Cint. eAp. mmunol. (1977) 28, 484-489.
DNA-synthesizing cells in the blood in coeliac disease and inflammatory bowel disease G. K. T. HOLMES, P. M. BRATT, N. R. LING & W. T. COOKE Birmingham General Hospital and Department of Experimental Pathology, University of Birmingham, Birmingham
(Received 21 December 1976)
SUMMARY
The level of 3H-labelled thymidine ([3H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the 'background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the [3H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [3H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [3H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis. I NTROD U CTI ON During an investigation into peripheral-blood-lymphocyte responses to gluten and mitogens in coeliac disease (Holmes, Asquith & Cooke, 1976) it was noted that unstimulated lymphocytes from these patients showed lower levels of tritiated-thymidine incorporation than comparable numbers of such cells from normal individuals. This phenomenon has been further examined in coeliac disease and the results compared with those obtained from patients with inflammatory bowel disease and from normal subjects. MATERIALS AND METHODS Patients studied. Two separate studies of patients with coeliac disease were made, the diagnosis hax ing been confirmed by a jejunal biopsy in each case. The definition of coeliac disease employed by the Nutritional and Intestinal Unit has been previously set out (Cooke & Asquith, 1974). Different patients and controls were used in the two studies. Study 1. Thirty-five patients were included, twenty-one of whom had been treated with a gluten-free diet for at least 3 months, while fourteen were taking a normal diet. Eighteen of the patients hav ing a gluten-free diet had shown an excellent clinical response, while the remaining three had shown only partial response. Sixteen had been re-biopsied whilst on the diet and the jejunal morphology in elex en had reverted to near normal. Twenty-sex en healthy individuals formed the control group. Cell-mediated responses to gluten Fraction III were investigated in this same series of patients and published elsewhere (Holmes et al., 1976). Study 2. This included patients with coeliac disease, Crohn's disease and ulcerative colitis. (a) Coeliac disease. Twenty-one of the twenty-eight patients studied were receiving a gluten-free diet (mean time 6-4 years). In this group only three patients had shown less than an excellent clinical response to gluten withdrawal. In all fourteen individuals who had undergone a further jejunal biopsy whilst on diet the jejunal mucosa had improved to near normal in all. A further patient was studied while on a conventional diet but after he had developed a reticulum cell sarcoma and has not been included in the statistical analyses. (b) Crohn's disease. Of nineteen subjects with Crohn's disease, eighteen were in remission with satisfactory haematoCorrespondence: Dr G. K. T. Holmes, Postgraduate Medical Centre, Selly Oak Hospital, Birmingham B29 6JD.
484
485
DNA-synthesizing cells in the blood
logical and biochemical indices. Only one patient was in relapse and she was studied while in the ward undergoing investigation, her results have not been included in the statistical analyses. None of the patients had received steroids within the last 2-5 years. The diagnosis of Crohn's disease was based on clinical, laboratory and radiological features. In eleven patients who had undergone operation, the resected specimens all had appearances characteristic of the diagnosis. (c) Ulcerative colitis. Sixteen patients with ulcerative colitis were studied. Two only were in relapse at the time of the investigation and one of these subsequently required total colectomy. Four of the fourteen patients in remission had undergone panpractocolectomy between 5 and 8 years prior to the study. No patient had received systemic steroids prior to the investigation. The diagnosis of ulcerative colitis was based on clinical, sigmoidoscopic and radiological appearances. In no instance was there any evidence of small intestinal disease. Only the ten unoperated patients in remission were analysed statistically. In study 2, nine healthy subjects were used as controls. Lymphocytes culture methods. (a) Leucocyte cultures (study 1). Peripheral blood was defibrinated by gentle stirring with sterile cherry sticks. The red cells were then sedimented by the addition of a one-third vol. of 3% gelatin in saline and incubated at 370C for 1 hr. Leucocytes were recovered from the leucocyte-rich serum-gelatin supernatant by centifugation at 600 g for 5 min. The cells were washed once in Eagle's medium and resuspended to 1 x 106 lymphocytes per ml in Eagle's medium supplemented with antibiotics and 20%. human serum-gelatin (obtained from a single large pool frozen in aliquots). Duplicate 1-ml cultures in sterile capped tubes were treated with 50 ug of PHA (Burroughs Wellcome) in 0 33 ml of phosphate-buffered saline or the same volume of phosphate-buffered saline without PHA in the case of the controls. The tubes were gassed with 5%4 CO2 in air cultured at 370C for 4, 5 or 6 days. Twenty-four hours before the end of the culture period 0-5 uCi of tritiated thymidine (150 mCi/mM) was added to each tube. Harvested cells were washed successively in salin 5% trichloracetic acid and methanol and then processed for scintillation counting as above. (b) Whole blood cultures (study 2). Blood (10 ml) was collected in heparin (preservative-free, 200 u). After removal of a sample for the white cell count and differential, 0-1 ml of the blood was added to 3 ml of RPMI-1640 medium (Biocult, Glasgow) supplemented with antibiotics and 5%4 foetal bovine serum. Cultures of each blood sample were set up in sextuplicate in bijou bottles. The bottles were sealed by an airtight cap and incubated, without gassing at 370C. Tritiated thymidine (6,uCi per culture, sp.act. 600 mCi/mM prepared from 3H-labelled methyl-thymidine obtained from the Radiochemical Centre, Amersham) was added 24 hr before harvesting. Two 1-ml samples were removed from three bijou bottles 24 hr after setting up the cultures and from the remaining three bottles at 72 hr. The tubes containing the 1 ml samples were treated with 1% acetic acid in saline (1 ml) and allowed to stand 30 min for the red cells to lyse. The cells were then spun down, washed in 5% trichloracetic acid (twice) and methanol (once), dissolved in 0-1 ml N NaOH and added to 1 ml of methanol and 10 ml of a triton-containing scintillation fluid for scintillation counting as previously described (Hardy, Knight & Ling, 1970). (c) Lymphocyte counts in peripheral blood. Films of blood were prepared and stained with Leishman's stain. From the total white count and differential, the numbers of lymphocytes were calculated.
RESULTS As can be seen from Fig. 2 and Table 2, the mean increments of response to PHA of lymphocytes from coeliac patients on days 4, 5 and 6 ofculture were lower than those ofthe control group but the differences TABLE 1. Mean tritiated thymidine incorporation in unstimulated leucocyte cultures
(study 1)
3H-labelled thymidine incorporation (d/min) Type and no. of cases Controls Coeliacs (ND)
28 14
Coeliacs (GFD)
21
Total coeliacs
35
Day 4
Day 5
Day 6
3171-6 1041-8 (P< 0 05) 1326-7
4540 5 1247-0 (P< 0-05) 1092-3 (P< 0-01) 1154-2 (p< o o01)
4726-2 1360-8 (P< 0-01) 1808-0 (P< 0-01) 1623-9 (p< o ool)
(P< 0-01) 1212-7
(p< o o01)
The P value shown refers to the difference of the mean 3H-labelled thymidine incorporation between control and coeliac lymphocytes. ND = Normal diet; GFD= gluten-free diet.
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Fia. 1 F iG. 2 FIG. 1. Thymidine incorporation of unstimulated lymphocytes cultured for 4, 5 or 6 days. Lymphocytes from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliacs (A). FIG. 2. (Study 1.) Thymidine incorporation of PHA-stimulated lymphocytes cultured for 4, 5 or 6 days. Lymphocytes from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliacs (A).
TABLE 2. Net tritiated thymidine incorporation (test minus control) in PHA-stimulated leucocyte cultures (study 1)
3H-labelled thymidine incorporation (d/min) Type and no. of cases
Controls Coeliacs (ND)
28 14
Coeliacs (GFD)
21
Total Cceliacs
35
Day 4
Day 5
Day 6
79204 50078
79322 56118 (P = n.s.) 62339 (P = n.s.) 59851 (P = n.s.)
70494 61958 (P = n.s.) 68676 (P = n.s.) 65910 (P = n.s.)
(P = n.s.) 71456
(P = n.s.) 62905
(P = n.s.)
The P value shown refers to the difference of the mean 3H-labelled thymidine incorporation between control and coeliac lymphocytes. n.s. = Not significant; ND = normal diet; GFD = gluten-free diet.
statistically significant. An incidental and unexpected finding in this study was that the 'background' thymidine incorporation in cultures not containing PHA was significantly lower than in the control group on all three days (see Table 1 and Fig. 1). The depression of response was significant both for coeliacs on a gluten-free diet and those on a normal diet but there were no significant differences between the two dietary groups. The second study was designed to find out whether the subnormal level of thymidine incorporation would also be found in samples of freshly-drawn, unfractionated blood. Accordingly whole blood cultures from coeliac patients, other clinical groups and controls were set up. The medium chosen was supplemented with foetal bovine instead of human serum since such a medium is known to be mildly stimulatory and to give progressively higher levels of background stimulation as the culture proceeds. Tritiated thymidine was added to some cultures immediately they were set up and to others after 2 days of culture. Only the results from the 0-24 hr interval are shown (Fig. 3 and Table 3) but the same trends were not
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Normals Coeioc Ulomtive C.rohn's do oolitis, dieas~e FIG. 3. (Study 2.) Thymidine incorporation by cells in whole blood over the 0-24-hr period. Cells from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliac (A); coeliac with lymphoma (L); ulcerative colitis treated medically, in remission (Z); in relapse (+); ulcerative colitis treated by panpractocolectomy (e); Crohn's disease, in remission (C); in relapse (a).
TABLE 3. Peripheral lymphocyte counts and relation to levels of DNA synthesis in whole blood cultures
Lymphocyte ct/mm3
3H-labelled thymidine incorporation (d/min)
Group and number
28 Cceliac (total) Coeliac (ND) 7 21 Coeliac (GFD) Crohn's disease 18 Ulcerative Colitis 10 Control 9
Mean
s.d.
Per cent of control
Mean
s.d.
Per cent of control
2024 1875 2077 2468 2320 2717
1047 502 1187 1039 1034 839
74 69 76 91 85 100
363 367 374 751 501 520
128 89 131 345 117 144
70 70 72 144 96 100
ND = Normal diet; GFD = gluten-free diet. were evident in the 48-72 hr cultures. The 3H-labelled thymidine incorporation of blood cells of the coeliac group was significantly lower than for the control group. The P values were
DNA-synthesizing cells in the blood in coeliac disease and inflammatory bowel disease G. K. T. HOLMES, P. M. BRATT, N. R. LING & W. T. COOKE Birmingham General Hospital and Department of Experimental Pathology, University of Birmingham, Birmingham
(Received 21 December 1976)
SUMMARY
The level of 3H-labelled thymidine ([3H]TdR) incorporation of blood cells of patients with coeliac disease was shown in two separate studies to be significantly lower than that of a normal control group. In the first study the 'background' DNA synthesis in unstimulated cultures containing a standard number of blood lymphocytes was measured on days 4, 5 and 6. In the second study a standard volume of freshly drawn whole blood was added to culture medium and the [3H]TdR incorporation measured over the first 24 hr and from 48 to 72 hr. In all cases the [3H]TdR incorporation of cells of coeliac patients on a normal or a gluten-free diet was lower than that of the control group. It is suggested that sequestration of DNA-synthesizing cells in the mucosa of the small bowel may partly explain these results. In whole-blood cultures from patients with inflammatory bowel disease in remission [3H]TdR incorporation over the first 24 hr was raised in Crohn's disease but normal in ulcerative colitis. I NTROD U CTI ON During an investigation into peripheral-blood-lymphocyte responses to gluten and mitogens in coeliac disease (Holmes, Asquith & Cooke, 1976) it was noted that unstimulated lymphocytes from these patients showed lower levels of tritiated-thymidine incorporation than comparable numbers of such cells from normal individuals. This phenomenon has been further examined in coeliac disease and the results compared with those obtained from patients with inflammatory bowel disease and from normal subjects. MATERIALS AND METHODS Patients studied. Two separate studies of patients with coeliac disease were made, the diagnosis hax ing been confirmed by a jejunal biopsy in each case. The definition of coeliac disease employed by the Nutritional and Intestinal Unit has been previously set out (Cooke & Asquith, 1974). Different patients and controls were used in the two studies. Study 1. Thirty-five patients were included, twenty-one of whom had been treated with a gluten-free diet for at least 3 months, while fourteen were taking a normal diet. Eighteen of the patients hav ing a gluten-free diet had shown an excellent clinical response, while the remaining three had shown only partial response. Sixteen had been re-biopsied whilst on the diet and the jejunal morphology in elex en had reverted to near normal. Twenty-sex en healthy individuals formed the control group. Cell-mediated responses to gluten Fraction III were investigated in this same series of patients and published elsewhere (Holmes et al., 1976). Study 2. This included patients with coeliac disease, Crohn's disease and ulcerative colitis. (a) Coeliac disease. Twenty-one of the twenty-eight patients studied were receiving a gluten-free diet (mean time 6-4 years). In this group only three patients had shown less than an excellent clinical response to gluten withdrawal. In all fourteen individuals who had undergone a further jejunal biopsy whilst on diet the jejunal mucosa had improved to near normal in all. A further patient was studied while on a conventional diet but after he had developed a reticulum cell sarcoma and has not been included in the statistical analyses. (b) Crohn's disease. Of nineteen subjects with Crohn's disease, eighteen were in remission with satisfactory haematoCorrespondence: Dr G. K. T. Holmes, Postgraduate Medical Centre, Selly Oak Hospital, Birmingham B29 6JD.
484
485
DNA-synthesizing cells in the blood
logical and biochemical indices. Only one patient was in relapse and she was studied while in the ward undergoing investigation, her results have not been included in the statistical analyses. None of the patients had received steroids within the last 2-5 years. The diagnosis of Crohn's disease was based on clinical, laboratory and radiological features. In eleven patients who had undergone operation, the resected specimens all had appearances characteristic of the diagnosis. (c) Ulcerative colitis. Sixteen patients with ulcerative colitis were studied. Two only were in relapse at the time of the investigation and one of these subsequently required total colectomy. Four of the fourteen patients in remission had undergone panpractocolectomy between 5 and 8 years prior to the study. No patient had received systemic steroids prior to the investigation. The diagnosis of ulcerative colitis was based on clinical, sigmoidoscopic and radiological appearances. In no instance was there any evidence of small intestinal disease. Only the ten unoperated patients in remission were analysed statistically. In study 2, nine healthy subjects were used as controls. Lymphocytes culture methods. (a) Leucocyte cultures (study 1). Peripheral blood was defibrinated by gentle stirring with sterile cherry sticks. The red cells were then sedimented by the addition of a one-third vol. of 3% gelatin in saline and incubated at 370C for 1 hr. Leucocytes were recovered from the leucocyte-rich serum-gelatin supernatant by centifugation at 600 g for 5 min. The cells were washed once in Eagle's medium and resuspended to 1 x 106 lymphocytes per ml in Eagle's medium supplemented with antibiotics and 20%. human serum-gelatin (obtained from a single large pool frozen in aliquots). Duplicate 1-ml cultures in sterile capped tubes were treated with 50 ug of PHA (Burroughs Wellcome) in 0 33 ml of phosphate-buffered saline or the same volume of phosphate-buffered saline without PHA in the case of the controls. The tubes were gassed with 5%4 CO2 in air cultured at 370C for 4, 5 or 6 days. Twenty-four hours before the end of the culture period 0-5 uCi of tritiated thymidine (150 mCi/mM) was added to each tube. Harvested cells were washed successively in salin 5% trichloracetic acid and methanol and then processed for scintillation counting as above. (b) Whole blood cultures (study 2). Blood (10 ml) was collected in heparin (preservative-free, 200 u). After removal of a sample for the white cell count and differential, 0-1 ml of the blood was added to 3 ml of RPMI-1640 medium (Biocult, Glasgow) supplemented with antibiotics and 5%4 foetal bovine serum. Cultures of each blood sample were set up in sextuplicate in bijou bottles. The bottles were sealed by an airtight cap and incubated, without gassing at 370C. Tritiated thymidine (6,uCi per culture, sp.act. 600 mCi/mM prepared from 3H-labelled methyl-thymidine obtained from the Radiochemical Centre, Amersham) was added 24 hr before harvesting. Two 1-ml samples were removed from three bijou bottles 24 hr after setting up the cultures and from the remaining three bottles at 72 hr. The tubes containing the 1 ml samples were treated with 1% acetic acid in saline (1 ml) and allowed to stand 30 min for the red cells to lyse. The cells were then spun down, washed in 5% trichloracetic acid (twice) and methanol (once), dissolved in 0-1 ml N NaOH and added to 1 ml of methanol and 10 ml of a triton-containing scintillation fluid for scintillation counting as previously described (Hardy, Knight & Ling, 1970). (c) Lymphocyte counts in peripheral blood. Films of blood were prepared and stained with Leishman's stain. From the total white count and differential, the numbers of lymphocytes were calculated.
RESULTS As can be seen from Fig. 2 and Table 2, the mean increments of response to PHA of lymphocytes from coeliac patients on days 4, 5 and 6 ofculture were lower than those ofthe control group but the differences TABLE 1. Mean tritiated thymidine incorporation in unstimulated leucocyte cultures
(study 1)
3H-labelled thymidine incorporation (d/min) Type and no. of cases Controls Coeliacs (ND)
28 14
Coeliacs (GFD)
21
Total coeliacs
35
Day 4
Day 5
Day 6
3171-6 1041-8 (P< 0 05) 1326-7
4540 5 1247-0 (P< 0-05) 1092-3 (P< 0-01) 1154-2 (p< o o01)
4726-2 1360-8 (P< 0-01) 1808-0 (P< 0-01) 1623-9 (p< o ool)
(P< 0-01) 1212-7
(p< o o01)
The P value shown refers to the difference of the mean 3H-labelled thymidine incorporation between control and coeliac lymphocytes. ND = Normal diet; GFD= gluten-free diet.
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Fia. 1 F iG. 2 FIG. 1. Thymidine incorporation of unstimulated lymphocytes cultured for 4, 5 or 6 days. Lymphocytes from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliacs (A). FIG. 2. (Study 1.) Thymidine incorporation of PHA-stimulated lymphocytes cultured for 4, 5 or 6 days. Lymphocytes from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliacs (A).
TABLE 2. Net tritiated thymidine incorporation (test minus control) in PHA-stimulated leucocyte cultures (study 1)
3H-labelled thymidine incorporation (d/min) Type and no. of cases
Controls Coeliacs (ND)
28 14
Coeliacs (GFD)
21
Total Cceliacs
35
Day 4
Day 5
Day 6
79204 50078
79322 56118 (P = n.s.) 62339 (P = n.s.) 59851 (P = n.s.)
70494 61958 (P = n.s.) 68676 (P = n.s.) 65910 (P = n.s.)
(P = n.s.) 71456
(P = n.s.) 62905
(P = n.s.)
The P value shown refers to the difference of the mean 3H-labelled thymidine incorporation between control and coeliac lymphocytes. n.s. = Not significant; ND = normal diet; GFD = gluten-free diet.
statistically significant. An incidental and unexpected finding in this study was that the 'background' thymidine incorporation in cultures not containing PHA was significantly lower than in the control group on all three days (see Table 1 and Fig. 1). The depression of response was significant both for coeliacs on a gluten-free diet and those on a normal diet but there were no significant differences between the two dietary groups. The second study was designed to find out whether the subnormal level of thymidine incorporation would also be found in samples of freshly-drawn, unfractionated blood. Accordingly whole blood cultures from coeliac patients, other clinical groups and controls were set up. The medium chosen was supplemented with foetal bovine instead of human serum since such a medium is known to be mildly stimulatory and to give progressively higher levels of background stimulation as the culture proceeds. Tritiated thymidine was added to some cultures immediately they were set up and to others after 2 days of culture. Only the results from the 0-24 hr interval are shown (Fig. 3 and Table 3) but the same trends were not
487
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Normals Coeioc Ulomtive C.rohn's do oolitis, dieas~e FIG. 3. (Study 2.) Thymidine incorporation by cells in whole blood over the 0-24-hr period. Cells from normal individuals (o); normal diet coeliacs (A); gluten-free diet coeliac (A); coeliac with lymphoma (L); ulcerative colitis treated medically, in remission (Z); in relapse (+); ulcerative colitis treated by panpractocolectomy (e); Crohn's disease, in remission (C); in relapse (a).
TABLE 3. Peripheral lymphocyte counts and relation to levels of DNA synthesis in whole blood cultures
Lymphocyte ct/mm3
3H-labelled thymidine incorporation (d/min)
Group and number
28 Cceliac (total) Coeliac (ND) 7 21 Coeliac (GFD) Crohn's disease 18 Ulcerative Colitis 10 Control 9
Mean
s.d.
Per cent of control
Mean
s.d.
Per cent of control
2024 1875 2077 2468 2320 2717
1047 502 1187 1039 1034 839
74 69 76 91 85 100
363 367 374 751 501 520
128 89 131 345 117 144
70 70 72 144 96 100
ND = Normal diet; GFD = gluten-free diet. were evident in the 48-72 hr cultures. The 3H-labelled thymidine incorporation of blood cells of the coeliac group was significantly lower than for the control group. The P values were
