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Spontaneous migration and chemotactic activity of neutrophil polymorphonuclear leukocytes in recurrent aphthous ulceration
Spontaneous migration and chemotactic activity of neutrophil polymorphonuclear leukocytes in recurrent aphthous ulceration P. Dagalis, D.D.S., J. Bagg, Ph.D., B.D.S., F.D.S.R.C.S., and D. M. Walker, M.D., F.D.S.R.C.S., M.R.C.Path., Cardif, Wales DEPARTMENT OF ORAL COLLEGE OF MEDICINE
MEDICINE
AND ORAL
PATHOLOGY,
DENTAL
SCHOOL,
UNIVERSITY
OF WALES
Chemotactic activity and spontaneous migration under agarose were assessed for peripheral blood neutrophil polymorphonuclear leukocytes from 22 patients with minor recurrent aphthous ulceration and 22 control subjects. The peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine was used as the standard chemotactic stimulus. Under the conditions of this study, no significant differences in either chemotaxis or spontaneous migration were evident between neutrophils from the patient and control groups. (ORAL SURG. ORAL MED. ORAL PATWOL. 1987;64:298-301)
T he cause of recurrent oral aphthous ulceration (RAU) remains unknown, but several workers have now detected circulating immune complexes in patients who have the disorder.le3 In addition, aphthous ulcers have histopathologic features suggestive of an immune complex-mediated vasculitis.4 Chemotactic split products of complement activated by immune complexes deposited in the oral mucosa may attract neutrophil polymorphonuclear leukocytes (PMNL), and these, in turn, may mediate the tissue damage in RAU, according to a recent hypothesis.s Enhanced chemotactic activity of PMNL has been demonstrated in Behcet’s syndrome (BS),6-8 which has the usual presenting feature of mouth ulceration and which may be regarded as part of an “aphthosis” spectrum.9 In our experience, colchicine, a strong inhibitor of PMNL chemotaxis,1° is effective in the treatment of BS6,8v1’ and also of major RAU, although this drug should be considered in only a few exceptional cases of RAU in view of the occasional adverse effects of colchicine. In the only previous study of PMNL chemotaxis in RAU, Abdulla and Lehnerlz investigated seven patients. Unlike other investigators,“8 they found that in BS and in patients with RAU, PMNL chemotaxis and random migration were normal. In the presence of autologous serum, these PMNL functions became depressed below those of controls.*2 298
In view of these contradictions, we considered it important to investigate PMNL chemotactic function in RAU in a larger series of patients. MATERIALS AND METHODS
Patients and control subjects. Twenty-two patients, each with minor recurrent aphthous ulceration as described by Lehner,” were studied. Twelve of the patients were women and ten were men, with ages ranging from 19 to 74 years and a mean age of 37.5 years. All of the patients were experiencing repeated crops of aphthous ulceration at the time of examination. Thirteen of the patients had active oral lesions at the time of venipuncture, and the other nine patients were studied between crops of ulcers. An age- and sex-matched control subject was simultaneously studied for each patient. Collection of blood and separation of PMNL fraction.
Venous blood (20 ml) was drawn from each participant, by standard venipuncture techniques, into heparinized tubes (Vacutainer Systems, Becton Dickinson, Rutherford, New Jersey). The PMNL fraction was separated by density gradient centrifugation over double-layer Ficoll-Hypaque columns,14 and the cells were washed twice in phosphatebuffered saline solution, followed by a final wash in
Volume 64 Number 3
Neutrophil
polymorphonuclear
leukocytes in recurrent aphthous ulceration
IO+M Aphrhous
Spontaneous migration (B) (umh-‘) (Mean F SD) Chemotaxis (A) (umh-I) (Mean + SD) Chemoiactic index (A/B) (Mean + SD)
322.9 (IL 169.4) 684.2 (k289.2) 2.7 (* 1.7)
299
10-M Conirol
Aphthous
354.6 (t- 179.73 683.1 (k313.4)
213.5 (k116.3) 605.6 (2 340.4)
( Z9)
,:;I,)
Control
246.2 (t- 119.0) 635.0 ( f 349.0) 2.7 (kO.8)
Dulbecco’s minimal essential medium. The PMNLs were then suspended in 10 ml of chemotaxis medium for counting and adjusted to a final concentration of lo* cells per milliliter. The chemotaxis medium comprised single-strength minimal essential medium, supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and antibiotics.
A), while spontaneous migration was assessed by movement toward the control medium (distance B). A chemotactic index, A/B,15 was derived from these values. Statistical analysis of results was performed according to the Student t test for paired samples.
Chemotaxis assay
Dose-response e#iect of FMLP on PM&L migration. Preliminary experiments revealed that a concentration of lo-‘M FMLP resulted in the greatest values for the chemotactic index. Thereafter, concentrations of 10m6M and 10-‘M FMLP were used routinely in all experiments. Chemotaxis (distance A) of PMNL from RAU suferers atid matched control subjects. At both concentrations of 10m6M and lo-‘M FMLP, there were no statistically significant differences between chemotaxis of PMNL from RAU patients and PMNL from control subjects (Table I) (p > 0.05). Spontaneous migration (distance B) of PMNL from RAU suflerers and matched control subjects. No statistically significant differences were evident between patient and control groups for this parameter (Table I). (p > 0.05). Chemofacti’c indices for PMNL from RAW suflerers and matched control subjects. Determination of the chemotactic index (A/B) revealed no statistically significant differences between RAU patients and control subjects (Table I) (p > 0.05).
Measurement of PMNL chemotaxis under agarose was performed as described by Nelson and associates.” Briefly, 7.5 ml of 2% 45°C agarose (electrophoresis grsde, supplied by Koch-Light, Ltd., Suffolk, Engiand), at a temperature of 48”C, was mixed with 7.35 ml of prewarmed double-strength MEM (supplemented with heat-inactivated fetal calf serum and antibiotics) and 150 ~1 of L-glutamine. Three-milliliter aliquots of this agarose medium were pipetted into individual sterile 35 X 10 mm polystyrene tissue culture dishes and allowed to set in a humidified 5% carbon dioxide atmosphere. Four series of three wells, 2.4 mm in diameter and spaced 2.4 mm apart, were cut into each plate, which had been prechilled to 4°C. An 8 ~1 aliquot of the PMNL suspension wsis placed in the central well of each series of three wells. The inner well of each triplet contained medium as a control, while each outer well was filled with the chemotactic factor N-formyl-L-methionyl-L-leucylL-phenylalanine (FMLP) at concentrations of 10m5M, 10m6M, 1O-‘M, or 10e8M, respectively. Each individual experiment was set up in quadruplicate. The dishes were incubated at 37°C in a humidified 5% carbon dioxide atmosphere for 2 hours. Each plate was then flooded with formalin and allowed to fix for 48 hours, after which it was stained with May-Griinwald-Giemsa stain. Cell migration was -..---rfC-_t -:r..Ao,n,;m 11~, hv mes(m nf an evtmiece
RiSULTS
DISCUSSION
There is now considerable evidence to support an autoimmune origin of recurrent oral aphthous ulceration (RAU).i6 Many workers have examined the possible role of lymphocytes in RAU,16 but, although the lvmnhocvte is histoloaicallv the initial cell’ in the
0m1surg, September 1981 of many cases of Behpt’s syndrome and, in our experience, major aphthous ulceration, at dose ranges over which the only pharmacologic effect is a depression of neutrophil polymorph chemotaxis. This strongly suggests an aberration of PMNL activity in the disorder. Furthermore, there is now ample evidence that PMNL, by exteriorization of free oxygen radicals, can cause tissue damage, particularly in autoimmune conditions.” It is, therefore, a cell worthy of study in RAU, especially in view of the enhanced chemotactic activity of PMNL in BS.6-8 None of the techniques used to measure chemotaxis in vitro are entirely without limitations, and it is important to compare data from different assay methods. Chemotaxis under agarosels was chosen because it is a rapid method, easy to quantitate, and in parallel studies gave measurements concordant with those based on membrane filter techniques,15 the latter having been used by previous workers studying PMNL chemotaxis in BS and RAU. We therefore feel confident in comparing our data with those from the earlier studies. For the chemotaxis assay we have used, previous workers18 have shown that the frequency distributions for both spontaneous migration and chemotaxis tend to be multimodal, and this was also true of our dat8. We have therefore derived the “chemotactic index” (A/B)15, I* which has a more normalized distribution and is of more value from a statistical standpoint. However, regardless of the method of presentation of the data, we have been unable to detect any significant differences in either random migration or chemotactic activity of peripheral PMNL from RAU patients, compared with control subjects. It would therefore appear that the enhanced chemotactic response of PMNL in BS6-* does not extend to patients with uncomplicated RAU. Abdulla and Lehner12 were unable to substantiate the findings of increased PMNL chemotaxis in BS. They found, conversely, that PMNL from patients with BS and, to a lesser extent, RAU showed a depressed response to the chemotactic stimulus of activated complement. I2 Their data did, however, reveal a significantly increased chemotactic activity in the sera of patients with BS when assayed against control PMNL, although sera from patients with RAU did not exhibit this enhanced activity.12 These results all appear, therefore, somewhat at variance. Abdulla and Lehner’* were unable to explain the reduced chemotaxis of PMNL in their BS patients as compared with the enhanced activity
minor, as opposed to major, RAU. The results for the equivalent subpopulation in the study by Abdulla and LehnerlZ indicate that those investigators also were unable to demonstrate any significant differences in chemotaxis or spontaneous migration of PMNL from patients and controls when assayed in the presence of control serum. Although we used a different chemotactic stimulus from Abdulla and Lehner’* and did not examine the effect of serum, our results, from a large population, would concur with their finding that inherent random migration and chemotactic activity of PMNL from patients with minor RAU are not siginficantly different from those of controls. Although no abnormality in PMNL function emerged in the present in vitro experiments, these leukocytes may still play a part in the pathogenesis of RAU. Locally deposited immune complexes in the oral mucosa could initiate the lesions by attracting normal PMNL with their capacity for releasing tissue-degrading enzymes, as described earlier. We are grateful to Dr. N. Matthews and Mrs. L. Neale, Department of Medical Microbiology, University of Wales College of Medicine, for advice regarding the chemotaxis assay and to Mrs. K.J. Davies for expert technical assistance. REFERENCES
1. Williams BD, Lehner T. Immune complexes in Behcet’s syndrome and recurrent oral ulceration. Br Med J 1977; 1:1387-9. 2. Levinsky RJ, Lehner T. Circulating soluble immune complexes in recurrent oral ulceration and Behcet’s syndrome. Clin Exp Immunol 1978;32: 193-8. 3. Gupta RC, O’Duffy JD, McDuffie FC, Meurer M, Jordon RE. Circulating immune complexes in active Behcet’s disease. Clin Exp Immunol 1978;34:213-8. 4. Lehner T. Pathology of recurrent oral ulceration and oral ulceration in Behcet’s syndrome: light, electron and fluorescence microscopy. J Path01 1969;97:48 l-94. 5. Lehner T: Immuno-pathology of Behcet’s syndrome. In: Lehner T, Barnes CG, eds. Behcet’s syndrome: Clinical and immunological features, London: Academic Press, 1979:12739. 6. Matsumara N, Mizushima Y. Leucocyte movement and colchicine treatment in Behcet’s disease. Lancet 1975;2:8 13. 7. Sobel JD. Haim S. Obedeanu T. Meshulam T. Merzbach D. Polymorihonuclea; leucocyte fuhction in Beh&t’s disease. J Clin Path01 1977;30:250-3. 8. Baum J. The hyperchemotaxis of Behcet’s disease and the effect of colchicine. J Rheumatol 198 1;8:17 1-2. 9. Touraine A. L’aphtose. Bull Sot Franc Dermat Syph 1941; 48:61-104. 10. Gallin JI, Gallin EK, Malech HL, Cramer EB. Structural and ionic events during leukocyte chemotaxis. In: Gallin JI, Quie PC, eds. Leukocyte chemotaxis; methods, physiology and clinical implications. New York: Raven Press, 1978: 123-41. 11. Miyachi Y, Taniguchi S, Ozaki M, Horio T. Colchicine in the treatment of the cutaneous manifestations of Behcet’s disease.
Volume 64 Number 3
13. 14.
15.
16. 17.
Neutrophil
polymorphonuclear
In: Lehner T, Barnes CG, eds. Behcet’s syndrome; clinical and immunological features. London: Academic Press, 1979: 55-66. Lehner T. Autoimmunity in oral diseases with special reference to recurrent oral ulceration. J R Sot Med 1968;61:5 15 24. Ferrante A, Thong YH. A rapid one-step procedure for purification of mononuclear and polymorphonuclear leucocytes from human blood using a modification of the hypaqueficoll technique. J Immunol Meth 1978;24:389-93. Nelson RD, Quie PG, Simmons RL. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 1975;115: 1650-6. Lehner T. Immunological aspects of recurrent oral ulceration and Behcet’s syndrome. J Oral Path01 1978;7:424-30. Fantone JC, Ward PA. Mechanisms of neutrophil-dependent
leukocytes in recurrent aphthous ulceration
301
lung injury. In: Ward PA, ed. Handbook of inflammation. Vol. 4. Immunology of inflammation. Amsterdam: Elsevier Biomedical Press, 1983:89- 119. 18. Nelson RD, McCormack RT, Fiegel VD: Chemotaxis of human leucocytes under agarose. In: Gallin JI, Quie PG, eds. Leukocyte chemotaxis: methods, physiology and clinical implications. New York, Raven Press, 1978:25-40. Reprint
requests
to:
Dr. J. Bagg Department of Oral Medicine and Oral Pathology Dental School University of Wales College of Medicine Heath Park Cardiff CF4 4XY, Wales
MEDICINE
AND ORAL
PATHOLOGY,
DENTAL
SCHOOL,
UNIVERSITY
OF WALES
Chemotactic activity and spontaneous migration under agarose were assessed for peripheral blood neutrophil polymorphonuclear leukocytes from 22 patients with minor recurrent aphthous ulceration and 22 control subjects. The peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine was used as the standard chemotactic stimulus. Under the conditions of this study, no significant differences in either chemotaxis or spontaneous migration were evident between neutrophils from the patient and control groups. (ORAL SURG. ORAL MED. ORAL PATWOL. 1987;64:298-301)
T he cause of recurrent oral aphthous ulceration (RAU) remains unknown, but several workers have now detected circulating immune complexes in patients who have the disorder.le3 In addition, aphthous ulcers have histopathologic features suggestive of an immune complex-mediated vasculitis.4 Chemotactic split products of complement activated by immune complexes deposited in the oral mucosa may attract neutrophil polymorphonuclear leukocytes (PMNL), and these, in turn, may mediate the tissue damage in RAU, according to a recent hypothesis.s Enhanced chemotactic activity of PMNL has been demonstrated in Behcet’s syndrome (BS),6-8 which has the usual presenting feature of mouth ulceration and which may be regarded as part of an “aphthosis” spectrum.9 In our experience, colchicine, a strong inhibitor of PMNL chemotaxis,1° is effective in the treatment of BS6,8v1’ and also of major RAU, although this drug should be considered in only a few exceptional cases of RAU in view of the occasional adverse effects of colchicine. In the only previous study of PMNL chemotaxis in RAU, Abdulla and Lehnerlz investigated seven patients. Unlike other investigators,“8 they found that in BS and in patients with RAU, PMNL chemotaxis and random migration were normal. In the presence of autologous serum, these PMNL functions became depressed below those of controls.*2 298
In view of these contradictions, we considered it important to investigate PMNL chemotactic function in RAU in a larger series of patients. MATERIALS AND METHODS
Patients and control subjects. Twenty-two patients, each with minor recurrent aphthous ulceration as described by Lehner,” were studied. Twelve of the patients were women and ten were men, with ages ranging from 19 to 74 years and a mean age of 37.5 years. All of the patients were experiencing repeated crops of aphthous ulceration at the time of examination. Thirteen of the patients had active oral lesions at the time of venipuncture, and the other nine patients were studied between crops of ulcers. An age- and sex-matched control subject was simultaneously studied for each patient. Collection of blood and separation of PMNL fraction.
Venous blood (20 ml) was drawn from each participant, by standard venipuncture techniques, into heparinized tubes (Vacutainer Systems, Becton Dickinson, Rutherford, New Jersey). The PMNL fraction was separated by density gradient centrifugation over double-layer Ficoll-Hypaque columns,14 and the cells were washed twice in phosphatebuffered saline solution, followed by a final wash in
Volume 64 Number 3
Neutrophil
polymorphonuclear
leukocytes in recurrent aphthous ulceration
IO+M Aphrhous
Spontaneous migration (B) (umh-‘) (Mean F SD) Chemotaxis (A) (umh-I) (Mean + SD) Chemoiactic index (A/B) (Mean + SD)
322.9 (IL 169.4) 684.2 (k289.2) 2.7 (* 1.7)
299
10-M Conirol
Aphthous
354.6 (t- 179.73 683.1 (k313.4)
213.5 (k116.3) 605.6 (2 340.4)
( Z9)
,:;I,)
Control
246.2 (t- 119.0) 635.0 ( f 349.0) 2.7 (kO.8)
Dulbecco’s minimal essential medium. The PMNLs were then suspended in 10 ml of chemotaxis medium for counting and adjusted to a final concentration of lo* cells per milliliter. The chemotaxis medium comprised single-strength minimal essential medium, supplemented with 10% heat-inactivated fetal calf serum, L-glutamine, and antibiotics.
A), while spontaneous migration was assessed by movement toward the control medium (distance B). A chemotactic index, A/B,15 was derived from these values. Statistical analysis of results was performed according to the Student t test for paired samples.
Chemotaxis assay
Dose-response e#iect of FMLP on PM&L migration. Preliminary experiments revealed that a concentration of lo-‘M FMLP resulted in the greatest values for the chemotactic index. Thereafter, concentrations of 10m6M and 10-‘M FMLP were used routinely in all experiments. Chemotaxis (distance A) of PMNL from RAU suferers atid matched control subjects. At both concentrations of 10m6M and lo-‘M FMLP, there were no statistically significant differences between chemotaxis of PMNL from RAU patients and PMNL from control subjects (Table I) (p > 0.05). Spontaneous migration (distance B) of PMNL from RAU suflerers and matched control subjects. No statistically significant differences were evident between patient and control groups for this parameter (Table I). (p > 0.05). Chemofacti’c indices for PMNL from RAW suflerers and matched control subjects. Determination of the chemotactic index (A/B) revealed no statistically significant differences between RAU patients and control subjects (Table I) (p > 0.05).
Measurement of PMNL chemotaxis under agarose was performed as described by Nelson and associates.” Briefly, 7.5 ml of 2% 45°C agarose (electrophoresis grsde, supplied by Koch-Light, Ltd., Suffolk, Engiand), at a temperature of 48”C, was mixed with 7.35 ml of prewarmed double-strength MEM (supplemented with heat-inactivated fetal calf serum and antibiotics) and 150 ~1 of L-glutamine. Three-milliliter aliquots of this agarose medium were pipetted into individual sterile 35 X 10 mm polystyrene tissue culture dishes and allowed to set in a humidified 5% carbon dioxide atmosphere. Four series of three wells, 2.4 mm in diameter and spaced 2.4 mm apart, were cut into each plate, which had been prechilled to 4°C. An 8 ~1 aliquot of the PMNL suspension wsis placed in the central well of each series of three wells. The inner well of each triplet contained medium as a control, while each outer well was filled with the chemotactic factor N-formyl-L-methionyl-L-leucylL-phenylalanine (FMLP) at concentrations of 10m5M, 10m6M, 1O-‘M, or 10e8M, respectively. Each individual experiment was set up in quadruplicate. The dishes were incubated at 37°C in a humidified 5% carbon dioxide atmosphere for 2 hours. Each plate was then flooded with formalin and allowed to fix for 48 hours, after which it was stained with May-Griinwald-Giemsa stain. Cell migration was -..---rfC-_t -:r..Ao,n,;m 11~, hv mes(m nf an evtmiece
RiSULTS
DISCUSSION
There is now considerable evidence to support an autoimmune origin of recurrent oral aphthous ulceration (RAU).i6 Many workers have examined the possible role of lymphocytes in RAU,16 but, although the lvmnhocvte is histoloaicallv the initial cell’ in the
0m1surg, September 1981 of many cases of Behpt’s syndrome and, in our experience, major aphthous ulceration, at dose ranges over which the only pharmacologic effect is a depression of neutrophil polymorph chemotaxis. This strongly suggests an aberration of PMNL activity in the disorder. Furthermore, there is now ample evidence that PMNL, by exteriorization of free oxygen radicals, can cause tissue damage, particularly in autoimmune conditions.” It is, therefore, a cell worthy of study in RAU, especially in view of the enhanced chemotactic activity of PMNL in BS.6-8 None of the techniques used to measure chemotaxis in vitro are entirely without limitations, and it is important to compare data from different assay methods. Chemotaxis under agarosels was chosen because it is a rapid method, easy to quantitate, and in parallel studies gave measurements concordant with those based on membrane filter techniques,15 the latter having been used by previous workers studying PMNL chemotaxis in BS and RAU. We therefore feel confident in comparing our data with those from the earlier studies. For the chemotaxis assay we have used, previous workers18 have shown that the frequency distributions for both spontaneous migration and chemotaxis tend to be multimodal, and this was also true of our dat8. We have therefore derived the “chemotactic index” (A/B)15, I* which has a more normalized distribution and is of more value from a statistical standpoint. However, regardless of the method of presentation of the data, we have been unable to detect any significant differences in either random migration or chemotactic activity of peripheral PMNL from RAU patients, compared with control subjects. It would therefore appear that the enhanced chemotactic response of PMNL in BS6-* does not extend to patients with uncomplicated RAU. Abdulla and Lehner12 were unable to substantiate the findings of increased PMNL chemotaxis in BS. They found, conversely, that PMNL from patients with BS and, to a lesser extent, RAU showed a depressed response to the chemotactic stimulus of activated complement. I2 Their data did, however, reveal a significantly increased chemotactic activity in the sera of patients with BS when assayed against control PMNL, although sera from patients with RAU did not exhibit this enhanced activity.12 These results all appear, therefore, somewhat at variance. Abdulla and Lehner’* were unable to explain the reduced chemotaxis of PMNL in their BS patients as compared with the enhanced activity
minor, as opposed to major, RAU. The results for the equivalent subpopulation in the study by Abdulla and LehnerlZ indicate that those investigators also were unable to demonstrate any significant differences in chemotaxis or spontaneous migration of PMNL from patients and controls when assayed in the presence of control serum. Although we used a different chemotactic stimulus from Abdulla and Lehner’* and did not examine the effect of serum, our results, from a large population, would concur with their finding that inherent random migration and chemotactic activity of PMNL from patients with minor RAU are not siginficantly different from those of controls. Although no abnormality in PMNL function emerged in the present in vitro experiments, these leukocytes may still play a part in the pathogenesis of RAU. Locally deposited immune complexes in the oral mucosa could initiate the lesions by attracting normal PMNL with their capacity for releasing tissue-degrading enzymes, as described earlier. We are grateful to Dr. N. Matthews and Mrs. L. Neale, Department of Medical Microbiology, University of Wales College of Medicine, for advice regarding the chemotaxis assay and to Mrs. K.J. Davies for expert technical assistance. REFERENCES
1. Williams BD, Lehner T. Immune complexes in Behcet’s syndrome and recurrent oral ulceration. Br Med J 1977; 1:1387-9. 2. Levinsky RJ, Lehner T. Circulating soluble immune complexes in recurrent oral ulceration and Behcet’s syndrome. Clin Exp Immunol 1978;32: 193-8. 3. Gupta RC, O’Duffy JD, McDuffie FC, Meurer M, Jordon RE. Circulating immune complexes in active Behcet’s disease. Clin Exp Immunol 1978;34:213-8. 4. Lehner T. Pathology of recurrent oral ulceration and oral ulceration in Behcet’s syndrome: light, electron and fluorescence microscopy. J Path01 1969;97:48 l-94. 5. Lehner T: Immuno-pathology of Behcet’s syndrome. In: Lehner T, Barnes CG, eds. Behcet’s syndrome: Clinical and immunological features, London: Academic Press, 1979:12739. 6. Matsumara N, Mizushima Y. Leucocyte movement and colchicine treatment in Behcet’s disease. Lancet 1975;2:8 13. 7. Sobel JD. Haim S. Obedeanu T. Meshulam T. Merzbach D. Polymorihonuclea; leucocyte fuhction in Beh&t’s disease. J Clin Path01 1977;30:250-3. 8. Baum J. The hyperchemotaxis of Behcet’s disease and the effect of colchicine. J Rheumatol 198 1;8:17 1-2. 9. Touraine A. L’aphtose. Bull Sot Franc Dermat Syph 1941; 48:61-104. 10. Gallin JI, Gallin EK, Malech HL, Cramer EB. Structural and ionic events during leukocyte chemotaxis. In: Gallin JI, Quie PC, eds. Leukocyte chemotaxis; methods, physiology and clinical implications. New York: Raven Press, 1978: 123-41. 11. Miyachi Y, Taniguchi S, Ozaki M, Horio T. Colchicine in the treatment of the cutaneous manifestations of Behcet’s disease.
Volume 64 Number 3
13. 14.
15.
16. 17.
Neutrophil
polymorphonuclear
In: Lehner T, Barnes CG, eds. Behcet’s syndrome; clinical and immunological features. London: Academic Press, 1979: 55-66. Lehner T. Autoimmunity in oral diseases with special reference to recurrent oral ulceration. J R Sot Med 1968;61:5 15 24. Ferrante A, Thong YH. A rapid one-step procedure for purification of mononuclear and polymorphonuclear leucocytes from human blood using a modification of the hypaqueficoll technique. J Immunol Meth 1978;24:389-93. Nelson RD, Quie PG, Simmons RL. Chemotaxis under agarose: a new and simple method for measuring chemotaxis and spontaneous migration of human polymorphonuclear leukocytes and monocytes. J Immunol 1975;115: 1650-6. Lehner T. Immunological aspects of recurrent oral ulceration and Behcet’s syndrome. J Oral Path01 1978;7:424-30. Fantone JC, Ward PA. Mechanisms of neutrophil-dependent
leukocytes in recurrent aphthous ulceration
301
lung injury. In: Ward PA, ed. Handbook of inflammation. Vol. 4. Immunology of inflammation. Amsterdam: Elsevier Biomedical Press, 1983:89- 119. 18. Nelson RD, McCormack RT, Fiegel VD: Chemotaxis of human leucocytes under agarose. In: Gallin JI, Quie PG, eds. Leukocyte chemotaxis: methods, physiology and clinical implications. New York, Raven Press, 1978:25-40. Reprint
requests
to:
Dr. J. Bagg Department of Oral Medicine and Oral Pathology Dental School University of Wales College of Medicine Heath Park Cardiff CF4 4XY, Wales