- Email: [email protected]
Activity and chronopharmacology of very low doses of physiological immune inducers
234
Tolerance and interleukin 2 deficiency SIR,
We read with interest the article by M. Malkovskp and P. B. Medawar (Immunol. Today 1984, 5, 340-343) in which they propose that tolerance will result from an immunological encounter when there is a deficiency of interleukin 2 (IL-2). Our recent experience in marrow transplantation for high-risk acute leukemia and aplastic anemia offers an interesting parallel to this postulate. Plasma and residual mononuclear cells were removed from 14 recipients undergoing marrow transplantation after high dose cyclophosphamide, arabinosyl cytosine and total body irradiation (1200 fads in 3 days) and prior to infusion of donor marrow in our transplantation unit between July 1983 and December 1984. The objective was firstly to remove lymphocytes from the recipient which might incite a response of donor lymphocytes against the recipient, and secondly to eliminate various tissue antigens cleared from the tissue and present in the plasma which the donor lymphocytes might recognize as foreign. Mononudear cells ranging from4 x 107to5 x 10S (average 2.1 x 10s) cells were removed by plasmalymphocytapheresis. In these 14 patients, graft vs host disease (GVHD) developed in only 2 (14%), and a mild generalized maculopapular rash in only 1. This contrasts with the 40-60% incidence seen in most transplant centers. We analysed the phenotype of cells recovered from 5 of the patients using monoclonal antibodies and an Ortho cytofluorograph. The differential phenotype as a percent of total cells is as follows: 55% T3 ÷, 45% T4 +, 22% T8 +, 27% T10 +, 17% Leu 11 ÷, 26% M1 + and4% M3 +. To test the functional ability of these cells removed from the recipient, we stimulated them with phytohemagglutinin and Daudi cells for 48 h and then assayed for IL-2 activity. These cells, after high-dose chemotherapy and irradiation, produced 1.5 times the amount of IL-2 generated by untreated, normal lymphocytes stimulated in the same way. Similarly, we have noted high IL-2 activity in lymphocytes of patients with head and neck cancer immediately after irradiation. Thus, plasma-lymphocytapheresis removed a very good source of IL-2 production in
Immunology Today, voL 6, No. 8, 1985 these patients. It may also have removed antigens broken down from the tissue. Recipients of marrow transplant for treatment of severe combined immune deficiency (SCID), leukemia and aplastic anemia have insignificant or no G V H D if their pretransplant natural killer (NK) activity against herpes simplex virus type 1 infected fibrohlasts is low or absent 1. A recent observation that N K activity depends on IL-2 suggests that the low incidence of G V H D in these patients may be due to low IL-2 production 2. The association betwen the low incidence of G V H D in our transplant patients with removal of residual lymphocytes and plasma may be significant. Removal of lymphocytes capable of producing IL-2, thus depriving donor lymphocytes of a stimulating factor in the initial period of
Activity and chronopharmacology of very low doses of physiological immune inducers SIR,
V l Bocci (Immunol. Today 1985, 6, 7-9) presents arguments in favor of his hypothesis that interferon 'is produced at a continuously low, physiological level that must be maintained for correct immune function to occur' and that 'trace amounts of exogenous and endogenous inducers act as physiological stimuli in the lymphoid tissue...'. In support of Bocci's hypothesis, we report here the effect of very low doses of interferon (IFN) on the humoral and cellular immune response of mice treated intraperitoneallyl,2. We observed that 8 international units (IU) and 8 x 10 -1° I U of murine a,fl IFN (kindly provided by M. Tovey) caused a significant increase in the humoral immune response of Swiss mice to sheep red blood cells (SRBC) using theJerne plaque-forming cell (PFC) technique (P < 0.01), and we found that 16 x 10 -1° IU of a,/~ IFN significantly stimulated (P < 0.001) the cellular immune response in C57BL/6 mice to P815 mastocytome cells using the 5'Crrelease technique 2. All control mice received solvent intraperitoneally under the same conditions as the test mice.
We also studied the action of thymic hormones on young Swiss and New Zealand Black (NZB) mice challenged with SRBC (a thymus-dependent antigen) by the PFC technique. We found that the very low doses studied, namely 400pg, 4 x 10-4pgand4 x 10-Spgof
marrow transplantation, may produce tolerance between the graft and the host. This interpretation supports the observation that donor marrow composition has little impact on the occurrence or severity o f G V H D 3. [~ ANDREW T. HUANG ROBERT F. HUNTER PATRICIA A. ROTH NELDA G. MOLD
Department ofMedicine, Duke UniversityMedical Center, Durham, NC27710, USA
References 1 Lopez, C., Kirkpatrick, D., Soren, M,, O'ReiUy, R., and Ching, C. (1979) Lanceti, 1103-1106 2 Domzig, W., Stadler, B. M., and Herberman, R. B. (1983)J. Immunol. 130, 1970-1973 3 Jansen, J., Goselink, H. M., Veenhof, W. F. J., Zwaan, F. E. and Blotkamp, C. (1983) Exp. Hematol. 11,967-973
thymulin per mouse, had an immunoregulatory action : the hormone was immunodepressive (P < 0.05) for the normal Swiss mice 3 and immunostimulating (P < 0.001) for immunodeficient NZB mice 4. In addition, we observed that 8 x 10- s pg of thymulin depressed the cellular immune response in C57BL/6 mice 2. Furthermore, we studied the effect on the humoral immune response over a 16-month period of the same low doses ofthymosin fraction 5 (kindly provided by A. Goldstein) on Swiss mice. Our results indicate that this hormone amplifies the circannual variation of the humoral immune response 5'6 at all doses studied 7. Analysis of these results using the Cosinor method showed that the acrophase was in the winter season (4 × 10- 4 pg : January 10 _+ 17 days and 4 x 10-a pg: December 25 _+ 24 days; P < 0.001). w e conclude that doses as low as 10-s IU or 10-8 p g of natural mediators such as IFN or the thymic hormones, respectively, are pharmacologically active and that they do not perturb but rather amplify the natural seasonal variation of the humoral immune response. We suggest that for best therapeutic efficacy in immunodisequilibrated subjects immune mediators should be used at very low doses so as to maintain homeostasis. These studies were supported by Dolisos Laboratories (J. Guillernain, Scientific Director). ~[~ M. BASTIDE M. DOUCET-JABOEUF V. DAURAT Universitt de Montpellier I, Unit~de Recherchesen Immunologic, Facult~de Pharmacie, 34060 Montpellier Cedex, France.
235
Immunology Today, vol. 6, No. 8, 1985 References 1 Doucet-Jaboeuf, M., Pelegrin, A., Sizes,
M., Guillemain,J. and Bastide, M. (1985) Int..]. ImmunopharmacoL 7,312 2 Daurat, V., Sizes,M., Pelegrin,A., DoucetJaboeuf, M., Guillemain,J. and Bastide, M. (.June 1985) Symposium Marqueurs Inflam., Lyon, France 3 Doucet-Jaboeuf, M., Guillemain, J.,
Piechaczyk, M., Karouby, Y. and Bastide, M. (1982) C.R. Acad. Sci. 295,283-286 4 Doucet-Jaboeuf,M., Pelegrin, A., Cot, M. C., Guillemain,J. and Bastide, M. Abstract~ Soc. Fr. ImmunoL, May 1984 5 Mac Murray, J., Barker,J., Armstrong,J., Bozzetti, L. and Kuhn, I. (1983)LifeSci. 32, 2363-2370
6 Reinberg, A., Schuller, E., Delasnerie, N., Clench, J. and Helary, M. (1977) La Nouv. PresseMed. 6, 3819-3823 7 Doueet-Jaboeuf,M., Pelegrin, A., Cot, M. C., Guillemain,J. and Bastide, M. (1984) Ann. Rev. Chronopharm. 1,231-234
) Interlcukin 1: amino acid sequencing reveals microheterogeneity and relationship with an interferon-inducing monokine A. Billiau, G. Opdenakker, J. Van Darnme, M. De Ley, G. Volckaert and J..Van Beeumen q" Interleukin 1 ( ! L - l ) is the n a m e give n in 19791 to a set of factors involved in the specific i m m u n e response and previously k n o w n u n d e r different names: mitogenic protein, T-cell replacing factor, B-cell activating factor, B-cell differentiation factor and lymphocyte activating factor (LAF). Currently, the t e r m IL- 1 is mostly used as an a c r o n y m for L A F , the latter being defined as the factor which, u n d e r physiological circumstances, is p r o d u c e d by antigen-presenting Inononuclear phagocytes (monocytes, macrophages) and which can activate antigenrecognizing T lymphocytes to produce interleukin 2 (IL2). IL-2, in turn, is the subsequent link in the chain of events leading to production of antibodies and cellmediated responses to the antigen. T h e r e is now growing evidence (for review, see Ref. 2) that IL-1, as a family name, also encompasses a n u m b e r of factors which are involved in nonspecific host resistance and inflammation: endogenous or leukocytic pyrogen (EP or LP), leukocytic endogenous mediators ( L E M ) , monocytic cell factor ( M C F ) , and others. E P or L P is defined by its ability to cause fever and alterations in white blood cell counts (leukopenia followed by hyperleukocytosis), both p h e n o m e n a resembling the effects of bacterial lipopolysaccharides. L E M is defined by its ability to induce acute phase components and M C F by its ability to induce production of collagenase and prostaglandin Fa by synovial cells. V a n D a m m e et al. 3 have isolated an antiviral factor (22K factor) from mitogen-stimulated m o n o n u c l e a r cells (Fig. 1). T h e antiviral effect of this factor was shown to be due to induction of interferon-fl in fibroblasts s'4. H o w e v e r , biological tests on the purified product led t h e m to conclude that it also had m a n y of the properties assigned to IL-1 (i.e. L A F , M C F and E P activities) 5. Rega Institute, University of Leuven, Belgium. tLaboratory of Microbiology, Faculty of Sciences, State University of Ghent, .Belgium.
I
MITOGENSor ANTIGENS
[
~ Monoeyte
T-lymphocyte
HulFN-~ fibroblasts
o
V
antiviralstate of tissue Fig. 1. An antiviral monokine related to IL-1, which acts by inducing interferon. The 22K factor was originally discovered as an interferon-like antiviral protein. Most significantly its antiviral effect could be blocked by antiserum against human interferon-/~ (HuIFN-/~). However, careful study revealed that the antiviral effect of the factor was due to its ability to induce HulFN-fl. Further study on the biological effects showed that the electrophoretically pure factor had several activities remindful of IL-1. Sequencing of the amino terminal revealed homology with sequences predicted from those ofIL-1 cDNAs. It can, therefore, be postulated that the interferon-inducing 22K factor belongs to the IL-1 family. © 1985, Elsevier Science Publishers B.V., Amsterdam
0167
4919/851502.00
Tolerance and interleukin 2 deficiency SIR,
We read with interest the article by M. Malkovskp and P. B. Medawar (Immunol. Today 1984, 5, 340-343) in which they propose that tolerance will result from an immunological encounter when there is a deficiency of interleukin 2 (IL-2). Our recent experience in marrow transplantation for high-risk acute leukemia and aplastic anemia offers an interesting parallel to this postulate. Plasma and residual mononuclear cells were removed from 14 recipients undergoing marrow transplantation after high dose cyclophosphamide, arabinosyl cytosine and total body irradiation (1200 fads in 3 days) and prior to infusion of donor marrow in our transplantation unit between July 1983 and December 1984. The objective was firstly to remove lymphocytes from the recipient which might incite a response of donor lymphocytes against the recipient, and secondly to eliminate various tissue antigens cleared from the tissue and present in the plasma which the donor lymphocytes might recognize as foreign. Mononudear cells ranging from4 x 107to5 x 10S (average 2.1 x 10s) cells were removed by plasmalymphocytapheresis. In these 14 patients, graft vs host disease (GVHD) developed in only 2 (14%), and a mild generalized maculopapular rash in only 1. This contrasts with the 40-60% incidence seen in most transplant centers. We analysed the phenotype of cells recovered from 5 of the patients using monoclonal antibodies and an Ortho cytofluorograph. The differential phenotype as a percent of total cells is as follows: 55% T3 ÷, 45% T4 +, 22% T8 +, 27% T10 +, 17% Leu 11 ÷, 26% M1 + and4% M3 +. To test the functional ability of these cells removed from the recipient, we stimulated them with phytohemagglutinin and Daudi cells for 48 h and then assayed for IL-2 activity. These cells, after high-dose chemotherapy and irradiation, produced 1.5 times the amount of IL-2 generated by untreated, normal lymphocytes stimulated in the same way. Similarly, we have noted high IL-2 activity in lymphocytes of patients with head and neck cancer immediately after irradiation. Thus, plasma-lymphocytapheresis removed a very good source of IL-2 production in
Immunology Today, voL 6, No. 8, 1985 these patients. It may also have removed antigens broken down from the tissue. Recipients of marrow transplant for treatment of severe combined immune deficiency (SCID), leukemia and aplastic anemia have insignificant or no G V H D if their pretransplant natural killer (NK) activity against herpes simplex virus type 1 infected fibrohlasts is low or absent 1. A recent observation that N K activity depends on IL-2 suggests that the low incidence of G V H D in these patients may be due to low IL-2 production 2. The association betwen the low incidence of G V H D in our transplant patients with removal of residual lymphocytes and plasma may be significant. Removal of lymphocytes capable of producing IL-2, thus depriving donor lymphocytes of a stimulating factor in the initial period of
Activity and chronopharmacology of very low doses of physiological immune inducers SIR,
V l Bocci (Immunol. Today 1985, 6, 7-9) presents arguments in favor of his hypothesis that interferon 'is produced at a continuously low, physiological level that must be maintained for correct immune function to occur' and that 'trace amounts of exogenous and endogenous inducers act as physiological stimuli in the lymphoid tissue...'. In support of Bocci's hypothesis, we report here the effect of very low doses of interferon (IFN) on the humoral and cellular immune response of mice treated intraperitoneallyl,2. We observed that 8 international units (IU) and 8 x 10 -1° I U of murine a,fl IFN (kindly provided by M. Tovey) caused a significant increase in the humoral immune response of Swiss mice to sheep red blood cells (SRBC) using theJerne plaque-forming cell (PFC) technique (P < 0.01), and we found that 16 x 10 -1° IU of a,/~ IFN significantly stimulated (P < 0.001) the cellular immune response in C57BL/6 mice to P815 mastocytome cells using the 5'Crrelease technique 2. All control mice received solvent intraperitoneally under the same conditions as the test mice.
We also studied the action of thymic hormones on young Swiss and New Zealand Black (NZB) mice challenged with SRBC (a thymus-dependent antigen) by the PFC technique. We found that the very low doses studied, namely 400pg, 4 x 10-4pgand4 x 10-Spgof
marrow transplantation, may produce tolerance between the graft and the host. This interpretation supports the observation that donor marrow composition has little impact on the occurrence or severity o f G V H D 3. [~ ANDREW T. HUANG ROBERT F. HUNTER PATRICIA A. ROTH NELDA G. MOLD
Department ofMedicine, Duke UniversityMedical Center, Durham, NC27710, USA
References 1 Lopez, C., Kirkpatrick, D., Soren, M,, O'ReiUy, R., and Ching, C. (1979) Lanceti, 1103-1106 2 Domzig, W., Stadler, B. M., and Herberman, R. B. (1983)J. Immunol. 130, 1970-1973 3 Jansen, J., Goselink, H. M., Veenhof, W. F. J., Zwaan, F. E. and Blotkamp, C. (1983) Exp. Hematol. 11,967-973
thymulin per mouse, had an immunoregulatory action : the hormone was immunodepressive (P < 0.05) for the normal Swiss mice 3 and immunostimulating (P < 0.001) for immunodeficient NZB mice 4. In addition, we observed that 8 x 10- s pg of thymulin depressed the cellular immune response in C57BL/6 mice 2. Furthermore, we studied the effect on the humoral immune response over a 16-month period of the same low doses ofthymosin fraction 5 (kindly provided by A. Goldstein) on Swiss mice. Our results indicate that this hormone amplifies the circannual variation of the humoral immune response 5'6 at all doses studied 7. Analysis of these results using the Cosinor method showed that the acrophase was in the winter season (4 × 10- 4 pg : January 10 _+ 17 days and 4 x 10-a pg: December 25 _+ 24 days; P < 0.001). w e conclude that doses as low as 10-s IU or 10-8 p g of natural mediators such as IFN or the thymic hormones, respectively, are pharmacologically active and that they do not perturb but rather amplify the natural seasonal variation of the humoral immune response. We suggest that for best therapeutic efficacy in immunodisequilibrated subjects immune mediators should be used at very low doses so as to maintain homeostasis. These studies were supported by Dolisos Laboratories (J. Guillernain, Scientific Director). ~[~ M. BASTIDE M. DOUCET-JABOEUF V. DAURAT Universitt de Montpellier I, Unit~de Recherchesen Immunologic, Facult~de Pharmacie, 34060 Montpellier Cedex, France.
235
Immunology Today, vol. 6, No. 8, 1985 References 1 Doucet-Jaboeuf, M., Pelegrin, A., Sizes,
M., Guillemain,J. and Bastide, M. (1985) Int..]. ImmunopharmacoL 7,312 2 Daurat, V., Sizes,M., Pelegrin,A., DoucetJaboeuf, M., Guillemain,J. and Bastide, M. (.June 1985) Symposium Marqueurs Inflam., Lyon, France 3 Doucet-Jaboeuf, M., Guillemain, J.,
Piechaczyk, M., Karouby, Y. and Bastide, M. (1982) C.R. Acad. Sci. 295,283-286 4 Doucet-Jaboeuf,M., Pelegrin, A., Cot, M. C., Guillemain,J. and Bastide, M. Abstract~ Soc. Fr. ImmunoL, May 1984 5 Mac Murray, J., Barker,J., Armstrong,J., Bozzetti, L. and Kuhn, I. (1983)LifeSci. 32, 2363-2370
6 Reinberg, A., Schuller, E., Delasnerie, N., Clench, J. and Helary, M. (1977) La Nouv. PresseMed. 6, 3819-3823 7 Doueet-Jaboeuf,M., Pelegrin, A., Cot, M. C., Guillemain,J. and Bastide, M. (1984) Ann. Rev. Chronopharm. 1,231-234
) Interlcukin 1: amino acid sequencing reveals microheterogeneity and relationship with an interferon-inducing monokine A. Billiau, G. Opdenakker, J. Van Darnme, M. De Ley, G. Volckaert and J..Van Beeumen q" Interleukin 1 ( ! L - l ) is the n a m e give n in 19791 to a set of factors involved in the specific i m m u n e response and previously k n o w n u n d e r different names: mitogenic protein, T-cell replacing factor, B-cell activating factor, B-cell differentiation factor and lymphocyte activating factor (LAF). Currently, the t e r m IL- 1 is mostly used as an a c r o n y m for L A F , the latter being defined as the factor which, u n d e r physiological circumstances, is p r o d u c e d by antigen-presenting Inononuclear phagocytes (monocytes, macrophages) and which can activate antigenrecognizing T lymphocytes to produce interleukin 2 (IL2). IL-2, in turn, is the subsequent link in the chain of events leading to production of antibodies and cellmediated responses to the antigen. T h e r e is now growing evidence (for review, see Ref. 2) that IL-1, as a family name, also encompasses a n u m b e r of factors which are involved in nonspecific host resistance and inflammation: endogenous or leukocytic pyrogen (EP or LP), leukocytic endogenous mediators ( L E M ) , monocytic cell factor ( M C F ) , and others. E P or L P is defined by its ability to cause fever and alterations in white blood cell counts (leukopenia followed by hyperleukocytosis), both p h e n o m e n a resembling the effects of bacterial lipopolysaccharides. L E M is defined by its ability to induce acute phase components and M C F by its ability to induce production of collagenase and prostaglandin Fa by synovial cells. V a n D a m m e et al. 3 have isolated an antiviral factor (22K factor) from mitogen-stimulated m o n o n u c l e a r cells (Fig. 1). T h e antiviral effect of this factor was shown to be due to induction of interferon-fl in fibroblasts s'4. H o w e v e r , biological tests on the purified product led t h e m to conclude that it also had m a n y of the properties assigned to IL-1 (i.e. L A F , M C F and E P activities) 5. Rega Institute, University of Leuven, Belgium. tLaboratory of Microbiology, Faculty of Sciences, State University of Ghent, .Belgium.
I
MITOGENSor ANTIGENS
[
~ Monoeyte
T-lymphocyte
HulFN-~ fibroblasts
o
V
antiviralstate of tissue Fig. 1. An antiviral monokine related to IL-1, which acts by inducing interferon. The 22K factor was originally discovered as an interferon-like antiviral protein. Most significantly its antiviral effect could be blocked by antiserum against human interferon-/~ (HuIFN-/~). However, careful study revealed that the antiviral effect of the factor was due to its ability to induce HulFN-fl. Further study on the biological effects showed that the electrophoretically pure factor had several activities remindful of IL-1. Sequencing of the amino terminal revealed homology with sequences predicted from those ofIL-1 cDNAs. It can, therefore, be postulated that the interferon-inducing 22K factor belongs to the IL-1 family. © 1985, Elsevier Science Publishers B.V., Amsterdam
0167
4919/851502.00