Aust. J. Derm. (1978) 19, 45
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY* I. SARKANY AND P . M . GAYLARDE
London, '•.
'
U.K.
SUMMARY
The role of lymphocyte transformation in the diagnosis of drug allergy is reviewed. Of 165 patients with clinically suspected drug allergy, 22 (13.3%) gave a positive lymphocyte transformation result. Reasons for this low incidence of positive reactions are put forward. Lymphocyte transformation testing in patients with halothane related hepatitis , was uniformly negative in 29 patients and this test has not been found useful in the diagnosis of this condition as against viral hepatitis. We have also found that a number of drugs including co-trimoxazole, piperazine, chlorpromazine, promethazine and griseofulvin inhibit DNA synthesis in lymphocytes in vitro. This may be the result of inhibition of transformation of lymphocytes, a cytotoxic effect or the effect of inhibition of thymidine incorporation. The in vitro suppression of DNA synthesis in lymphocytes by co-trimoxazole, rifampicin and miconazole has been suggested as being related to their possible in vivo immunosuppressive activity. The incidence of side efEects to drugs is variously given as between 2-2Q%.^'--''^ This great discrepancy is the result of differences in criteria for labelling unwanted effects of drugs. Subjective symptoms, such as nausea, dizziness, headaches, insomnia, constipation, sweating, tension etc. are difficult to assess and are frequent even in patients receiving placebo treatment. Objective features, especially those associated with skin abnormalities or liver function changes are less common and more likely clinically reliable. In a study of 5,410 in-patients, Lloyd' found an incidence of 2 per cent side effects, mainly referable to the skin, liver and haemopoietic system. Of these, only a proportion was based on allergic mechanisms and there is clearly a need for objective laboratory conffrmation in these cases. The alternative of re-administration of the drug may not be safe, feasible or acceptable to the patient. Laboratory tests for allergic sensitivity * Presented by I. Sarkany at the Annual Meeting of the Australasian College of Dermatologists Brisbane. May, 1978. Address for reprints: Dr. I. Sarkany, Consultant Dermatologist, Royal Free Hospital, London, N.W.3, England.
to systemically administered drugs are therefore needed. We have used lymphocyte transformation testing for some years^' ° and this is a review of our results. The methods of lymphocyte culture are now widely used and have become fairly standardized. In brief, lymphocytes from heparinized blood are incubated in the presence of the putative antigen for six days. Transformation of the cells is measured by examining smears microscopically and by the measurement of uptake of labelled thymidine by the transformed cells. The results are compared with those obtained from controls not exposed to the antigen. RESULTS
Of 165 patients with clinically suspected drug allergy, 22 patients gave a positive result to the lymphocyte transformation test (13.3%). The details of drugs tested, the drug concentrations used in the test and the results are given in Tables 1 and 2. Several of the drugs tested in vitro caused suppression of thymidine uptake at concentrations known to be safely tolerated in the peripheral blood under therapeutic conditions (Table 3).
I. SARKANY AND P , M . GAYLARDE
46
TABLE 2—contd.
TABLE 1
Positive Lymphocyte Transformation Test Number of Patients
Drugs Allopurinol Ampicilfin Carbamazepine Chlorpropamide Chlorpromazine Oxyphenisatin Oxytetracycline Para-aminosalicylie acid Penicillin Piperazine Rifampicin Streptomycin Succinyl sulphathiazole Sulphadiazine Sulphadimidine Thiobendazole Trimethoprim-sulphamethoxazole
Total :
1
1 1 1 1 1 1 2 4 1 1 2 1 1 2 1 2
24 drugs in 22 patients
TABLE 2
Drug Concentrations Used in Culture and Results
Drug
Positive Cases
Allopurinol Amethocaine Amphotericin Ampicillin Benzocaine Bupivacaine Carbamazepine Carbroma] Chlorothiazide Chlorpromazine Chlorpropamide Cinehoeaine Codeine Demethylchlortetracycline Digoxin Dothiepin Erythromycin Gelfoam Gold aurothiomalate Griseofulvin Halothane Hygroton Hydrochlorolhiazide Isoniazid Ketoprofen Lignocaine Meprobamate Mersalyl Metformin Methyldopa Nalidixie acid
1 1
Negative Cases 1 1 I 7 1 1
1
V ^ L 4 J V,' ± vV 1 1 1 L 4 1
1 1
3 1 5 1 1
2 1 1 2 1 1
2
29 1 1 10 1 1 1 1 1 1 1
Concentration per ml 10 Mg 15 Mg l,12,5Mg 50, 25 Mg 15Mg 15 Mg 10 Mg 40, 4, 0,4, 0,04 Mg 100, 10, I Mg 150,45, 15, 5,0,5 Mg 1,0,1,0.01 mg 15 Mg 0,5 Mg 75, 15,7,5Mg 0,025 Mg 10 Mg 10 Mg solid material 0.5 Mg 12,5, 1 Mg 100, 50, 25, 10, 5 Mg 10 Mg 1.2 Mg 10 Mg 5 Mg 5 Mg 7,5 Mg 50, 5 Mg 50 Mg 12,5 Mg 5Mg
Drug
Positive 1Cases
Neomercazole Neomycin Nitrofurantoin Oxyphenisatin Oxyphenbutazone Oxytetracycline Para-aminosalicyclic acid Paracetamol Penicillin Perchlorethylene Phenobarbitone Phenylbutazone Phenytoin Piperazine Prilocaine Primidone Promethazine hydrochloride Promethazine theoclate Propranolol Quinidine Quinine Rifampicin Salazopyrine Streptomycin Succinyl sulphathiazole Sulphadiazine Sulphadimidine Sulphafurazole Sulphapyridine Tetracycline Thiobendazole Thiopentone sodium Tolazamide Tolbulamide Trifluoperazine Trimethoprimsulphamethoxazole
1
Negative Cases 2 1 3 2 2
1 2 4
7 2 16 1 2 3 1 1 4
5, 1,0.25, 0,1 Mg
1 2 1 1
6.25 Mg lOMg 3, 1.5 Mg 0.8 Mg 15Mg 12 Mg 100, 50Mg
2
1
1 1 2
1 12 8 3 5 1 1 1
1 1 1 1 1 2
0,5 Mg 10, 1 Mg 50 Mg 12,5, 2 Mg 12,5, 10 Mg 25 Mg 750, 75, 7,5 Mg 50 Mg 250, 100 units 50, lOmMl 0,8, 0,08 Mg 12,5 Mg 25,5Mg -^ ._•: 0,25 Mg 15 Mg 40, 0,4 Mg
1
2
Concentration per ml
12
^
:-
2 5 0 , 1 2 5 Mg 2 5 0 , 1 2 5 Mg 2 5 0 , 1 2 5 Mg lOOMg 10 Mg 5Mg 15Mg 10, 1,0,1 Mg 250, 25 Mg 12,5 Mg 0,5 Mg 50, 1 0 , 5 , iMg
DISCUSSION
We have now had experience of the use of lymphocyte transformation and its role in the diagnosis of drug allergy for the past 13 years. Of 165 patients tested, 22 were found to give a positive response in culture. This represents 13,3% of all patients studied. The work has involved testing of 67 drugs or related substances, of which 17 preparations yielded a positive reaction. The most common sensitivity reactions confirmed in this way were those due to sulphonamides and antibiotics. At first sight, the low percentage of positive findings would suggest that the test is of little value in confirmation of drug allergy. However, initially we included in the test series a number of
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY TABLE 3
Drags Causing in vitro Toxicity
Drug
Minimal Concentratioi at which Toxic Effect Observed {iJ-glmt)
Chlorpromazine
1
Co-trimoxazole
1.5
Griseofulvin
1
Neomercazole
1
Nitrofiirantoin
2.5
Oxyphenisatin
2
Quinidine Penieillin
1.5 125
Phenylbutazone
10
Phenytoin
5
Piperazine
2.5
Promethazine Sulphapyridine
125 10
Effect and Comments Inhibition of thymidine uptake. Inhibition of thymidine uptake; components act synergistically. Inhibition of thymidine uptake. Inhibition of thymidine uptake. Haemolysis, also inhibition of thymidine uptake at 5 ."g/ml Inhibition of thymidine uptake. Cell necrosis. Inhibition of uptake in patient with agranulocytosis. Inhibition not observed up to 1600/ig/ml in 8 normal subjects. Haemolysis. Suppression of thymidine uptake 1 subject only. Inhibition of thymidine uptake. Inhibition of thymidine uptake. Cell necrosis. Haemolysis.
conditions not necessarily based on delayed type of sensitivity reactions, such as fixed drug eruptions, urticaria, purpura etc. All of these were negative and swelled the number of negative findings. Similarly, we found a negative response to halothane in patients with halothane induced jaundice in 29 cases." Even discounting this largely expected negative group of conditions, we failed to define with any degree of likelihood the clinical patterns of cases of suspected drug reactions which gave a positive lymphocyte transformation test result. Perhaps a more promising indication of the value of this test is shown by the fact that of the last 28 patients tested, eight have given a positive result (29% ).
47
The test picks up a certain, if small, proportion of cases where it is of clinical value. It seems, therefore, that there is a fairly large and as yet ill-defined number of mechanisms responsible for drug allergy, possibly based on humoral and cellular enzymatic abnormalities in addition to the various known types of allergic mechanisms, as defined by Gell. The lymphocyte transformation reaction is valuable in those based on cell mediated allergy only. The clinical conditions in which a positive lymphocyte transformation test was obtained were various forms of cutaneous eruptions, including exfoliative dermatitis, epidermal necrolysis, light sensitivity reactions, fever, hepatitis and neurological conditions, such as peripheral neuropathy and encephalopathy. Specific areas where disturbance of immunity has been implicated, were separately evaluated in greater detail. A summary of our experiences is presented. HALOTHANE-RELATED HEPATITIS
Paronetto and Popper^ described stimulation of lymphocyte transformation in 10 out of 15 patients with suspected halothane hepatitis. We" have failed to confirm this finding in a study of 17 patients suspected of halothane sensitivity and in a subsequent series of 12 cases the results were also negative. Similar negative conclusions in this condition were independently reported by Walton et ab'*- « and Mathieu et al." We observed and reported that halothane at dose levels of 50 ^.g per ml and above caused suppression of thymidine incorporation in PHA stimulated culture in both normal and halothane hepatitis sensitivity suspected cases. Such suppression has also been noted by Bruce" in vitro and Viljanen.'- Hepatotoxicity associated with halothane anaesthesia following administration of a polychlorinated biphenyl (PCB) to rats was reported by Sipes and Brown^-^ who failed to produce liver necrosis in these animals with either of these agents alone. The administration of PCB caused an increase in cytochrome P-450 in these animals and indicated an induction of drug metabolizing enzymes. Similarly, drugs causing an increase in liver cytochrome P-450 levels are known to increase the biotransformation of carbon tetrachloride in both man and rats and to decrease the threshold dose causing hepatotoxicity in rats.^* CO-TRIMOXAZOLE
In both patients (Table 1 and 2) with a positive reaction to co-trimoxazole, lymphocyte
48
I. SARKANY AND P, M . GAYLARDE
transformation was positive to both trimethoprim and sulphamethoxazole. This double sensitivity reaction in the positive cases is intriguing and raises the question of an adjuvant effect of the drugs towards each other. We have also demonstrated^" that cotrimoxazolc caused suppression of -^H thymidine uptake in PHA stimulated cultures in 15 of 25 normal subjects at a dose level of 1 fig per ml trimethoprim and 5 |Ug per ml sulphamethoxazole. In the 15 individuals, the average suppression was 84% and represented a marked suppressive effect. This phenomenon was due to a synergistic suppressive effect of the two drug components, and was not inhibited by folic acid. The distribution of the degree of suppression was found to be separated into two discrete groups and suggested the possibility of a genetic factor. Co-trimoxazole also inhibits thymidine uptake in unstimulated cultures and appears to act directly on DNA synthesis and not by inhibition of lymphocyte transformation. PIPERAZINE
lymphocytes (125 /xg/ml) but does not suppress thymidine uptake at 5 /xg/ml. Ferguson et aV'- *' * reported that rat thymocyte transformation in response to concanavalin-A is inhibited by 5 x 10""M chlorpromazine and Trowell'" noted that promethazine was toxic to rat lymph node lymphocytes in vitro at 10 x 10-''M, These figures show that rat lymphocytes are sensitive to phenothiazines at concentrations about an order of magnitude less than those causing inhibition of human peripheral lymphocytes. GRISEOFULVIN
Griseofulvin produced very marked reduction of ^H thymidine in vitro at concentrations of 1, 10 and 100 /xg per ml. In the rat, 100 mg per kg intravenously or 2 g per kg intraperitoneally, caused arrest of mitoses in metaphase in certain tissues,-" In vitro, 20 ftg per ml inhibited growth of epithelial cells.^i' -2 DRUG TOXICITY IN VITRO
We have shown that a number of drugs Piperazine has been widely used as an antisuppress thymidine uptake by lymphocytes in helminthic since its first description in the treatment of roundworm and threadworm in- vitro at dose levels apparently well tolerated festation. Adverse side effects have been in vivo. This effect was first noted by Lajtha-^ reported but hepatotoxicity had not been who observed that barbiturates were toxic to previously noted. We described a patient with human blood and bone marrow cells at levels piperazine hepatitis with a positive lymphocyte less than those normally tolerated in vivo. transformation reaction,^" A second patient Diphenylhydantoin and cortisone have also with a similar history gave a negative result. been recognized as toxic against lymphocytes Piperazine suppressed thymidine uptake in vitro in vitro at very low dose levels. In this context, at a level of 25 /xg per ml in five normal subjects it is worth noting that lymphocytes are much and at 2,5 /xg per ml in four other control more sensitive to ionizing radiation in vitro subjects tested. The positive lymphocyte trans- than they appear to be in vivo. In a recent report,-* the death certificates of formation result was obtained at a concentration of 0.25 (Ug per ml. Suppression of all 17 patients who died of agranulocytosis in thymidine uptake in this patient was observed England and Wales from October 1974 to October 1975 were examined and 13 of these at 2.5 and 25 /j.g per ml. deaths were attributed to co-trimoxazole. In CHLORPROMAZINE AND PROMETHAZINE the Committee on Safety of Medicines Report, Suppression of thymidine uptake was ob- 1977,-''' the four most common drugs reserved at concentrations of 50 and 100 fxg per sponsible for granulocytopenia and agranuml with chlorpromazine. PHA stimulated locytosis were phenylbutazone, oxyphenlymphocyte cultures from five normal vol- butazone, chlorpromazine and co-trimoxazole. unteers showed suppression of ''H thymidine Of these, chlorpromazine and co-trimoxazole incorporation of between 82 and 99,3% when were found to be associated with in vitro toxic exposed to chlorpromazine at a concentration effects against lymphocytes causing suppression of 50 /ig/ml. Chlorpromazine 5 /xg/ml caused of ^H thymidine uptake in vitro. A number of no significant suppression in a single normal non-steroid anti-inflammatory agents including subject tested, but suppression has been noted aspirin, indomethacin and phenylbutazone have at 1 /tg/ml in a patient with suspected hyper- been shown to inhibit lymphocyte transsensitivity, Promethazine, another drug based formation induced by PHA and to inhibit hi^"^" on phenothiazine, is also toxic to human protein
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY
Immunosuppressive agents also inhibit lymphocyte transformation-' and for this reason the inhibition of PHA induced lymphocyte transformation by rifampicin'^' and miconazole^-'' ''^ has been presented as further evidence that these drugs have immunosuppressive properties. In view of the inhibition of transformation of lymphocytes by chlorpromazine,'''' local anaesthetics'^'' and anti-inflammatory drugs, additional evidence is required to confirm their immunosuppressive activity. Phenylbutazone causes marked suppression of PHA induced thymidine uptake at a level of 5 /ig/ml, but our own findings show that no suppression occurs up to 12.5 /ig/ml in unstimulated cultures, although haemolysis is usually observed. In lymphocyte cultures, even in the absence of lymphoblastic transformation, there is a low level of thymidine uptake by the cells, representing both DNA synthesis and repair. This is usually of a low level and statistically significant suppression of thymidine uptake is not demonstrable, unless it is of the order of 20 to 3 0 % . Transformation of lymphocytes into blast cells can be inhibited by a number of substances including non-steroid antiinfiammatory agents. This suppression of transformation leads to a decrease in thymidine uptake by the cells, although there is no evidence of direct interference with DNA synthesis. This implies that chlorpromazine and co-trimoxazole inhibit DNA synthesis by means other than interference with lymphoblastic transformation. The inhibition of DNA synthesis is accompanied by cytotoxic eifects on the cells in the case of cfilorpromazine, but it appears likely that co-trimoxazole inhibits DNA synthesis directly. ACKNOWLEDGEMENTS
We are grateful to the Herbert E. Dunhill Trust for generous support. REFERENCES
1 Lloyd, M. (1969): "A Drug Reaction Survey", Trans. St. Jotin's Hosp. Derm. Soc, 55 : 64. 2 Cluff, L. E.. Thornton, G. F., and Seidl, L. G. (1964): "Studies on the Epidemiology of Adverse Drug Reactions", J. Amer. Med. Assoc. 188 : 976. •'' Schimmel, E. M. (1964) : "Hazards of Hospitalization", Annats of Int. Med., 60 : 100. i Caron, G. A., and Sarkany, I. (1965) : "Lymphoblast Transformation in Sulphonamide Sensitivity", Brit. J. Derm., 11 : 556. •"' Sarkany, I. (1968): "Clinical and Laboratory Aspects of Drug Allergy", Proc. Roy. Soc. Med., 61 : 891.
49
' Moult. P. T. A.. Adjukiewicz, A. B.. Gaylarde, P. M., Sarkany, L, and Sherlock, S. (1975): "Lymphocyte Transformation in Halothane Related Hepatitis", Brit. Med. J., ii : 69. Paronetto, F., and Popper, H. (1970): "Lymphocyte Stimulation Induced by Halothane in Patients with Hepatitis Following Exposure to Halothane", New Engt. J. Med., 283 : 277. ' Walton, B., Dumonde, D. C , Williams, C . Jones, D., Strunin, J. M., Layton, J. M., Strunin, L., and Simpson, R. (l973) : "Lymphocyte Transformation. Absence of Increased Responses in Alleged Halothane Jaundice", J. Amer. Med. Assoc, 225 : 494. Walton. B., Hamblin, A.. Dumonde, D, C . and Simpson, R. (1976): "Absence of Cellular Hypersensitivity in Patients with Unexplained Hepatitis Following Halothane", Anesthesiotogy, 44 : 391. Mathicu, A., Walger, S., and Di Padua, D. (1973) : "Test de transformation lymphocyte dans deux cas d'hepatite apres anesthesia a l'halothane", Anesthesie Anatgesie Reanimation, 30 : 109. Bruce. D. L. (1976): "Halothane Action on Lymphocytes Does Not Involve Cylic AMP", Anesthesiotogy, 44 : 151. Viljanen, M. K., Kanto, J., Vapaavuori, M., and Toivanen. P. (1973) : "Immunosuppression by Halothane", Brit. Med. J., iii : 499. Sipes. ]. G., and Brown, B. R. (1976): "An Animal Model of Hepatotoxicity Associated with Halothane Anesthesia", Anesthesioiogy, 45 : 622. McLean. A. E. M.. and McLean, E. K. (1966): "The Effect of Diet and l,l,l,-Trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) on Microsomal Hydroxylating Enzymes and on Sensitivity of Rats to Carbon Tetrachloride Poisoning", Biochem. J., 100 : 564. Gaylarde, P. M., and Sarkany, I. (1972): "Suppression of Thymidine Uptake of Human Lymphocytes by Co-trimoxazole", Brit. Med. J.. iii : 144. Hamlyn, A. N., Morris, J. S., Sarkany, I., and Sherlock, S. (1976): "Piperazine Hepatitis", Gastroenterotogy, 70 : 1144. Ferguson, R. M., Schmidtke, J. R.. and Simmons R. L. (1975): "Concurrent Inhibition of Concanavilin-A Induced Lymphocyte Aggregation and Mitogenesis by Chlorpromazine", Nature (Lond.), 256 : 744. Ferguson. R. M., Schmidtke, J. R., and Simmons, R. L. (1976): "Differential Effects of Chlorpromazine on the in vitro Generation and Effector Function of Cytotoxic Lymphocytes", J. Exper. Med.. 143 : 232. Trowell, O. A. (1965): Cetls and Tissues in Culture, Volume 2, Ed. E. N. Willmer, Academic Press, London. Paget. G. E., and Walpole, A. L. (1958): "Some Cytological Effects of Griseofulvin". Nature (Lond.), 182 : 1320. Demis. D. J., Davis, M. J., and Campbell, J. C. (1960) : "The Effects of Griseofulvin on Epithelial Cells in Tissue Culture", /. invest. Derm., 34 : 99. Sarkany, L, quoted by Hildick-Smith, G., Blank, H., and Sarkany, I. (1964) in Fungus Diseases and Their Treatment, Little, Brown and Company, Boston.
50
I. SARKANY AND P. M. GAYLARDE
23 Lajtha, L. G., quoted by Trowell, O. A. (1965) in Celts and Tissues in Culture, Volume 2, Ed. E. N. Willmer, Academic Press, London. 2-t Inman. W. H. W. (1977): "Study of Fatal Bone Marrow Depression with Special Reference to Phenylbutazone and Oxyphenbutazone", Brit. Med. J., ii : 1500. 2i> Committee on Safety of Medicines. Extracts from the Register of Adverse Reactions (1977). -0 Schneider, W., Pappas, A., and Scheurlen, P. G. (1973) : "Suppression of Lymphocyte Transformation by Salicylates", Lancet, ii : 676. 27 Forbes, L J., and Smith, J. L. (1967) : "Effects of Anti-Inflammatory Drugs on Lymphocytes", Lancet, ii : 334. 2H Ambrose, C. T. (1966) : "Inhibition of the Secondary Antibody Response in vitro by Salicylate and Gentisate", I. Exper. Med., 124 : 461. 29 Opelz, G., Terasaki, P. I., and Hirata, A. A.
(1973) : "Suppression of Lymphocyte Stimulation by Aspirin". Lancet, ii : 478. :^" Panayi. G. S., and Rise, A. (1974): "The EflFect of Fhenylbutazone, Fndomethacin and Ibuprofen on Lymphocyte Stimulation by Phytohaemagglutinin in vitro". Rheum, and Rehcib., 13 : 179. •^1 Ruben, F. L., Winkelstein, A., and Fotiadis, I. G. (1974): "Immunological Responsiveness of Tubsrculous Patients Receiving Rifampicin", Antimicrobial Agents and Chemotherapy, 5 : 383. •'2 Thong, Y. H., and Rowan-Kelly, B. (1978): "Inhibitory Effect of Miconazole on Mitogeninduced Lymphocyte Proliferative Responses", Brit. Med. J., i : 149.
•^s Nilsson, B. S. (1971): "Rifampicin: an Immunosuppressant". Lancet, ii : 374. «4 Ferguson, R. M., Schmidtke, J. R., and Simmons, R. L. (1976): "Inhibition of Mitogen-induced Lymphocyte Transformation by Local Anesthetics", /. Immunol., 116 : 627.
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY* I. SARKANY AND P . M . GAYLARDE
London, '•.
'
U.K.
SUMMARY
The role of lymphocyte transformation in the diagnosis of drug allergy is reviewed. Of 165 patients with clinically suspected drug allergy, 22 (13.3%) gave a positive lymphocyte transformation result. Reasons for this low incidence of positive reactions are put forward. Lymphocyte transformation testing in patients with halothane related hepatitis , was uniformly negative in 29 patients and this test has not been found useful in the diagnosis of this condition as against viral hepatitis. We have also found that a number of drugs including co-trimoxazole, piperazine, chlorpromazine, promethazine and griseofulvin inhibit DNA synthesis in lymphocytes in vitro. This may be the result of inhibition of transformation of lymphocytes, a cytotoxic effect or the effect of inhibition of thymidine incorporation. The in vitro suppression of DNA synthesis in lymphocytes by co-trimoxazole, rifampicin and miconazole has been suggested as being related to their possible in vivo immunosuppressive activity. The incidence of side efEects to drugs is variously given as between 2-2Q%.^'--''^ This great discrepancy is the result of differences in criteria for labelling unwanted effects of drugs. Subjective symptoms, such as nausea, dizziness, headaches, insomnia, constipation, sweating, tension etc. are difficult to assess and are frequent even in patients receiving placebo treatment. Objective features, especially those associated with skin abnormalities or liver function changes are less common and more likely clinically reliable. In a study of 5,410 in-patients, Lloyd' found an incidence of 2 per cent side effects, mainly referable to the skin, liver and haemopoietic system. Of these, only a proportion was based on allergic mechanisms and there is clearly a need for objective laboratory conffrmation in these cases. The alternative of re-administration of the drug may not be safe, feasible or acceptable to the patient. Laboratory tests for allergic sensitivity * Presented by I. Sarkany at the Annual Meeting of the Australasian College of Dermatologists Brisbane. May, 1978. Address for reprints: Dr. I. Sarkany, Consultant Dermatologist, Royal Free Hospital, London, N.W.3, England.
to systemically administered drugs are therefore needed. We have used lymphocyte transformation testing for some years^' ° and this is a review of our results. The methods of lymphocyte culture are now widely used and have become fairly standardized. In brief, lymphocytes from heparinized blood are incubated in the presence of the putative antigen for six days. Transformation of the cells is measured by examining smears microscopically and by the measurement of uptake of labelled thymidine by the transformed cells. The results are compared with those obtained from controls not exposed to the antigen. RESULTS
Of 165 patients with clinically suspected drug allergy, 22 patients gave a positive result to the lymphocyte transformation test (13.3%). The details of drugs tested, the drug concentrations used in the test and the results are given in Tables 1 and 2. Several of the drugs tested in vitro caused suppression of thymidine uptake at concentrations known to be safely tolerated in the peripheral blood under therapeutic conditions (Table 3).
I. SARKANY AND P , M . GAYLARDE
46
TABLE 2—contd.
TABLE 1
Positive Lymphocyte Transformation Test Number of Patients
Drugs Allopurinol Ampicilfin Carbamazepine Chlorpropamide Chlorpromazine Oxyphenisatin Oxytetracycline Para-aminosalicylie acid Penicillin Piperazine Rifampicin Streptomycin Succinyl sulphathiazole Sulphadiazine Sulphadimidine Thiobendazole Trimethoprim-sulphamethoxazole
Total :
1
1 1 1 1 1 1 2 4 1 1 2 1 1 2 1 2
24 drugs in 22 patients
TABLE 2
Drug Concentrations Used in Culture and Results
Drug
Positive Cases
Allopurinol Amethocaine Amphotericin Ampicillin Benzocaine Bupivacaine Carbamazepine Carbroma] Chlorothiazide Chlorpromazine Chlorpropamide Cinehoeaine Codeine Demethylchlortetracycline Digoxin Dothiepin Erythromycin Gelfoam Gold aurothiomalate Griseofulvin Halothane Hygroton Hydrochlorolhiazide Isoniazid Ketoprofen Lignocaine Meprobamate Mersalyl Metformin Methyldopa Nalidixie acid
1 1
Negative Cases 1 1 I 7 1 1
1
V ^ L 4 J V,' ± vV 1 1 1 L 4 1
1 1
3 1 5 1 1
2 1 1 2 1 1
2
29 1 1 10 1 1 1 1 1 1 1
Concentration per ml 10 Mg 15 Mg l,12,5Mg 50, 25 Mg 15Mg 15 Mg 10 Mg 40, 4, 0,4, 0,04 Mg 100, 10, I Mg 150,45, 15, 5,0,5 Mg 1,0,1,0.01 mg 15 Mg 0,5 Mg 75, 15,7,5Mg 0,025 Mg 10 Mg 10 Mg solid material 0.5 Mg 12,5, 1 Mg 100, 50, 25, 10, 5 Mg 10 Mg 1.2 Mg 10 Mg 5 Mg 5 Mg 7,5 Mg 50, 5 Mg 50 Mg 12,5 Mg 5Mg
Drug
Positive 1Cases
Neomercazole Neomycin Nitrofurantoin Oxyphenisatin Oxyphenbutazone Oxytetracycline Para-aminosalicyclic acid Paracetamol Penicillin Perchlorethylene Phenobarbitone Phenylbutazone Phenytoin Piperazine Prilocaine Primidone Promethazine hydrochloride Promethazine theoclate Propranolol Quinidine Quinine Rifampicin Salazopyrine Streptomycin Succinyl sulphathiazole Sulphadiazine Sulphadimidine Sulphafurazole Sulphapyridine Tetracycline Thiobendazole Thiopentone sodium Tolazamide Tolbulamide Trifluoperazine Trimethoprimsulphamethoxazole
1
Negative Cases 2 1 3 2 2
1 2 4
7 2 16 1 2 3 1 1 4
5, 1,0.25, 0,1 Mg
1 2 1 1
6.25 Mg lOMg 3, 1.5 Mg 0.8 Mg 15Mg 12 Mg 100, 50Mg
2
1
1 1 2
1 12 8 3 5 1 1 1
1 1 1 1 1 2
0,5 Mg 10, 1 Mg 50 Mg 12,5, 2 Mg 12,5, 10 Mg 25 Mg 750, 75, 7,5 Mg 50 Mg 250, 100 units 50, lOmMl 0,8, 0,08 Mg 12,5 Mg 25,5Mg -^ ._•: 0,25 Mg 15 Mg 40, 0,4 Mg
1
2
Concentration per ml
12
^
:-
2 5 0 , 1 2 5 Mg 2 5 0 , 1 2 5 Mg 2 5 0 , 1 2 5 Mg lOOMg 10 Mg 5Mg 15Mg 10, 1,0,1 Mg 250, 25 Mg 12,5 Mg 0,5 Mg 50, 1 0 , 5 , iMg
DISCUSSION
We have now had experience of the use of lymphocyte transformation and its role in the diagnosis of drug allergy for the past 13 years. Of 165 patients tested, 22 were found to give a positive response in culture. This represents 13,3% of all patients studied. The work has involved testing of 67 drugs or related substances, of which 17 preparations yielded a positive reaction. The most common sensitivity reactions confirmed in this way were those due to sulphonamides and antibiotics. At first sight, the low percentage of positive findings would suggest that the test is of little value in confirmation of drug allergy. However, initially we included in the test series a number of
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY TABLE 3
Drags Causing in vitro Toxicity
Drug
Minimal Concentratioi at which Toxic Effect Observed {iJ-glmt)
Chlorpromazine
1
Co-trimoxazole
1.5
Griseofulvin
1
Neomercazole
1
Nitrofiirantoin
2.5
Oxyphenisatin
2
Quinidine Penieillin
1.5 125
Phenylbutazone
10
Phenytoin
5
Piperazine
2.5
Promethazine Sulphapyridine
125 10
Effect and Comments Inhibition of thymidine uptake. Inhibition of thymidine uptake; components act synergistically. Inhibition of thymidine uptake. Inhibition of thymidine uptake. Haemolysis, also inhibition of thymidine uptake at 5 ."g/ml Inhibition of thymidine uptake. Cell necrosis. Inhibition of uptake in patient with agranulocytosis. Inhibition not observed up to 1600/ig/ml in 8 normal subjects. Haemolysis. Suppression of thymidine uptake 1 subject only. Inhibition of thymidine uptake. Inhibition of thymidine uptake. Cell necrosis. Haemolysis.
conditions not necessarily based on delayed type of sensitivity reactions, such as fixed drug eruptions, urticaria, purpura etc. All of these were negative and swelled the number of negative findings. Similarly, we found a negative response to halothane in patients with halothane induced jaundice in 29 cases." Even discounting this largely expected negative group of conditions, we failed to define with any degree of likelihood the clinical patterns of cases of suspected drug reactions which gave a positive lymphocyte transformation test result. Perhaps a more promising indication of the value of this test is shown by the fact that of the last 28 patients tested, eight have given a positive result (29% ).
47
The test picks up a certain, if small, proportion of cases where it is of clinical value. It seems, therefore, that there is a fairly large and as yet ill-defined number of mechanisms responsible for drug allergy, possibly based on humoral and cellular enzymatic abnormalities in addition to the various known types of allergic mechanisms, as defined by Gell. The lymphocyte transformation reaction is valuable in those based on cell mediated allergy only. The clinical conditions in which a positive lymphocyte transformation test was obtained were various forms of cutaneous eruptions, including exfoliative dermatitis, epidermal necrolysis, light sensitivity reactions, fever, hepatitis and neurological conditions, such as peripheral neuropathy and encephalopathy. Specific areas where disturbance of immunity has been implicated, were separately evaluated in greater detail. A summary of our experiences is presented. HALOTHANE-RELATED HEPATITIS
Paronetto and Popper^ described stimulation of lymphocyte transformation in 10 out of 15 patients with suspected halothane hepatitis. We" have failed to confirm this finding in a study of 17 patients suspected of halothane sensitivity and in a subsequent series of 12 cases the results were also negative. Similar negative conclusions in this condition were independently reported by Walton et ab'*- « and Mathieu et al." We observed and reported that halothane at dose levels of 50 ^.g per ml and above caused suppression of thymidine incorporation in PHA stimulated culture in both normal and halothane hepatitis sensitivity suspected cases. Such suppression has also been noted by Bruce" in vitro and Viljanen.'- Hepatotoxicity associated with halothane anaesthesia following administration of a polychlorinated biphenyl (PCB) to rats was reported by Sipes and Brown^-^ who failed to produce liver necrosis in these animals with either of these agents alone. The administration of PCB caused an increase in cytochrome P-450 in these animals and indicated an induction of drug metabolizing enzymes. Similarly, drugs causing an increase in liver cytochrome P-450 levels are known to increase the biotransformation of carbon tetrachloride in both man and rats and to decrease the threshold dose causing hepatotoxicity in rats.^* CO-TRIMOXAZOLE
In both patients (Table 1 and 2) with a positive reaction to co-trimoxazole, lymphocyte
48
I. SARKANY AND P, M . GAYLARDE
transformation was positive to both trimethoprim and sulphamethoxazole. This double sensitivity reaction in the positive cases is intriguing and raises the question of an adjuvant effect of the drugs towards each other. We have also demonstrated^" that cotrimoxazolc caused suppression of -^H thymidine uptake in PHA stimulated cultures in 15 of 25 normal subjects at a dose level of 1 fig per ml trimethoprim and 5 |Ug per ml sulphamethoxazole. In the 15 individuals, the average suppression was 84% and represented a marked suppressive effect. This phenomenon was due to a synergistic suppressive effect of the two drug components, and was not inhibited by folic acid. The distribution of the degree of suppression was found to be separated into two discrete groups and suggested the possibility of a genetic factor. Co-trimoxazole also inhibits thymidine uptake in unstimulated cultures and appears to act directly on DNA synthesis and not by inhibition of lymphocyte transformation. PIPERAZINE
lymphocytes (125 /xg/ml) but does not suppress thymidine uptake at 5 /xg/ml. Ferguson et aV'- *' * reported that rat thymocyte transformation in response to concanavalin-A is inhibited by 5 x 10""M chlorpromazine and Trowell'" noted that promethazine was toxic to rat lymph node lymphocytes in vitro at 10 x 10-''M, These figures show that rat lymphocytes are sensitive to phenothiazines at concentrations about an order of magnitude less than those causing inhibition of human peripheral lymphocytes. GRISEOFULVIN
Griseofulvin produced very marked reduction of ^H thymidine in vitro at concentrations of 1, 10 and 100 /xg per ml. In the rat, 100 mg per kg intravenously or 2 g per kg intraperitoneally, caused arrest of mitoses in metaphase in certain tissues,-" In vitro, 20 ftg per ml inhibited growth of epithelial cells.^i' -2 DRUG TOXICITY IN VITRO
We have shown that a number of drugs Piperazine has been widely used as an antisuppress thymidine uptake by lymphocytes in helminthic since its first description in the treatment of roundworm and threadworm in- vitro at dose levels apparently well tolerated festation. Adverse side effects have been in vivo. This effect was first noted by Lajtha-^ reported but hepatotoxicity had not been who observed that barbiturates were toxic to previously noted. We described a patient with human blood and bone marrow cells at levels piperazine hepatitis with a positive lymphocyte less than those normally tolerated in vivo. transformation reaction,^" A second patient Diphenylhydantoin and cortisone have also with a similar history gave a negative result. been recognized as toxic against lymphocytes Piperazine suppressed thymidine uptake in vitro in vitro at very low dose levels. In this context, at a level of 25 /xg per ml in five normal subjects it is worth noting that lymphocytes are much and at 2,5 /xg per ml in four other control more sensitive to ionizing radiation in vitro subjects tested. The positive lymphocyte trans- than they appear to be in vivo. In a recent report,-* the death certificates of formation result was obtained at a concentration of 0.25 (Ug per ml. Suppression of all 17 patients who died of agranulocytosis in thymidine uptake in this patient was observed England and Wales from October 1974 to October 1975 were examined and 13 of these at 2.5 and 25 /j.g per ml. deaths were attributed to co-trimoxazole. In CHLORPROMAZINE AND PROMETHAZINE the Committee on Safety of Medicines Report, Suppression of thymidine uptake was ob- 1977,-''' the four most common drugs reserved at concentrations of 50 and 100 fxg per sponsible for granulocytopenia and agranuml with chlorpromazine. PHA stimulated locytosis were phenylbutazone, oxyphenlymphocyte cultures from five normal vol- butazone, chlorpromazine and co-trimoxazole. unteers showed suppression of ''H thymidine Of these, chlorpromazine and co-trimoxazole incorporation of between 82 and 99,3% when were found to be associated with in vitro toxic exposed to chlorpromazine at a concentration effects against lymphocytes causing suppression of 50 /ig/ml. Chlorpromazine 5 /xg/ml caused of ^H thymidine uptake in vitro. A number of no significant suppression in a single normal non-steroid anti-inflammatory agents including subject tested, but suppression has been noted aspirin, indomethacin and phenylbutazone have at 1 /tg/ml in a patient with suspected hyper- been shown to inhibit lymphocyte transsensitivity, Promethazine, another drug based formation induced by PHA and to inhibit hi^"^" on phenothiazine, is also toxic to human protein
ROLE OF LYMPHOCYTE TRANSFORMATION IN DRUG ALLERGY
Immunosuppressive agents also inhibit lymphocyte transformation-' and for this reason the inhibition of PHA induced lymphocyte transformation by rifampicin'^' and miconazole^-'' ''^ has been presented as further evidence that these drugs have immunosuppressive properties. In view of the inhibition of transformation of lymphocytes by chlorpromazine,'''' local anaesthetics'^'' and anti-inflammatory drugs, additional evidence is required to confirm their immunosuppressive activity. Phenylbutazone causes marked suppression of PHA induced thymidine uptake at a level of 5 /ig/ml, but our own findings show that no suppression occurs up to 12.5 /ig/ml in unstimulated cultures, although haemolysis is usually observed. In lymphocyte cultures, even in the absence of lymphoblastic transformation, there is a low level of thymidine uptake by the cells, representing both DNA synthesis and repair. This is usually of a low level and statistically significant suppression of thymidine uptake is not demonstrable, unless it is of the order of 20 to 3 0 % . Transformation of lymphocytes into blast cells can be inhibited by a number of substances including non-steroid antiinfiammatory agents. This suppression of transformation leads to a decrease in thymidine uptake by the cells, although there is no evidence of direct interference with DNA synthesis. This implies that chlorpromazine and co-trimoxazole inhibit DNA synthesis by means other than interference with lymphoblastic transformation. The inhibition of DNA synthesis is accompanied by cytotoxic eifects on the cells in the case of cfilorpromazine, but it appears likely that co-trimoxazole inhibits DNA synthesis directly. ACKNOWLEDGEMENTS
We are grateful to the Herbert E. Dunhill Trust for generous support. REFERENCES
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' Moult. P. T. A.. Adjukiewicz, A. B.. Gaylarde, P. M., Sarkany, L, and Sherlock, S. (1975): "Lymphocyte Transformation in Halothane Related Hepatitis", Brit. Med. J., ii : 69. Paronetto, F., and Popper, H. (1970): "Lymphocyte Stimulation Induced by Halothane in Patients with Hepatitis Following Exposure to Halothane", New Engt. J. Med., 283 : 277. ' Walton, B., Dumonde, D. C , Williams, C . Jones, D., Strunin, J. M., Layton, J. M., Strunin, L., and Simpson, R. (l973) : "Lymphocyte Transformation. Absence of Increased Responses in Alleged Halothane Jaundice", J. Amer. Med. Assoc, 225 : 494. Walton. B., Hamblin, A.. Dumonde, D, C . and Simpson, R. (1976): "Absence of Cellular Hypersensitivity in Patients with Unexplained Hepatitis Following Halothane", Anesthesiotogy, 44 : 391. Mathicu, A., Walger, S., and Di Padua, D. (1973) : "Test de transformation lymphocyte dans deux cas d'hepatite apres anesthesia a l'halothane", Anesthesie Anatgesie Reanimation, 30 : 109. Bruce. D. L. (1976): "Halothane Action on Lymphocytes Does Not Involve Cylic AMP", Anesthesiotogy, 44 : 151. Viljanen, M. K., Kanto, J., Vapaavuori, M., and Toivanen. P. (1973) : "Immunosuppression by Halothane", Brit. Med. J., iii : 499. Sipes. ]. G., and Brown, B. R. (1976): "An Animal Model of Hepatotoxicity Associated with Halothane Anesthesia", Anesthesioiogy, 45 : 622. McLean. A. E. M.. and McLean, E. K. (1966): "The Effect of Diet and l,l,l,-Trichloro-2,2-bis(p-chlorophenyl) ethane (DDT) on Microsomal Hydroxylating Enzymes and on Sensitivity of Rats to Carbon Tetrachloride Poisoning", Biochem. J., 100 : 564. Gaylarde, P. M., and Sarkany, I. (1972): "Suppression of Thymidine Uptake of Human Lymphocytes by Co-trimoxazole", Brit. Med. J.. iii : 144. Hamlyn, A. N., Morris, J. S., Sarkany, I., and Sherlock, S. (1976): "Piperazine Hepatitis", Gastroenterotogy, 70 : 1144. Ferguson, R. M., Schmidtke, J. R.. and Simmons R. L. (1975): "Concurrent Inhibition of Concanavilin-A Induced Lymphocyte Aggregation and Mitogenesis by Chlorpromazine", Nature (Lond.), 256 : 744. Ferguson. R. M., Schmidtke, J. R., and Simmons, R. L. (1976): "Differential Effects of Chlorpromazine on the in vitro Generation and Effector Function of Cytotoxic Lymphocytes", J. Exper. Med.. 143 : 232. Trowell, O. A. (1965): Cetls and Tissues in Culture, Volume 2, Ed. E. N. Willmer, Academic Press, London. Paget. G. E., and Walpole, A. L. (1958): "Some Cytological Effects of Griseofulvin". Nature (Lond.), 182 : 1320. Demis. D. J., Davis, M. J., and Campbell, J. C. (1960) : "The Effects of Griseofulvin on Epithelial Cells in Tissue Culture", /. invest. Derm., 34 : 99. Sarkany, L, quoted by Hildick-Smith, G., Blank, H., and Sarkany, I. (1964) in Fungus Diseases and Their Treatment, Little, Brown and Company, Boston.
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•^s Nilsson, B. S. (1971): "Rifampicin: an Immunosuppressant". Lancet, ii : 374. «4 Ferguson, R. M., Schmidtke, J. R., and Simmons, R. L. (1976): "Inhibition of Mitogen-induced Lymphocyte Transformation by Local Anesthetics", /. Immunol., 116 : 627.
