Mechanisms of Idiopathic Constipation: Outlet Obstruction

00 16-5085/ 78/ 7504-0632$02. 00!0 7.5:632-637, 1978 Copyright tD 1978 by the American Gastroenterological Association

Vol. 75, No.4

GASTROENTEROLOGY

Printed in U.S .A.

INDUCTION OF GRANULOMAS IN MICE BY CROHN'S DISEASE TISSUES DAviD R. CAvE, M.R.C.P., PH.D., DoNALD N. MITCHELL, M.D., AND BRYAN N. BROOKE, M.D., M.CHIR., F.R.C.S. Departm ent of' Surgery, St. George's Hospital, and MRC Tuberculosis and Chest Diseases Unit , Brompton Hospital, London, England

Normal and immunodeficient CBA and A 2G strain mice were inoculated with crude (100 or 20 to 30 p.m), cell-free (0.2 p.m) filtrates of Crohn's or noninflammatory bowel disease tissue homogenates, which were either fresh or frozen to -70°C. Mice of each strain developed epithelioid and giant cell granulomas both locally at the site of injection and systemically in response to crude and cell-free filtrates of Crohn's tissues. Control mice did not develop such changes. The granulomas evolved slowly, predominantly between 9 and 27 months. The granuloma-inciting agent has been shown to be present in ileum, colon, and mesenteric lymph nodes of patients with Crohn's disease and it withstands freezing to -70°C. The use of Crohn's tissues common to this study and one in rabbits previously reported, suggests that the induction of granulomas by this agent is not strain- or species-specific, and is independent of the immune status of CBAmice. Evidence of a transmissible agent from Crohn's disease is accumulating. 1-:1 Transmission studies in CBA strain mice~ and New Zealand white (NZW) 5 rabbits have provided evidence for an agent that will pass a 220 nm filter and will induce slowly evolving and persistent granulomatous changes both locally at the injection site and systemically. The studies with NZW rabbits have been partially confirmed by two other laboratories. u, 7 However, additional workers using T.O. strain mice, Dunkin-Hartley guinea pigs, Sprague-Dawley rats and NZW rabbitsH, 9 but using different techniques have failed to reproduce the findings. The question of specificity has been raised by the short term experiments by Taub et aL, 10 who have reported transient granulomatous changes induced by crude tissue homogenates from patients with gastrointestinal diseases other than inflammatory bowel disease. Our current study was carried out to investigate the susceptibility of normal and immunodeficient CBA and A2G strain mice to the transmissible agent from Crohn's disease tissues by the use of donor tissues common to our previous study with NZW rabbits. Additional experiments were included to investigate the specificity, rate of evolution, and distribution of the induced granulomatous changes. Received July 21 , 1977. Accepted April 12, 1978. Address requests for r eprints to: Dr. David R. Cave, Box 400, The University of Chicago Hospitals a nd Clinics, 950 East 59th Street, Chicago, Illinois 60637. This study was supported by th e British Medica l Research Council. Dr. Cave was a Research Fellow of the British Digestive Founda tion. 632

Methods Animals. Normal female CBA strain mice of the same age (12 weeks) and stock were used; 90 mice were inoculated with Crohn's and 66 with control tissue homogenates. Immunodeficient female CBA strain mice were prepared by thymectomy at 6 weeks of age and sequential whole body irradiation at 2week intervals to a total of 600 rads. Thirty such mice were injected with Crohn's and 15 with control homogenates. The mice were caged in groups of 6, were fed on a standard laboratory pellet diet, and allowed water ad libitum. Additiona lly 30 A 2 G strain mice, inbred for 42 generations, were injected with Crohn's disease tissue homogenates at 12 weeks of age. Control A 2 G mice were injected with ileal tissue homogenates from patients with ulcerative colitis and are reported elsewhere. 11 Homogenate origin and preparation . Tissues were obtained from 9 patients with a clinical and pathological diagnosis of Crohn's disease. The patients all had ileocolitis with the exception of donors 2 and 8 who had ileal and colonic disease, respectively; all tissues were taken from areas of active disease and contained granulomas. Control tissues were obtained from 5 patients and included ileal tissue resected with a hemangioma of cecum, familial polyposis coli, and carcinoma of the cecum; colonic tissue from the hemangioma specimen; and lymph nodes removed at two cholecystectomies. All tissues were cultured for acid- and alcohol-fast bacilli and none were detected. Homogenates were prepared immediately after resection either by mincing full thickness of bowel wall or lymph node with scissors and placing in a VirTis homogenizer (VirTis Co., Inc., Gardiner, N.Y.) for 4 min at 4°C in isotonic saline (10% w/v), or by snap-freezing in liquid nitrogen, storing at -70°C, and subsequent homogenization in a Biotech X press (L. K. B. Producter, Fack S-161, 25 Bromma-1, Sweden) at - 30°C with subsequent dilution (10% w/v). All homogenates were then passed through a 100 !Lm sintered glass filter under low

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IND UCTION OF GRANULOMAS IN MICE BY CROHN'S DISEASE TISSUES

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vacuum to remove coarse debris. Some were additionally passed through a 20 to 30 J.Lm filter to provide an intermediatesized filtrate. Finally, four of these were passed through a 0.2 J.Lm Millipore filter to obtain a cell-free filtrate. The resulting filtrates were injected into both hind footpads, 0.03 ml in each, or 0.5 ml intraperitoneally immediately after preparation. Biopsy and autopsy. Sequential pairs of mice in each group of 6 had one footpad punch biopsy under ether anesthesia at three monthly intervals. After 9 months had elapsed mice were autopsied if sick or at 15 to 27 months after inoculation. At autopsy the remaining foot pad, ileum and/or colon, liver, and spleen were submitted for histological examination: sections were stained with hematoxylin and eosin, read "blind" from coded sections, and a ll were examined under polarized light to assist in detection of foreign body granulomas. Microscopic definitions. For a positive response the essential feature was the presence of one or more granulomas composed principally of epithelioid cells with occasional Langhans-type giant cells. In equivocal responses either a diffuse arrangement of epithelioid cells was seen with n o true epithelioid cell granuloma, or focal collections of histiocytes (with less abundant cytoplasm and smaller rounded nuclei) with few or no epithelioid cells. A negative response included the presence of nonspecific inflammatory cells, mononuclear cells, lymphocytes, neutrophils, plasma cells, eosinophils, foreign body reaction, scar with fibroblasts or fibrocytes, or normal tissue.

Results Tables 1 to 3 detail the number of mice developing granulomas and equivocal or negative responses in

their footpads, at biopsy or autopsy, with respect to individual donors. The biopsy results at 3, 6, and 9 months are accumulated in one table because of the small incidence of changes in the first 9 months. Numbers associated with superior letters refer to mice developing changes in liver, spleen, terminal ileum or colon. The correlation of footpad results with systemic spread is not included, because the results for each group of 6 mice were pooled. Granulomatous changes evolved slowly. In the fresh tissue-injected CBA mice, only 1 developed definite and 10 developed equivocal changes within the first 9 months. Subsequently, the numbers increased to 2 definite and 17 equivocal, with some mice developing systemic granulomatous changes. Both footpad and intraperitoneal inject ion achieved transmission. None of <-l-. 8 mice injected with frozen homogenates developed :mlomatous ch anges within 9 m onths, although 4 ~dowed equivocal responses. In those autopsied between 23 and 25 months after inoculation, 5 had granulomas and 5 were equivocal, and again systemic spread was seen. The immunosuppressed CBA mice showed a comparable low response rate at biopsy to the normal mice. Autopsy revealed a higher rate of positivity than in normals, but with a low rate of equivocal findings. However only one-third of the initial number were autopsied. No positive or equivocal changes were seen in any of

TABLE 1. Microscopic findings at biopsy and autopsy in normal CBA mice injected via the footpads or intraperitoneally w ith fres h or frozen Crohn's tissue homogenates" Histological changes Donor

Tissue, fresh

+

Avg surNo. of mice

Autopsy

Biopsy

Filtrate

+

Eq

0 1, 1" 1

3 1 2 3 1, 'l'

2 5 0 2

~

Eq

Injected

Autopsied

mo

p.m

1*

2* 2 3* 6 7 7

Node Ileum Ileum Ileum Ileum Colon Ileum Ileum Ileum

100 100, 20-30 0. 22 100 100 ip 100, 20- 30 100 100 0.22

Total footpads % footpads

viva! of mice autopsied

1 0 2 0 1 1

4 12

1 0 0 0 0 0 0 0 0

1 3

5 3

1 2

10 16

55

0 0 0 0

1 0 0 0

5 6 6 6

0 0

4

23 96

4 6 5 11

5

82

f' 1'"· 0 0 0 0

d

2 5

3 3, '2!' 1 17 43

5 1 3 2

6 12 6 6 6 12 6 6 6

5 7 3 5 2 5

21 52

66 100

40 61

0 2 1 3

6 6 6 6

3

6 38

24 100

4 6 3

25 27 27 27 20 9 27 22 23

Tissue, frozen

1

4* 4 4

Colon Ileum Node Node

Total footpads % footpads

100 100 100 0.22

1, 1d 1 2 1 5 31

2 1, 1b 2, 'l' 0 5 31

4 5

4 16 67

"See Results for explanation of table. Abbreviations used are:+ , posit ive; Eq, equivocal; - , negative; ip, intraperitoneally. "Bowel. '"Liver. b Spleen. * Donors common to rabbit study.:'

25 23 23

TABLE

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2. Microscopic findings at biopsy and autopsy in immunodeficient CBA mice injected uia the footpads with fres h Crohn's tissue

homogenate" Histological changes Donor

Biopsy

Filtrate

Tissue

+

No. ofmice

Autopsy

Eq

Injected

Eq

+

Autopsied

mo

JLm

5* 5 9 9 9

Ileum Node Node Node Ileum

100 100 100 0.22 100

Total footpads %footpads

Avg survival of mice autopsied

0 0 0 0 0

0 2 0 0 0

4 2 4 4 4

0 0

2 10

18 90

0

0

1

'2!'

0

0 1, 1'" 0

3

30

1 10

0 1 3 2 0

6 6 6 6 6

0 2 3 4 1

6 60

30 100

10 33

18 18 20 15

" See table 1, footnote a. & Bowel. ,. Spleen . * Donor common to rabbit study.·'

3. Microscopic findings at biopsy and autopsy in normal A 2G mice injected uia the footpads or intraperitoneally with fresh or frozen . Crohn's tissue homogenates" Histological changes Avg survival Autopsy No. of mice Donor Tissue Filtrate Biopsy of mice Eq Eq Injected Autopsied autopsied + +

TABLE

mo

JLm

4* 4 4 8

Ileum Ileum Ileum colon

100 100ip 0.22 ip 100

Total footpads % footpads

0 0 0 0

0 0 0

6 6 6 5

0 0

1 4

23 96

1, 1b 1b 0 1, 1" 3 22

l b,

c, d

0 1 1, 1d 2 14

5 2 0 2

12 6 6 6

6 2 1 4

23 21 24 22

9 64

30 100

13 43

22.5

" See table 1, footnote a. Bowel. " Liver. 11

<~Spleen.

* Donors common to rabbit study."

the control mice, normal or immunosuppressed, in either footpad , bowel, liver, or spleen in the first 9 months or between 16 and 27 months after inoculation. The mortality rates in the normal control mice and Crohn's-injected mice were similar. Approximately onethird of the mice in each category were not autopsied because of autolysis and/or cannibalism by cagemates. Crohn's tissues from donor 4 (ileocolitis) and 8 (colitis), induced an equivocal r esponse in 1 of 24 A2 G mouse footpads in the first 9 months. Between 21 and 24 months, 2 of 13 footpads showed granulomatous changes, and 3 of 13 were equivocal. Of 13 mice, 3 showed granulomatous changes in ileum, liver, and spleen. Crohn's donors 1 and 2, whose tissues induced changes in the previously reported rabbit study, also induced changes in normal CBA mice. Donors 5 and 9 induced changes in immunodeficient mice and donor 4 did so in A2 G mice . Discussion This study further confirms that Crohn's disease tissues are able to induce slowly evolving granulomatous changes in normal and immunodeficient CBA

strain mice. Of the Crohn's tissue donors used in this study, 5 were also used in our previously reported study involving the use of NZW rabbits. 5 Four of these five donor tissues induced granulomatous changes in mice, suggesting that this phenomenon is not species-specific. Furthermore, the use of the A2 G strain mouse, although uncontrolled in this study, has shown that it is susceptible to the agent from Crohn's disease tissues. Slowly evolving granuloma were induced by injecting terminal ileal homogenates into A2G mice from patients with ulcerative colitis. 11 This phenomenon is therefore not specific to the CBA strain mouse. Granulomatous changes were induced in this study using 100 f.tm , 20 to 30 f.Lm, and 0.22 f.tm filtrates, either fresh or frozen. The mice that were injected with frozen tissue homogenates showed more positive changes than did those receiving fresh tissue (31 a nd 5%, respectively). Whether this difference is real or attributable to variations between donors, or to more efficient homogenization by the Biotech X press, remains to be determined. Footpad inoculation resulted in both local and systemic granulomatous changes, a nd two equivocal r esponses were seen in footpads after intraperitoneal injection. This systemic spread concurs with the find-

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INDUCTION OF GRANULOMAS IN MICE BY CROHN'S DISEASE TISSUES

635

FIG. 1. Granulomatous changes in the small intestinal wall of a CBA mouse 23 months after the injection, into the footpads, of a homogenate of fresh ileum (100 f.Lm) from Crohn's donor 2 (H & E , x 140).

FIG. 2. Granulomatous infiltration of the muscle layers of the colon of a CBA mouse 23 months after intraperitoneal injection of a filtrate (100 f.Lm) of fresh ileal tissue from Crohn's donor 2 (H & E , x 140).

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CAVE ET AL.

FIG. 3. Granulomatous infiltration of the small intestine of a CBA mouse 27 months after footpad injection of a filtrate (20 to 30 fresh ileal tissue from Crohn's donor 1 (H & E, x 140).

ings of Mitchell and Rees, 4 but does not demonstrate the intestinal selectivity reported by them. However, the spread of histological change beyond the limits of the local site of injection suggests a more active process than a simple foreign body reaction. The histology of the lesions was granulomatous (figs. 1 to 3). The lymphoid aggregates frequently seen in Crohn's disease in man have not been noted. Furthermore, the process appeared to be independent of the immune status of the animals, and again this concurs with Mitchell and Rees's work. 1' 4 The significance of the very slow evolution and lack of overt relationship to immunological status is currently unclear but may be evidence for a specific mode of pathogenesis of granuloma formation. The specificity of the findings reported here is at variance with the findings of Taub et al. 10 that both inflammatory bowel disease tissues and some control tissues produced granulomatous changes within 25 days, and all but one had disappeared by 200 days; in addition, no systemic spread was noted. The size of their homogenate, filtered through a 25- or 26-gauge needle, is coarser than that reported here (approximately 450 to 100 ,urn). Comparison therefore, is difficult, and differences need to be resolved by refining the homogenates, using long incubation periods and passage of tissues. This study and others, although suggestive of an infectious etiology for Crohn's disease, are not as yet conclusive. Diseases, such as kuru and Creutzfeldt-Ja-

J.Lm)

of

kob disease, 12 have been shown to have an infectious etiology by using animal transmission experiments. However, no conventional virus has been seen and infectivity is associated with cell membranes. Furthermore, infectivity can be maintained in cell culture, and produces no visible effect. Subsequent injection of these cells into a suitable animal host produces disease. With these considerations in mind, further studies are in progress to define more clearly the nature of the agent in Crohn's disease tissue and the relationship of the induced granuloma to the pathogenesis of Crohn's disease in man. REFERENCES 1. Mitchell DN, Rees RJW: Agent transmissible from Crohn's disease tissue. Lancet 2:168-171, 1970

2. Cave DR, Mitchell DN, Kane SP, et al: Further animal evidence of a transmissible agent in Crohn's disease. Lancet 2:1120-1122, 1973

3. Taub RN, Sachar D, Siltzbach LE, et al: Transmission of ileitis and sarcoid granulomas to mice. Trans Assoc Am Physicians 87:219-224, 1974

4. Mitchell DN, Rees RJW: Further observations on the transmissibility of Crohn's disease. Ann NY Acad Sci 278:546-559, 1976 5. Cave DR, Mitchell DN, Brooke BN: Experimental animal studies of the etiology and pathogenesis of Crohn's disease. Gastroenterology 69:618-624, 1975 6. Simonowitz D, Block GE, Riddell R, et al: The production of an

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7. 8.

9. 10.

INDUCTION OF GRANULOMAS IN MICE BY CROHN'S DISEASE TISSUES

unusual tissue reaction in rabbit bowel injected with Crohn's disease homogenates. Surgery 82:211-218, 1977 Donnelly BJ, Delaney PV, Healy TM: Evidence for a transmissible factor in Crohn's disease. Gut 18:360-363, 1977 Bolton PM, Heatley RV, Owen E, et al: Negative findings in laboratory animals for a transmissible agent in Crohn's disease. Lancet 2:1122-1124, 1973 Heatley RV, Bolton PM, Owen E, et al: A search for a transmissible agent in Crohn's disease. Gut 16:523-532, 1975 Taub RN, Sachar D, Janowitz H, et al: Induction of granulomas

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in mice by inoculation of tissue homogenates from patients with inflammatory bowel disease and sarcoidosis. Ann NY Acad Sci 278:560- 564, 1976 11. Cave DR, Mitchell DN, Brooke BN: Preliminary evidence of a transmissible agent from ulcerative colitis tissue. Lancet 1:13111316, 1976 12. Gibbs CJ, Gajdusek C: Isolation and characterization of the subacute spongiform virus encephalopathies of man: kuru and Creutzfeld-Jakob disease. J Clin Pathol (suppl) 25 (Roy Coli Path) 6:84-96, 1972