicrotome and The histiocytes in the lamina propria were found to mounted on carbon-coated copper grids. Tissues were contain various types of inclusions, some of which stained with lead citrate and/or a saturated solution of were relativelylarge andhomogeneous on high magni- uranyl nitrate in 50 % methanol (Watson, 1958; Zobel and fication (Figs. 1Oa and 10b). Other smaller inclusions Beer, 1961). Specimens were examined with an A.E.I. were aggregated into irregular masses and occasion- EM 6 electron microscope. Electron micrographs were ally contained indistinct membranous particles taken at magnifications ranging between 1,500 and demonstrated on high magnification (Figs. 10c and 50,000 on Kodak B.10 high-contrast plates. Further enlargement was obtained photographically. 1Od). Although extracellular structures resembling the larger inclusions were found in the vicinity of one RESULTS of the histiocytes, there was no convincing evidence of active phagocytosis. The inclusions were unlike JEJUNAL MUCOSA IN WHIPPLE'S DISEASE The electron those found in the cytoplasm of histiocytes in the microcsopic appearances were similar to those jejunum in Whipple's disease. Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from 462 Fred E. Pittman, W. Thomas Smith, Abraham Mizrahi, William A. Blanc, and Joan C. Pittman http://gut.bmj.com/ FIG. 5. Electron micrograph: Whipple's disease ofjejunal mucosa. Large numbers of extracellular bacilliform bodies are seen in the lamina propria. The margins of several histiocytes are also included.

x 15,000. on October 1, 2021 by guest. Protected copyright.

FIGS. 6a and 6b. Electron micrographs: Whipple's disease of jejunal mucosa. Longitudinal and transverse sections through extracellular bacilliform bodies, demonstrating capsular membranes and internal structure. (Fig. 6a, x 70,000; Fig. 6b, x 140,000.)

FIG. 7. Electron micrograph: Whipple's disease ofjejunal mucosa. An inclusion (arrows) in the cytoplasm of a histiocyte consists of a clump ofphagocytosed bacilliform bodies. x 40,000. Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from Clinical, histochemical, and electron microscopic study of colonic histiocytosis 463

FIG. 8. Electron micrograph (case 1) of rectal mucosa. Rod-shaped electron-dense bodies are present near the base of a goblet cell. LP = lamina propria. x 60,000. http://gut.bmj.com/ on October 1, 2021 by guest. Protected copyright.

FIG. 9. Electron micrograph (case 2) ofrectal mucosa. Mainly spherical electron-dense bodies near the base of a goblet cell (GC). The lamina propria (LP) is at the lower left ofthe micrograph. x 13,000. Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from 464 Fred E. Pittman, W. Thomas Smith, Abraham Hizrahi, William A. Blanc, and Joan C. Pittman

FIG. lOa. Electron micrograph (case 1) of rectal mucosa. Histiocytes (H) in the lamina propria. Large inclusions (1) and aggregates of small inclusions (2) are present in the cytoplasm. The cells are surrounded by connective tissue (CT). x 3,000. http://gut.bmj.com/

...CT

Anil~ ~ ~

FIG. lOb. Electron micrograph (case 1) ofrectal mucosa. Higher magnification ofthe histiocytes (H) shown in the lower portion of Figure IQa. Large inclusions (1) and aggregates of small inclusions (2) are present in the cytoplasm. The cells are surrounded by connective tissue (CT). x 12,000. Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from Clinical, histochemical, and electron microscopic study of colonic histiocytosis 465

FIG. IOC. FIG.1Od. FIG. 1 Oc. Electron micrograph (case 1) ofrectal mucosa. Part ofan aggregate ofsmall inclusions. x 32,000. FIG. lOd. Electron micrograph (case 1) ofrectal mucosa. Higher magnification ofsmall inclusions showing membranous http://gut.bmj.com/ particles. x 112,000.

DISCUSSION and we can do no more than record their presence. It is also difficult to account for the cytoplasmic inclu- The results of these studies support the view that sions found in therectal histiocytes ofthetwo patients. these two patients with chronic diarrhoea and The failure to observe evidence of active phagocyto- P.A.S.-positive histiocytes in the lamina propria of sis does not necessarily indicate that these inclusions on October 1, 2021 by guest. Protected copyright. the rectal mucosa probably had the disorder previ- were formed within the cytoplasm of the histio- ously described as colonic histiocytosis (Rowlands cytes. and Landing, 1960) and did not have classical The reason for the infiltration of the rectal mucosa Whipple's disease. Treatment with tetracycline failed with P.A.S.-positive histiocytes and the relation of to relieve the diarrhoea. Histochemical comparison this infiltration to the chronic diarrhoea has not been of the cytoplasm of these histiocytes with similar determined. Nevertheless, our observations indicate P.A.S.-positive histiocytes in the lamina propria of that such infiltration is not necessarily diagnostic of the jejunal mucosa of patients with Whipple's disease Whipple's disease, which is more reliably diagnosed revealed a difference of affinity for the mucicarmine by peroral jejunal biopsy. The frequency and rele- stain. Finally, electron microscopic examination of vance of rectal or colonic histiocytosis can only be rectal mucosa from these patients failed to reveal determined by the routine use of rectal biopsy with either extracellular encapsulated bacilliform bodies P.A.S. staining of the tissue in patients with chronic or histiocytes with cytoplasmic inclusions similar to diarrhoea of obscure aetiology. those found in the histiocytes present in jejunal The therapeutic effect of diphenoxylate hydro- mucosa of patients with Whipple's disease. chloride in the control of the diarrhoea is a result of The nature and significance of the electron-dense its chemical similarity to morphine. The successful bodies found at the base of some of the goblet cells use of this drug with atropine sulphate in the two in the rectal mucosa of the two patients is not clear, patients reported here presumably reflects only the Gut: first published as 10.1136/gut.7.5.458 on 1 October 1966. Downloaded from 466 Fred E. Pittman, W. Thomas Smith, Abraham Mizrahi, William A. Blanc, and Joan C. Pittman effectiveness of the anti-peristaltic activity of the ment throughout this study and for his assistance with the drugs. preparation of the manuscript.

SUMMARY REFERENCES

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