219
characterisation studies’ revealed that the agent passes through a SO nm millipore filter. The agent contains R.N.A. since growth has been inhibited by 5-iodo-2-deoxyuridine. The virus is stable both to acid and to ether but reacts with formalin.s The agent has been shown to be heat stable when heated at 50°C for one hour but can be made heat labile by treatment with magnesium chloride. The viral agent has been shown to be pathogenic for suckling CBA mice. Previous studies have also demonstrated failure of neutralisation using antisera to polio viruses i to ill, the group A coxsackie viruses 1 to 22 and 24, the group B coxsackie viruses 1 to 6, and echoviruses 1 to 7, 9, 11-27, and 29 to 33.7 The previous characterisation studies together with the electron microscopic appearance of the virus suggest that the agent is a picornavirus. Further studies will be necessary to idenify properly the viral agent which has now been isolated from Crohn’s disease ileal tissue, cultivated in tissue culture, and partially characterised.
previous
We thank Mrs Marika Arthur and Miss Sue Hertweck for their valuable assistance.
Requests for reprints should be addressed to G.L.G., Department of Medicine, Division of Gastroenterology, U.C.L.A. Center for the Health Sciences, Los Angeles, California 90024, U.S.A. REFERENCES 1. Mitchell, D. N., Rees, R. J. W. Lancet, 1970, ii, 168. 2. Cave, D. R., Mitchell, D. N., Kane, S. P., Brooke, B. N. ibid. 1973, ii, 1120. 3. Farmer, G. W., Vincent, M. M., Fucillo, L., Horta-Barbosa, L., Sever, J. L., Gitnick, G. L. Gastroenterology 1973, 65, 8. 4. Aronson, M. D., Phillips, C. A., Becker, W. L. ibid. 1974, 66, 661. 5. Gitnick, G. L. ibid. 1976, 70, A29. 6. Gitnick, G. L., Arthur, M. A., Shibata, I. Lancet, 1976, ii, 215. 7. Aronson, M. D., Phillips, C. A., Beeken, W. L., Forsyth, B. R. Prog. med. Virol. 1976, 21, 165.
NEUTROPHIL DYSFUNCTION IN CROHN’S DISEASE A. W. SEGAL
Royal Postgraduate Medical School, Du Cane Road, London W12 0HS
G. LOEWI Clinical Research Centre, Watford Road, Harrow, Middlesex
The migration of neutrophils into skin windows has been examined in with Crohn’s disease and rheumatoid arthritis patients and compared with normal. Leucocyte migration was significantly lower in patients with Crohn’s disease than in others. This abnormality of neutrophil function in Crohn’s disease appears to be secondary to a defective acute inflammatory response as the neutrophils themselves were found to behave normally on in-vitro
tract. Evidence is accumulating which the that persistence of particulate or antigenic suggests material can result in damage to surrounding tissues by the release of toxic lysosomal enzymes from autologous macrophages.1 The intestinal lumen contains large quantities of microorganisms and ingested protein some of which gain access to the submucosa. A poor acute inflammatory response or relatively indigestible material could delay the clearance of this antigenic material, persistence of which would predispose to chronic inflammation. This study has been performed to investigate the acute inflammatory response in patients with Crohn’s disease and to compare it with that of normal subjects and patients with another chronic inflammatory condition.
gastrointestinal
Subjects and Methods 25 patients with Crohn’s disease were studied (see accompanying table). Barium studies were compatible with this diagnosis in all the patients and demonstrated small-bowel disease in 12, large-bowel disease in 2, and both small and large bowel disease in 11. The diagnosis was confirmed histologically by the demonstration of granulomas on rectal biopsy in 10, and in operative specimens from a further 5. 2 patients had had surgical removal of all overtly affected bowel. 12 of the patients had no clinical or laboratory evidence of active disease and only 2 of these patients received drug therapy-with 10 mg of prednisolone daily, and with 7 mg of prednisone and 75 mg of amitryptiline daily, respectively. The other 13 patients had abdominal pain, diarrhoea, or malaise or had objective evidence of disease activity, but none was ill enough to require confinement to bed and none was on any therapy. The number of neutrophils passing within 5 hours from forearm skin abrasions into covering plastic chambers (skin windows, modified from Senn2) filled with autologous serum was investigated. In-vitro chemotaxis was studied by the double-filter technique in a modified Boyden chamber3 with 0-5% casein, autologous serum activated with endotoxin, and autologous plasma as attractants. All tests were done in duplicate, and compared with a tissue-culture-medium control. Non-motile functions of peripheral blood neutrophils from 12 patients were screened by the nylon column dye test.4 In 3 Crohn’s patients the migration of monocytes in 24 hours from skin abrasions on to attached glass cover-slips was determined.5 Neutrophil migration from skin windows and in-vitro chemotaxis was also studied in 31 patients with rheumatoid
arthritis selected to encompass a wide range of disease activity and therapy, including glucocorticoids and anti-inflammatory agents, and in 24 normal subjects.
Summary
A weak acute inflammatory response to particulate or antigenic material in the bowel wall could result in the chronic inflammation observed in this condition.
testing.
Results
Neutrophils of patients
with Crohn’s disease migrated of skin windows in much smaller numbers (mean 0-25±0-15 x 106) than were observed in normal subjects (mean 1-3±0-0-87 x 106) or patients with rheumatoid arthritis (mean 1.15±1-13 x 106) (see This was unrelated to the defect accompanying figure). site or extent of inflammatory bowel lesions, or treatment, and there was no obvious difference between all the Crohn’s patients and the subgroup with inactive disease (table). There was no statistically significant relationship between the number of cells that migrated from skin windows in Crohn’s patients and the E.s.R.(r 0.154), serum-albumin (r 0.065), haemoglobin r 0.223), white-blood-cell count (rO-224), or neutrophil
out
=
=
=
=
Introduction disease is inflammatory condition CROH’S
chronic
granulomatous affecting predominantly the a
=
count(r= 0-238).
=
220 DETAILS OF PATIENTS WITH
CROHN’S
Amongst this group of patients was a subgroup without Results are expressed as mean ( 1 S.E.M.) values.
DISEASE ON WHOM SKIN WINDOW AND CHEMOTAXIS STUDIES WERE PERFORMED
overt
evidence of active
inflammatory
Neutrophils from patients with Crohn’s disease normally on in-vitro testing. Chemotaxis was entirely normal and dye reduction in the nylon column dye test, a screening test of the non-motile functions of neutrophils, was normal (3-37 ±0-32 fmol form-
behaved
azan/phagocyte,490-2± 1.73%
of which
trophils [mean±1S.E.M., n=12]). Monocyte migration from skin
windows
slides after 24 hours was normal in the 3 Crohn’s disease investigated.
were
neu-
on to
glass
patients
with
Discussion
and efficient removal of particulate and anfrom an inflammatory lesion is essential material tigenic in preventing progress to a self-perpetuating state of chronic inflammation.6 Neutrophils accumulate most rapidly at foci of tissue damage and may be important for the clearance or modification of foreign material before the appearance of macrophages,’ which have been implicated in6 the pathogenesis of chronic inflammatory conditions.1 Silicosis serves as an example where damage to surrounding tissue may occur through the release of lysosomal enzymes.6 The present study shows that neutrophil migration into areas of acute inflammation is defective in patients The
rapid
disease
(clinically
well Crohn’s
patients).
with Crohn’s disease. This abnormal neutrophil function was observed in all the patients with Crohn’s disease and was independent of disease activity, extent of bowel involvement, and treatment, and even persisted after surgical resection of overtly affected bowel. Normal neutrophil migration in patients with rheumatoid arthritis indicates that the abnormal results in Crohn’s patients are not directly attributable to the presence of chronic inflammatory disease. Normal function of the neutrophils of Crohn’s patients on in-vitro testing shows that the cells themselves are competent. The defective migration of neutrophils into skin windows in vivo indicates an imperfect inflammatory response, probably on the basis of inadequate or inappropriate release or activation of chemotactic mediators. However, this defective chemotactic response does not appear to extend to macrophages as these cells showed the characteristic delayed migration out of skin windows on to glass coverslips.5 The postulated mechanism of tissue damage by the release of lysosomal enzymes from macrophages as a result of "frustrated phagocytosis" of particulate material appears feasible.16 A similar mechanism could have been responsible for the bowel lesions, closely resembling those of Crohn’s disease, produced experimentally in animals fed on indigestible particulate matter,899 which resulted in the hypothesis that toothpaste ingestion could play a pathogenic role in
humans.1o
generalised disorder of this kind would be expected manifest itself most commonly in tissues which experience the greatest antigenic exposure. The gut contains both bacterial and ingested antigens, both of which penetrate the mucosa,11-13 Why the disease should involve predominantly the distal small bowel is uncertain. Possibly the mucosa of the large bowel is more highly specialised to resist invasion by its bacterial contents, or the combination of a high bacterial concentration and A
to
stool
liquidity
is
most
disadvantageous
in this
area.
studies have shown that in Crohn’s disHowever, ease the whole gut is microscopically abnormal despite the absence of overt lesions,14 and that surgical resection of grossly affected bowel does not modify the overall course of the disease,15 which also suggests diffuse involvement. The skip areas of inflammation separated by recent
-
I
I
R.A.
I
Controls
I
I
Crohn’s
Migration out of skin windows of neutrophils from patients with rheumatoid arthritis, controls, and patients with Crohn’s disease.
relatively normal-looking bowel, which occur commonly in this condition, may just be more overtly affected areas of a generally damaged mucosa. If the underlying lesion in Crohn’s disease is a defective acute inflammatory response, then treatment with immunosuppressive drugs such as glucocorticoids and azathioprine may temporarily relieve symptoms but promote chronicity. We may yet be required to heed the
221
advice offered by George Bernard Shaw in The Doctor’s Dilemma that "There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes." We thank Ms
Jean de Luca for typing the manuscript.
-
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Cardella, C. J., Davies, P., Allison, A. C. Nature, 1974, 247, 46. Senn, H., Fektalswehr bei Hömoblastosen; p. 36. Berlin, 1972. Keller, H. V., Hess, M. W., Cottier, H. in Chemotaxis: Its Biology and Biochemistry (edited by E. Sorkin); p. 19. Basic, 1974. Segal, A. W., Peters, T. J. Clin. Sci. molec. Med. 1975, 49, 591. Rebuck, J. W. Anat. Rec. 1940, 76, 46. Allison, A. C. Ann. N.Y. Acad. Sci. 1974, 221, 299. Metchinikoff, E. Immunity in Infective Diseases (translated by F. G. Binnie).
London, 1905. 8. Chess, S., Chess, D., Olander, G., Benner, W., Cole, W. H. Surgery St. Louis, 1950, 27, 221. 9. Reichert, F. L., Mathes, M. E. Ann. Surg. 1936, 104, 601. 10. Edwards, H. C. in Modern Trends in Gastroenterology (edited by F. Avery Jones); p. 17. London, 1958. 11. Staley, T. E., Corley, L. D., Wynn Jones, E. Am. J. dig. Dis. 1970, 15, 923. 12. Aluwihare, A. P. R. Gut, 1971, 12, 509. 13. Walker, W. A., Isselbacher, K. J. Gastroenterology, 1974, 67, 531. 14. Goodman, M. J., Skinner, J. M., Truelove, S. C. Lancet, 1976, i, 275. 15. Greenstein, A. J., Sachar, D. B., Pasternack, B. S., Janowitz, H. D. New Engl. J. Med. 1975, 293, 685.
THE RISK OF PELVIC INFLAMMATORY DISEASE IN WOMEN USING INTRAUTERINE
CONTRACEPTIVE DEVICES AS COMPARED TO NON-USERS LARS PHILIP BENGTSSON LARS WESTRÖM PER-ANDERS MÅRDH
Department of Obstetrics and Gynæcology, University Hospital, and Institute of Medical Microbiology, University of Lund, Sweden The frequency of women using an intrauterine contraceptive device (I.U.D.) among 515 patients presenting with acute, laparoscopically verified salpingitis, has been compared with the corresponding frequency in 741 sexually active women who were matched to the patients by date of birth, domicile, and socioeconomic status. The frequency of I.U.D. use was significantly (p
characterisation studies’ revealed that the agent passes through a SO nm millipore filter. The agent contains R.N.A. since growth has been inhibited by 5-iodo-2-deoxyuridine. The virus is stable both to acid and to ether but reacts with formalin.s The agent has been shown to be heat stable when heated at 50°C for one hour but can be made heat labile by treatment with magnesium chloride. The viral agent has been shown to be pathogenic for suckling CBA mice. Previous studies have also demonstrated failure of neutralisation using antisera to polio viruses i to ill, the group A coxsackie viruses 1 to 22 and 24, the group B coxsackie viruses 1 to 6, and echoviruses 1 to 7, 9, 11-27, and 29 to 33.7 The previous characterisation studies together with the electron microscopic appearance of the virus suggest that the agent is a picornavirus. Further studies will be necessary to idenify properly the viral agent which has now been isolated from Crohn’s disease ileal tissue, cultivated in tissue culture, and partially characterised.
previous
We thank Mrs Marika Arthur and Miss Sue Hertweck for their valuable assistance.
Requests for reprints should be addressed to G.L.G., Department of Medicine, Division of Gastroenterology, U.C.L.A. Center for the Health Sciences, Los Angeles, California 90024, U.S.A. REFERENCES 1. Mitchell, D. N., Rees, R. J. W. Lancet, 1970, ii, 168. 2. Cave, D. R., Mitchell, D. N., Kane, S. P., Brooke, B. N. ibid. 1973, ii, 1120. 3. Farmer, G. W., Vincent, M. M., Fucillo, L., Horta-Barbosa, L., Sever, J. L., Gitnick, G. L. Gastroenterology 1973, 65, 8. 4. Aronson, M. D., Phillips, C. A., Becker, W. L. ibid. 1974, 66, 661. 5. Gitnick, G. L. ibid. 1976, 70, A29. 6. Gitnick, G. L., Arthur, M. A., Shibata, I. Lancet, 1976, ii, 215. 7. Aronson, M. D., Phillips, C. A., Beeken, W. L., Forsyth, B. R. Prog. med. Virol. 1976, 21, 165.
NEUTROPHIL DYSFUNCTION IN CROHN’S DISEASE A. W. SEGAL
Royal Postgraduate Medical School, Du Cane Road, London W12 0HS
G. LOEWI Clinical Research Centre, Watford Road, Harrow, Middlesex
The migration of neutrophils into skin windows has been examined in with Crohn’s disease and rheumatoid arthritis patients and compared with normal. Leucocyte migration was significantly lower in patients with Crohn’s disease than in others. This abnormality of neutrophil function in Crohn’s disease appears to be secondary to a defective acute inflammatory response as the neutrophils themselves were found to behave normally on in-vitro
tract. Evidence is accumulating which the that persistence of particulate or antigenic suggests material can result in damage to surrounding tissues by the release of toxic lysosomal enzymes from autologous macrophages.1 The intestinal lumen contains large quantities of microorganisms and ingested protein some of which gain access to the submucosa. A poor acute inflammatory response or relatively indigestible material could delay the clearance of this antigenic material, persistence of which would predispose to chronic inflammation. This study has been performed to investigate the acute inflammatory response in patients with Crohn’s disease and to compare it with that of normal subjects and patients with another chronic inflammatory condition.
gastrointestinal
Subjects and Methods 25 patients with Crohn’s disease were studied (see accompanying table). Barium studies were compatible with this diagnosis in all the patients and demonstrated small-bowel disease in 12, large-bowel disease in 2, and both small and large bowel disease in 11. The diagnosis was confirmed histologically by the demonstration of granulomas on rectal biopsy in 10, and in operative specimens from a further 5. 2 patients had had surgical removal of all overtly affected bowel. 12 of the patients had no clinical or laboratory evidence of active disease and only 2 of these patients received drug therapy-with 10 mg of prednisolone daily, and with 7 mg of prednisone and 75 mg of amitryptiline daily, respectively. The other 13 patients had abdominal pain, diarrhoea, or malaise or had objective evidence of disease activity, but none was ill enough to require confinement to bed and none was on any therapy. The number of neutrophils passing within 5 hours from forearm skin abrasions into covering plastic chambers (skin windows, modified from Senn2) filled with autologous serum was investigated. In-vitro chemotaxis was studied by the double-filter technique in a modified Boyden chamber3 with 0-5% casein, autologous serum activated with endotoxin, and autologous plasma as attractants. All tests were done in duplicate, and compared with a tissue-culture-medium control. Non-motile functions of peripheral blood neutrophils from 12 patients were screened by the nylon column dye test.4 In 3 Crohn’s patients the migration of monocytes in 24 hours from skin abrasions on to attached glass cover-slips was determined.5 Neutrophil migration from skin windows and in-vitro chemotaxis was also studied in 31 patients with rheumatoid
arthritis selected to encompass a wide range of disease activity and therapy, including glucocorticoids and anti-inflammatory agents, and in 24 normal subjects.
Summary
A weak acute inflammatory response to particulate or antigenic material in the bowel wall could result in the chronic inflammation observed in this condition.
testing.
Results
Neutrophils of patients
with Crohn’s disease migrated of skin windows in much smaller numbers (mean 0-25±0-15 x 106) than were observed in normal subjects (mean 1-3±0-0-87 x 106) or patients with rheumatoid arthritis (mean 1.15±1-13 x 106) (see This was unrelated to the defect accompanying figure). site or extent of inflammatory bowel lesions, or treatment, and there was no obvious difference between all the Crohn’s patients and the subgroup with inactive disease (table). There was no statistically significant relationship between the number of cells that migrated from skin windows in Crohn’s patients and the E.s.R.(r 0.154), serum-albumin (r 0.065), haemoglobin r 0.223), white-blood-cell count (rO-224), or neutrophil
out
=
=
=
=
Introduction disease is inflammatory condition CROH’S
chronic
granulomatous affecting predominantly the a
=
count(r= 0-238).
=
220 DETAILS OF PATIENTS WITH
CROHN’S
Amongst this group of patients was a subgroup without Results are expressed as mean ( 1 S.E.M.) values.
DISEASE ON WHOM SKIN WINDOW AND CHEMOTAXIS STUDIES WERE PERFORMED
overt
evidence of active
inflammatory
Neutrophils from patients with Crohn’s disease normally on in-vitro testing. Chemotaxis was entirely normal and dye reduction in the nylon column dye test, a screening test of the non-motile functions of neutrophils, was normal (3-37 ±0-32 fmol form-
behaved
azan/phagocyte,490-2± 1.73%
of which
trophils [mean±1S.E.M., n=12]). Monocyte migration from skin
windows
slides after 24 hours was normal in the 3 Crohn’s disease investigated.
were
neu-
on to
glass
patients
with
Discussion
and efficient removal of particulate and anfrom an inflammatory lesion is essential material tigenic in preventing progress to a self-perpetuating state of chronic inflammation.6 Neutrophils accumulate most rapidly at foci of tissue damage and may be important for the clearance or modification of foreign material before the appearance of macrophages,’ which have been implicated in6 the pathogenesis of chronic inflammatory conditions.1 Silicosis serves as an example where damage to surrounding tissue may occur through the release of lysosomal enzymes.6 The present study shows that neutrophil migration into areas of acute inflammation is defective in patients The
rapid
disease
(clinically
well Crohn’s
patients).
with Crohn’s disease. This abnormal neutrophil function was observed in all the patients with Crohn’s disease and was independent of disease activity, extent of bowel involvement, and treatment, and even persisted after surgical resection of overtly affected bowel. Normal neutrophil migration in patients with rheumatoid arthritis indicates that the abnormal results in Crohn’s patients are not directly attributable to the presence of chronic inflammatory disease. Normal function of the neutrophils of Crohn’s patients on in-vitro testing shows that the cells themselves are competent. The defective migration of neutrophils into skin windows in vivo indicates an imperfect inflammatory response, probably on the basis of inadequate or inappropriate release or activation of chemotactic mediators. However, this defective chemotactic response does not appear to extend to macrophages as these cells showed the characteristic delayed migration out of skin windows on to glass coverslips.5 The postulated mechanism of tissue damage by the release of lysosomal enzymes from macrophages as a result of "frustrated phagocytosis" of particulate material appears feasible.16 A similar mechanism could have been responsible for the bowel lesions, closely resembling those of Crohn’s disease, produced experimentally in animals fed on indigestible particulate matter,899 which resulted in the hypothesis that toothpaste ingestion could play a pathogenic role in
humans.1o
generalised disorder of this kind would be expected manifest itself most commonly in tissues which experience the greatest antigenic exposure. The gut contains both bacterial and ingested antigens, both of which penetrate the mucosa,11-13 Why the disease should involve predominantly the distal small bowel is uncertain. Possibly the mucosa of the large bowel is more highly specialised to resist invasion by its bacterial contents, or the combination of a high bacterial concentration and A
to
stool
liquidity
is
most
disadvantageous
in this
area.
studies have shown that in Crohn’s disHowever, ease the whole gut is microscopically abnormal despite the absence of overt lesions,14 and that surgical resection of grossly affected bowel does not modify the overall course of the disease,15 which also suggests diffuse involvement. The skip areas of inflammation separated by recent
-
I
I
R.A.
I
Controls
I
I
Crohn’s
Migration out of skin windows of neutrophils from patients with rheumatoid arthritis, controls, and patients with Crohn’s disease.
relatively normal-looking bowel, which occur commonly in this condition, may just be more overtly affected areas of a generally damaged mucosa. If the underlying lesion in Crohn’s disease is a defective acute inflammatory response, then treatment with immunosuppressive drugs such as glucocorticoids and azathioprine may temporarily relieve symptoms but promote chronicity. We may yet be required to heed the
221
advice offered by George Bernard Shaw in The Doctor’s Dilemma that "There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes." We thank Ms
Jean de Luca for typing the manuscript.
-
REFERENCES 1. 2. 3. 4. 5. 6. 7.
Cardella, C. J., Davies, P., Allison, A. C. Nature, 1974, 247, 46. Senn, H., Fektalswehr bei Hömoblastosen; p. 36. Berlin, 1972. Keller, H. V., Hess, M. W., Cottier, H. in Chemotaxis: Its Biology and Biochemistry (edited by E. Sorkin); p. 19. Basic, 1974. Segal, A. W., Peters, T. J. Clin. Sci. molec. Med. 1975, 49, 591. Rebuck, J. W. Anat. Rec. 1940, 76, 46. Allison, A. C. Ann. N.Y. Acad. Sci. 1974, 221, 299. Metchinikoff, E. Immunity in Infective Diseases (translated by F. G. Binnie).
London, 1905. 8. Chess, S., Chess, D., Olander, G., Benner, W., Cole, W. H. Surgery St. Louis, 1950, 27, 221. 9. Reichert, F. L., Mathes, M. E. Ann. Surg. 1936, 104, 601. 10. Edwards, H. C. in Modern Trends in Gastroenterology (edited by F. Avery Jones); p. 17. London, 1958. 11. Staley, T. E., Corley, L. D., Wynn Jones, E. Am. J. dig. Dis. 1970, 15, 923. 12. Aluwihare, A. P. R. Gut, 1971, 12, 509. 13. Walker, W. A., Isselbacher, K. J. Gastroenterology, 1974, 67, 531. 14. Goodman, M. J., Skinner, J. M., Truelove, S. C. Lancet, 1976, i, 275. 15. Greenstein, A. J., Sachar, D. B., Pasternack, B. S., Janowitz, H. D. New Engl. J. Med. 1975, 293, 685.
THE RISK OF PELVIC INFLAMMATORY DISEASE IN WOMEN USING INTRAUTERINE
CONTRACEPTIVE DEVICES AS COMPARED TO NON-USERS LARS PHILIP BENGTSSON LARS WESTRÖM PER-ANDERS MÅRDH
Department of Obstetrics and Gynæcology, University Hospital, and Institute of Medical Microbiology, University of Lund, Sweden The frequency of women using an intrauterine contraceptive device (I.U.D.) among 515 patients presenting with acute, laparoscopically verified salpingitis, has been compared with the corresponding frequency in 741 sexually active women who were matched to the patients by date of birth, domicile, and socioeconomic status. The frequency of I.U.D. use was significantly (p
