CELLULAR
IMMUNOLOGY
Transfer
42,424-421
(1979)
of Delayed Hypersensitivity to Leishmanin (Montenegro Reaction) ERNESTO
Se&o de Alergia. Medicina Tropical,
MENDES
Departamento de Clinica Mkdica do Hospital das Ciinicas and the lnstituto de Faculdade de Medicina da Universidade de So Pa&o, Caixa Postal 8091, Brasil Received
August
2, 1978
It was possible to transfer the cutaneous leishmanin hypersensitivity (Montenegro reaction) to 7 out of 12 recipients. The diameter of the indurations observed ranged from 8 to 25 mm. Histological examination of skin biopsies from the site of three positive Montenegro reactions showed intense mononuclear infiltrate of lymphocytes and histiocytes, and one biopsy showed the feature of tuberculoid granuloma. Four recipients retested with leishmanin after 11, 22, 25, and 32 days, still showed positive reactions.
INTRODUCTION Transfer of delayed hypersensitivity to Leishmania donovani in guinea pigs was reported by Boysia (1) using lymph node cells from animals immunized with L. donovani (promastigotes) in Freund’s adjuvant. Delayed hypersensitivity has also been transferred to normal guinea pigs by peritoneal cells or lymph node cells from guinea pigs infected with Leishmania enrietti or from animals sensitized with promastigote antigen in Freund’s complete adjuvant. Adler and Nelkin (2) were unable to obtain the transfer with leukocytes or whole blood from a patient highly sensitive to leishmanin (Montenegro reaction). Attempts were made by Bryceson (4) to transfer sensitivity to leishmanin negative patients with disseminated cutaneous leishmaniasis and to three leishmanin-negative normal volunteers using leukocytes from four Montenegro-positive donors with cutaneous leishmaniasis. Transient hypersensitivity with small indurations was achieved for up to 3 weeks in three normal volunteers and in three patients. Long-lasting sensitization was not achieved and the course of the infection was not altered. In view of the ability to transfer delayed hypersensitivity to other pathogens with lymphoid cells (3,5) or with cells extracts (“transfer factor”) (6) it might be expected that delayed hypersensitivity to leishmanin in man could be transferred by this way. For this reason we attempted the transfer of delayed hypersensitivity from donors highly sensitive to leishmanin to previously Montenegro-negative patients with various diseases. PATIENTS Twelve previously transfer experiments:
AND METHODS
anergic patients to leishmanin were studied as recipients in six patients with South American blastomycosis; two with 424
0008-8749/79/020424-04$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
425
chronic ulcer of the legs; one with lepromatous leprosy; one with allergic vasculitis; one with chromomycosis, and one with kala-azar. Montenegro antigen (leishmanin) containing 2-3 x lo6 leishmanias per milliliter prepared by Instituto Adolph0 Lutz was used as antigen. The Montenegro antigen was injected intracutaneously in O.lO-ml doses and the reaction was read 48 hr later: the diameter of the induration area was then measured in millimeters. The criteria by which the recipient patients were judged anergic to leishmanin were the following: (a) the recipients were skin-tested with 0.1 ml of Montenegro antigen for the first time in our laboratory and the results were negative; (b) the dermatologic examination excluded previous cutaneous leishmaniasis; (c) the recipients never have lived in endemic areas of leishmaniasis, except the patient with kala-azar. Transfer of delayed hypersensitivity. Forty milliliters of venous blood from seven patients highly sensitive to Montenegro antigen (leishmanin) was drawn into sterile syringes containing 400 units of heparin. The blood was transferred to sterile glass tubes and allowed to settle for 1 hr at 37°C. The leukocyte-rich plasma was passed through a sterile nylon column and the effluent lymphocytes (90% pure) were counted in a hemocytometer and resuspended in autologous plasma. The viability, evaluated by trypan blue exclusion, was always greater than 90%. Donors with positive skin reactions to leishmanin (Montenegro antigen) were selected among patients with cutaneous leishmaniasis considered cured by chemotherapy. Conventional tests to detect HbAg and serological tests for syphilis were performed before the transfer. Lymphocytes (12-20 x 106) were suspended in 1 ml of plasma. The suspension (0.2 ml) was injected intradermally and the remaining 0.8 ml subcutaneously in the same place without withdrawing the needle. Beginning 5 days later, the recipient patients were skin-tested with 0.1 ml of Montenegro antigen in areas distant from the site previously injected with lymphocytes and the reactions were read as described. Three positive reactions were followed by histologic examination of skin biopsies. Seven recipients were retested with leishmanin after the transference of lymphocytes, in order to evaluate the duration of the transference and the influence of repeated skin tests. RESULTS It was possible to transfer the cutaneous leishmanin hypersensitivity (Montenegro reaction) to 7 out of 12 recipients as follows: one out of six patients with South American blastomycosis, one with allergic vasculitis, one with kala-azar, one with lepromatous leprosy, one with chromomycosis and two with chronic ulcer of legs (Table 1). All these recipients, except the patient with kala-azar, showed delayed cutaneous reactions to leishmanin read between 6 and 7 days after the lymphocyte transfer. The diameter of the indurations observed ranged from 8 to 25 mm. The cutaneous induration lasted for a period from 4 to 7 days. Histologic examination of skin biopsies from the site of three positive Montenegro reaction showed intense mononuclear infiltrate of lymphocytes and histiocytes, and one biopsy showed the aspect of tuberculoid granuloma. The patient with kala-azar accepted only the second transfer of Montenegro hypersensitivity, 2 months after the first transfer, when a different donor was used.
SHORT COMMUNICATIONS
426
TABLE
1
Results of Leishmanin Transference to 12 Recipients Skin tests with leishmanin After Recipients Diagnostic
Name
Before the transference
the transference induration (diameter in mm)
1. D.H.
Allergic
vasculitis
2. I.C.L.
Chronic
ulcer of legs
8 x IO
Chronic ulcer of legs Blastomycosis Blastomycosis Blastomycosis Blastomycosis Blastomycosis Lepromatous leprosy Chromomycosis Biastomycosis Kda-azar
12 x 18 -
3. 4. 5. 6. 7. 8. 9. 10. II. 12.
M.V.S. J.H. A.R. J.M.C. R.A.O. A.J.S. I.T. I.S.M. R.S. S.H.S.
16 x 25
IO x 8 8X8 I5 x I5 10 x 10
Retest (days after the transference) 16, 25, 32
IS, 25
11.22 14. 21, 28, 42, 52 13, 18. 26
Histology Intense mononuclear infiltrate of lymphocytes and histiocytes Intense mononuclear infiltrate of lymphocytesand histiocytes Tuberculoidgranuloma
12. I8
30, 60,66,78,
150
Five and twelve days after this transfer the induration was 10 mm in diameter, but the retest after 5 months was negative. Four other recipients retested with leishmanin after 11,22,25 and 32 days respectively still showed positive reactions. Two recipients remained negative in spite of repeated retests with leishmanin (patients 4 and 5, Table 1). The lymphocytes from one donor failed to transfer the Montenegro reaction to two patients with South American blastomycosis but the lymphocytes from the same donor could transfer the hypersensitivity to a patient with lepromatous leprosy. DISCUSSION The possibility of transferring delayed hypersensitivity to leishmanin (Montenegro reaction) will probably contribute to the better understanding of the disease and to stimulate the immunotherapy of leishmaniasis. Kala-azar is considered an anergic form of leishmaniasis with high levels of immunoglobulins and circulating antibodies, with impairment of cell-mediated immunity (7). Usually the Montenegro test is negative during the active phase of the disease and it usually becomes positive after convenient recovery of the patient by chemotherapy. The transfer of delayed hypersensitivity to different microbial antigens to patients with paracoccidioidomycosis has been reported (8,9). In the present study only one out of six recipients with paracoccidioidomycosis accepted the transfer of hypersensitivity to leishmanin. The transfer of delayed hypersensitivity reactions to recipients with lepromatous leprosy has been demonstrated previously (10). The possibility of transferring the Montenegro reaction to one recipient with lepromatous leprosy constitutes another evidence on the possibility to induce delayed cutaneous reactions in previously negative lepromatous leprosy patients. One assumes that repeated intracutaneous tests with leishmanin by itself never has been mentioned to induce a positive reaction, according to several papers
SHORT COMMUNICATIONS
427
on Montenegro reaction (11). The various retests with leishmanin carried out in two negative recipients seem to confirm this statement. An important point to discuss is whether the transfer of Montenegro reaction could be considered specific or not. The recipients had never lived in areas of endemic leishmaniasis and therefore the possibility of an anamnestic responses was not probable, although some of them had lived in rural areas. The lymphocyte transfer and “transferfactor” possess antigen-specific activities according to the reports of transfer of delayed hypersensitivity responses to nonenvironmental antigens such as denatured human albumin (12), coccidioidin (13), keyhole limpet hemocyanin (14), and transfer of acellerated allograft rejection (15). On the other hand, the transfer of immune responses that were not present in the donor suggests that the lymphocyte transfer possesses adjuvant-like, antigen independent activities (16). A possibility to be considered is that lymphocyte transfer contains both nonspecific T-cell-activating factors, as well as antigen specific factors. The discussion of our results based on these hypotheses suggests that the transference of leishmanin hypersensitivity seems to be induced by antigen-specific factors. An interesting observation was seen when the lymphocytes from the same donor transferred the Montenegro reaction to a leprosy patient but failed to transfer the reaction to two paracoccidioidomycosis patients. This observation seems to demonstrate that the transfer of hypersensitivity depends on both donor and recipient. According to Bryceson (4) long-lasting transfer to leishmanin was never achieved. Our recipients retested after 11, 22, 25, and 32 days respectively still showed positive reactions to leishmanin. It remains to be established whether induction of cell-mediated immunity to leishmanin by lymphocyte transfer alters the clinical course of visceral leishmaniasis. REFERENCES 1. Boysia, F. T., Ph.D. Thesis, Graduate School. Rutgers University, New Brunswick, N.J. 1968. Apud Bryceson, A.D.M. 2. Adler, S., and Nelkin, D., Trans. Roy. Sot. Trap. Med. Hyg. 59, 59, 1965. 3. Bray, R. S., Bryceson, A. D. M., and Dumonde, D. C., Clin. Exp. Ztnmunol. 16, 189, 1974. 4. Bryceson, A. D. M., Trans. Roy. Sot. Trop. Med. Hyg. 64, 380, 1970. 5. Landsteiner, R., and Chase, N. W., Proc. Sot. Exp. Biol. Med. 49, 688, 1942. 6. Lawrence, H. S., Adv. Zmmunol. 11, 195, 1969. 7. Mendes, E., Allergology. Proc. VIII Int. Congr. Allergol. Tokyo, p. 230. ExcerptaMedica, Amsterdam, 1973. 8. Mendes, E., and Raphael, A., J. Allergy 47, 17, 1971. 9. Musatti, C. C., Rezkallah, M. T., Mendes, E., and Mendes, N. F., Cell. Zmmunol. 24, 365, 1976. 10. Mendes, E., Raphael, A., Mota, N. G. S., and Mendes, N. F., J. Allergy Clin. Zmmunol. 53, 223, 1974. 11. Pessoa, S. B., and Martins, A. V., “Parasitologia Mbdica.” 9th ed. Editora Guanabara Koogan S.A., Rio de Janeiro, 1974. 12. Maurer, P. H., J. Exp. Med. 113, 1029, 1961. 13. Rappaport, F. T., Lawrence, H. S., Miller, J. W., Pappagianis, D., and Smith, C. E., J. Zmmunol. 84, 358, 1960. 14. Zuckerman, K. S., Neidhort, J. A., Balcerzak, S. P., and Lobuglio, A. F., J. Clin. Invest. 54,997, 1974. 15. Lawrence, H. S., Rappaport, F. T., Converse, J. M., and Tillet, W. S., J. Clin. Invest. 39, 185, 1960. 16. Bloom, B. R., N. Engl. J. Med. 288, 908, 1973.
IMMUNOLOGY
Transfer
42,424-421
(1979)
of Delayed Hypersensitivity to Leishmanin (Montenegro Reaction) ERNESTO
Se&o de Alergia. Medicina Tropical,
MENDES
Departamento de Clinica Mkdica do Hospital das Ciinicas and the lnstituto de Faculdade de Medicina da Universidade de So Pa&o, Caixa Postal 8091, Brasil Received
August
2, 1978
It was possible to transfer the cutaneous leishmanin hypersensitivity (Montenegro reaction) to 7 out of 12 recipients. The diameter of the indurations observed ranged from 8 to 25 mm. Histological examination of skin biopsies from the site of three positive Montenegro reactions showed intense mononuclear infiltrate of lymphocytes and histiocytes, and one biopsy showed the feature of tuberculoid granuloma. Four recipients retested with leishmanin after 11, 22, 25, and 32 days, still showed positive reactions.
INTRODUCTION Transfer of delayed hypersensitivity to Leishmania donovani in guinea pigs was reported by Boysia (1) using lymph node cells from animals immunized with L. donovani (promastigotes) in Freund’s adjuvant. Delayed hypersensitivity has also been transferred to normal guinea pigs by peritoneal cells or lymph node cells from guinea pigs infected with Leishmania enrietti or from animals sensitized with promastigote antigen in Freund’s complete adjuvant. Adler and Nelkin (2) were unable to obtain the transfer with leukocytes or whole blood from a patient highly sensitive to leishmanin (Montenegro reaction). Attempts were made by Bryceson (4) to transfer sensitivity to leishmanin negative patients with disseminated cutaneous leishmaniasis and to three leishmanin-negative normal volunteers using leukocytes from four Montenegro-positive donors with cutaneous leishmaniasis. Transient hypersensitivity with small indurations was achieved for up to 3 weeks in three normal volunteers and in three patients. Long-lasting sensitization was not achieved and the course of the infection was not altered. In view of the ability to transfer delayed hypersensitivity to other pathogens with lymphoid cells (3,5) or with cells extracts (“transfer factor”) (6) it might be expected that delayed hypersensitivity to leishmanin in man could be transferred by this way. For this reason we attempted the transfer of delayed hypersensitivity from donors highly sensitive to leishmanin to previously Montenegro-negative patients with various diseases. PATIENTS Twelve previously transfer experiments:
AND METHODS
anergic patients to leishmanin were studied as recipients in six patients with South American blastomycosis; two with 424
0008-8749/79/020424-04$02.00/O Copyright 8 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
SHORT
COMMUNICATIONS
425
chronic ulcer of the legs; one with lepromatous leprosy; one with allergic vasculitis; one with chromomycosis, and one with kala-azar. Montenegro antigen (leishmanin) containing 2-3 x lo6 leishmanias per milliliter prepared by Instituto Adolph0 Lutz was used as antigen. The Montenegro antigen was injected intracutaneously in O.lO-ml doses and the reaction was read 48 hr later: the diameter of the induration area was then measured in millimeters. The criteria by which the recipient patients were judged anergic to leishmanin were the following: (a) the recipients were skin-tested with 0.1 ml of Montenegro antigen for the first time in our laboratory and the results were negative; (b) the dermatologic examination excluded previous cutaneous leishmaniasis; (c) the recipients never have lived in endemic areas of leishmaniasis, except the patient with kala-azar. Transfer of delayed hypersensitivity. Forty milliliters of venous blood from seven patients highly sensitive to Montenegro antigen (leishmanin) was drawn into sterile syringes containing 400 units of heparin. The blood was transferred to sterile glass tubes and allowed to settle for 1 hr at 37°C. The leukocyte-rich plasma was passed through a sterile nylon column and the effluent lymphocytes (90% pure) were counted in a hemocytometer and resuspended in autologous plasma. The viability, evaluated by trypan blue exclusion, was always greater than 90%. Donors with positive skin reactions to leishmanin (Montenegro antigen) were selected among patients with cutaneous leishmaniasis considered cured by chemotherapy. Conventional tests to detect HbAg and serological tests for syphilis were performed before the transfer. Lymphocytes (12-20 x 106) were suspended in 1 ml of plasma. The suspension (0.2 ml) was injected intradermally and the remaining 0.8 ml subcutaneously in the same place without withdrawing the needle. Beginning 5 days later, the recipient patients were skin-tested with 0.1 ml of Montenegro antigen in areas distant from the site previously injected with lymphocytes and the reactions were read as described. Three positive reactions were followed by histologic examination of skin biopsies. Seven recipients were retested with leishmanin after the transference of lymphocytes, in order to evaluate the duration of the transference and the influence of repeated skin tests. RESULTS It was possible to transfer the cutaneous leishmanin hypersensitivity (Montenegro reaction) to 7 out of 12 recipients as follows: one out of six patients with South American blastomycosis, one with allergic vasculitis, one with kala-azar, one with lepromatous leprosy, one with chromomycosis and two with chronic ulcer of legs (Table 1). All these recipients, except the patient with kala-azar, showed delayed cutaneous reactions to leishmanin read between 6 and 7 days after the lymphocyte transfer. The diameter of the indurations observed ranged from 8 to 25 mm. The cutaneous induration lasted for a period from 4 to 7 days. Histologic examination of skin biopsies from the site of three positive Montenegro reaction showed intense mononuclear infiltrate of lymphocytes and histiocytes, and one biopsy showed the aspect of tuberculoid granuloma. The patient with kala-azar accepted only the second transfer of Montenegro hypersensitivity, 2 months after the first transfer, when a different donor was used.
SHORT COMMUNICATIONS
426
TABLE
1
Results of Leishmanin Transference to 12 Recipients Skin tests with leishmanin After Recipients Diagnostic
Name
Before the transference
the transference induration (diameter in mm)
1. D.H.
Allergic
vasculitis
2. I.C.L.
Chronic
ulcer of legs
8 x IO
Chronic ulcer of legs Blastomycosis Blastomycosis Blastomycosis Blastomycosis Blastomycosis Lepromatous leprosy Chromomycosis Biastomycosis Kda-azar
12 x 18 -
3. 4. 5. 6. 7. 8. 9. 10. II. 12.
M.V.S. J.H. A.R. J.M.C. R.A.O. A.J.S. I.T. I.S.M. R.S. S.H.S.
16 x 25
IO x 8 8X8 I5 x I5 10 x 10
Retest (days after the transference) 16, 25, 32
IS, 25
11.22 14. 21, 28, 42, 52 13, 18. 26
Histology Intense mononuclear infiltrate of lymphocytes and histiocytes Intense mononuclear infiltrate of lymphocytesand histiocytes Tuberculoidgranuloma
12. I8
30, 60,66,78,
150
Five and twelve days after this transfer the induration was 10 mm in diameter, but the retest after 5 months was negative. Four other recipients retested with leishmanin after 11,22,25 and 32 days respectively still showed positive reactions. Two recipients remained negative in spite of repeated retests with leishmanin (patients 4 and 5, Table 1). The lymphocytes from one donor failed to transfer the Montenegro reaction to two patients with South American blastomycosis but the lymphocytes from the same donor could transfer the hypersensitivity to a patient with lepromatous leprosy. DISCUSSION The possibility of transferring delayed hypersensitivity to leishmanin (Montenegro reaction) will probably contribute to the better understanding of the disease and to stimulate the immunotherapy of leishmaniasis. Kala-azar is considered an anergic form of leishmaniasis with high levels of immunoglobulins and circulating antibodies, with impairment of cell-mediated immunity (7). Usually the Montenegro test is negative during the active phase of the disease and it usually becomes positive after convenient recovery of the patient by chemotherapy. The transfer of delayed hypersensitivity to different microbial antigens to patients with paracoccidioidomycosis has been reported (8,9). In the present study only one out of six recipients with paracoccidioidomycosis accepted the transfer of hypersensitivity to leishmanin. The transfer of delayed hypersensitivity reactions to recipients with lepromatous leprosy has been demonstrated previously (10). The possibility of transferring the Montenegro reaction to one recipient with lepromatous leprosy constitutes another evidence on the possibility to induce delayed cutaneous reactions in previously negative lepromatous leprosy patients. One assumes that repeated intracutaneous tests with leishmanin by itself never has been mentioned to induce a positive reaction, according to several papers
SHORT COMMUNICATIONS
427
on Montenegro reaction (11). The various retests with leishmanin carried out in two negative recipients seem to confirm this statement. An important point to discuss is whether the transfer of Montenegro reaction could be considered specific or not. The recipients had never lived in areas of endemic leishmaniasis and therefore the possibility of an anamnestic responses was not probable, although some of them had lived in rural areas. The lymphocyte transfer and “transferfactor” possess antigen-specific activities according to the reports of transfer of delayed hypersensitivity responses to nonenvironmental antigens such as denatured human albumin (12), coccidioidin (13), keyhole limpet hemocyanin (14), and transfer of acellerated allograft rejection (15). On the other hand, the transfer of immune responses that were not present in the donor suggests that the lymphocyte transfer possesses adjuvant-like, antigen independent activities (16). A possibility to be considered is that lymphocyte transfer contains both nonspecific T-cell-activating factors, as well as antigen specific factors. The discussion of our results based on these hypotheses suggests that the transference of leishmanin hypersensitivity seems to be induced by antigen-specific factors. An interesting observation was seen when the lymphocytes from the same donor transferred the Montenegro reaction to a leprosy patient but failed to transfer the reaction to two paracoccidioidomycosis patients. This observation seems to demonstrate that the transfer of hypersensitivity depends on both donor and recipient. According to Bryceson (4) long-lasting transfer to leishmanin was never achieved. Our recipients retested after 11, 22, 25, and 32 days respectively still showed positive reactions to leishmanin. It remains to be established whether induction of cell-mediated immunity to leishmanin by lymphocyte transfer alters the clinical course of visceral leishmaniasis. REFERENCES 1. Boysia, F. T., Ph.D. Thesis, Graduate School. Rutgers University, New Brunswick, N.J. 1968. Apud Bryceson, A.D.M. 2. Adler, S., and Nelkin, D., Trans. Roy. Sot. Trap. Med. Hyg. 59, 59, 1965. 3. Bray, R. S., Bryceson, A. D. M., and Dumonde, D. C., Clin. Exp. Ztnmunol. 16, 189, 1974. 4. Bryceson, A. D. M., Trans. Roy. Sot. Trop. Med. Hyg. 64, 380, 1970. 5. Landsteiner, R., and Chase, N. W., Proc. Sot. Exp. Biol. Med. 49, 688, 1942. 6. Lawrence, H. S., Adv. Zmmunol. 11, 195, 1969. 7. Mendes, E., Allergology. Proc. VIII Int. Congr. Allergol. Tokyo, p. 230. ExcerptaMedica, Amsterdam, 1973. 8. Mendes, E., and Raphael, A., J. Allergy 47, 17, 1971. 9. Musatti, C. C., Rezkallah, M. T., Mendes, E., and Mendes, N. F., Cell. Zmmunol. 24, 365, 1976. 10. Mendes, E., Raphael, A., Mota, N. G. S., and Mendes, N. F., J. Allergy Clin. Zmmunol. 53, 223, 1974. 11. Pessoa, S. B., and Martins, A. V., “Parasitologia Mbdica.” 9th ed. Editora Guanabara Koogan S.A., Rio de Janeiro, 1974. 12. Maurer, P. H., J. Exp. Med. 113, 1029, 1961. 13. Rappaport, F. T., Lawrence, H. S., Miller, J. W., Pappagianis, D., and Smith, C. E., J. Zmmunol. 84, 358, 1960. 14. Zuckerman, K. S., Neidhort, J. A., Balcerzak, S. P., and Lobuglio, A. F., J. Clin. Invest. 54,997, 1974. 15. Lawrence, H. S., Rappaport, F. T., Converse, J. M., and Tillet, W. S., J. Clin. Invest. 39, 185, 1960. 16. Bloom, B. R., N. Engl. J. Med. 288, 908, 1973.
