Journal of the Royal Society of Medicine Volume 76 June 1983 473
Immunosuppression with glucocorticoids - a possible immunological explanation for interpatient variation in sensitivity: discussion paper'
J Swanson Beck FRCPEd FRcPath Margaret C K Browning BSC Departments of Pathology and Biochemical Medicine, University of Dundee
Glucocorticoids are widely used in clinical medicine for their potent immunosuppressive and anti-inflammatory effects. The mode of action of these drugs is very complicated, so it is regrettable that most of the extensive literature on animal experimental work is irrelevant to human therapeutics since many species respond in a very different manner from man (Claman 1972). Consequently this discussion paper will concentrate, wherever possible, on results from human experiments.
The immune response Antigenic challenge may occur at any site of the body. The immune response at the portal of entry is very limited and most of the changes are concentrated in the local lymph nodes or in the central lymphoid organs (such as spleen or bone marrow), where the antigen is effectively concentrated by lymphatic drainage from damaged tissue or by selective removal from the circulation. Moreover, these tissues have specialized mechanisms for continuous removal of recirculating lymphocytes from the blood and the microanatomical conditions ensure that there is a high probability that a potentially responsive lymphocyte will meet the foreign antigen, so that the lymphocytes will be used to the best advantage of the individual (Figure 1).
Prior to antigenic stimulation, there are relatively few lymphoid cells capable of responding to any particular epitope (the region of the antigen molecule recognized by the antibody or lymphocytic receptor). The immune response has two essential components: first, by repeated division, the cells of appropriate specificity become much more numerous, the so-called 'clonal expansion' (for economy of effort, irrelevant clones remain dormant); and secondly, a large proportion of the progeny differentiate into effector cells while the remainder continue to recirculate as 'memory' cells. Attachment of antigen or hapten to the cell membrane receptors provokes proliferation, but the signal for differentiation is not yet clearly defined. The complicated cellular interactions are summarized in Figure 2. The extent of the response is partly determined by the local concentration of antigenic material (decreased by macrophage digestion and possibly accentuated by appropriate presentation on dendritic cells). However, the immune response is also controlled by various regulatory subpopulations of helper and suppressor T cells and by variations in the local concentrations of messenger molecules, some of which are antigen-specific and others nonspecific: moreover, some are stimulatory, whereas others are inhibitory (Altman 1980). The relative importance of the many humoral regulatory systems that have been described has not yet been evaluated. Currently the best understood stimulating system (T-cell growth factor) is non-antigen specific and it may prove to be the main driving force for cell proliferation (Watson et al. 1980). Stimulated macrophages release interleukin 1 (Il 1) which in turn stimulates certain T cells to synthesize and secrete Il 2, which then binds to T cells which have had the appropriate receptor induced by antigenic stimulation: binding of Il 2 to its receptor is a powerful stimulus to replication (Figure 3). Il 2 appears to be essential for the target cell to pass from early GI-phase into S-phase (Sekaly et al. 1982).
'Accepted 12 Janurary 1983
0 141-0768/83/060473-07/$O 1.00/0 ,3( 1983 The Royal Society of Medicine
474 Journal of the Royal Society of Medicine Volume 76 June 1983
Figure 1. Cell traffic through a lymph node
As the immune response develops, an increasing proportion of the cells produced by clonal expansion differentiate into effector cells. The B cells become plasma cells and synthesize and secrete specific antibody (predominantly as IgM, IgG or IgA) which can participate in various forms of reaction, such as precipitation of antigen, killing of target cells (usually involving complement), stimulation of phagocytosis by granulocytes or macrophages, or induction of tissue inflammation. The effector T cells may be cytotoxic for target cells, but their main response on exposure to antigen is the synthesis and release of a wide range of lymphokines that modify the behaviour of macrophages and other connective tissue cells to produce the complicated changes of chronic inflammation.
Effects of glucocorticoids on the immune response Glucocorticoids induce a lymphopenia with selective depletion of T cells from the circulating blood (Haynes & Fauci 1978): many of these cells appear to be sequestered in the bone marrow (Fauci 1975) but not in the spleen, since previous splenectomy does not influence the process (Beardsley & Cohen 1978). The explanation for the modification of lymphocyte recirculation by glucocorticoids is not yet clear; pharmacological concentrations reduce
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Journal of the Royal Society of Medicine Volume 76 June 1983 475
lymphocyte responses in chemotactic tests (Beer & Center 1980), but it is not known whether glucocorticoids affect cellular progress through post-capilla'ry venule or movement through the lymph nodes. Nevertheless, it is now recognized that interference with T-cell recirculation diminishes the probability of lymphocyte: antigen interaction and so is an important component of the immunosuppressive action of glucocorticoids in man.
Glucocorticoids have a relatively limited capacity to reduce an established immune response, and cell-mediated reactions are more susceptible than antibody formation. Moreover, these agents are much more potent if administered at, or just before, the time of first exposure to antigen when both cell-mediated reactions and-humoral antibody formation can be suppressed almost completely (Craddock 1978). Most of the human experimental work on the mode of action of glucocorticoids as
immunosuppressives has been performed on lymphocytes isolated from the peripheral blood, since they are easily accessible for repeated testing. Most of the studies have examined the effect of glucocorticoids on lymphocyte growth (as a measure of clonal expansion) in preference to the study of differentiation. Moreover, purified antigens have been used in a minority of the studies and the usual approach has been to assess proliferation induced by purified plant proteins (mitogens) which stimulate large subpopulations: this artificial stimulus has been used because the response can be measured much more easily and precisely.
There is strong evidence (summarized by Cupps & Fauci 1982) that glucocorticoids administered in vivo or in vitro depress the response of peripheral blood lymphocytes to various forms of stimulation (mitogen, mixed lymphocyte reaction and antigen): the slowing of cell proliferation would interfere with the immune response and must be an important component of glucocorticoid-induced immunosuppression. Glucocorticoids block the production of Il 1 and I1 2, but neither the generation of Il 2 receptors on the stimulated cells (Gillis et al. 1979a,b, Larsson 1980) nor the intrinsic lymphocyte growth mechanisms are affected. T cells appear to be more sensitive than B cells to suppression of in vitro growth by glucocorticoids (Blomgren & Andersson 1976), but it is possible that this may reflect the relative sensitivities of the different lymphokines and monokines. Evidence that the accessory cells may be the major target for glucocorticoids is provided by the observation that lymphocytes in culture are more sensitive to glucocorticoids during the initial period in culture when interleukins are being generated, than at later periods when the accessory cells have a more subservient role (Ramer & Yu 1978, Neifeld & Tormey 1979). Experimental studies of the action of glucocorticoids on suppressor cells are conflicting with reports of abrogation (Haynes & Fauci 1979) and of augmentation (Hirschberg et al. 1980) of various forms of concanavalin A-induced suppressor cell function. Since relatively little is known of the mechanisms of induction of differentiation into plasma cells, it is hardly surprising that the mechanism by which glucocorticoids suppress antibody production has not yet been explained, but it is currently thought that again the effect is mediated through the accessory cells, as well as by direct action on the B-cells (Cupps & Fauci 1982). It is abundantly clear that glucocorticoids do not have a single action on the immune response, and that these drugs interfere with several interacting processes to cause particularly powerful suppression of the early response to antigens.
Cellular basis of glucocorticoid hormone action During the last decade there have been important advances in clarifying the mode of action of glucocorticoid hormones at the cellular level (Chan & O'Malley 1978, Baxter & Funder 1979). It now appears that these hormones act in a similar manner on all cell types. The processes are summarized in Figure 4: (1) After diffusion through the cell membrane into the cytoplasm, the glucocorticoid binds to cytoplasmic receptors. (2) The glucocorticoid-receptor complex translocates into the cell nucleus; this process has recently been visualized (Papamichail et al. 1980).
476 Journal of the Royal Society of Medicine Volume 76 June 1983
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Journal of the Royal Society of Medicine Volume 76 June 1983 477
Hydrocortisone Dose Response
a
20 p Subject K(+) Slope = + 0.92 Subject L (*) Slope = - 6.67
3.5 x 104 5.6 x 10.8 1 x 10.6 1.5 x 10-5 2.3 x 104
Log M Dose
Figure 5. Method for calculation of a patient's dose- response to hydrocortisone
3.5x109 5.6x108 1x104 1.5x105 2.3x 104 Log M Dose
Figure 6. Dose-response curves for two patients at the extreme ends of the observed distribution in a study of normal subjects
this response curve, and assays carried out on blood samples removed at intervals over a
period of 4-6 months from normal individuals have been shown to give consistent results. In a study of 125 normal subjects, the slope of the log-dose response to hydrocortisone was shown to have an approximately normal distribution. Peripheral blood mononuclear cells from patients with a wide range of diseases (rheumatoid arthritis, systemic lupus erythematosus, polymyalgia rheumatica, sarcoidosis, Crohn's disease and ulcerative colitis) also show a similar distribution of slopes of the log-dose response. At the extremes of the population (Figure 6) there are some subjects who have a very flat response so that cell growth is virtually unaffected, even at concentrations far greater than those likely to be achieved by pharmacological doses of glucocorticoids. There is also a small number of subjects whose cells are very sensitive and in whom growth is very seriously curtailed even at
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478 Journal of the Royal Society of Medicine Volume 76 June 1983
moderate concentrations. Most subjects show an intermediate response with some effect becoming obvious at 10-' mol/l and with only 60-70% cells growing at 10'7 mol/l. Assuming that lymphocytes in the main lymphoid organs behave in a similar manner to
peripheral mononuclear cells, that cells from different individuals behave similarly with regard to the rate of growth and life span, and that similar blood levels of glucocorticoid are attained during the course of treatment, then it is possible to calculate the rate of increase of numbers of stimulated cells for any subject. Figure 7 shows the results of calculations of the effect of reduction in percentage of replicating cells on the build up of cell numbers in a population where the total cell cycle time is one day. Curve A shows the behaviour of cells without growth suppression. Curves B and C show the effect of 20% and 40% suppression of growth fraction: these conditions were selected since they correspond to the extent of growth inhibition encountered at the lower and upper blood concentrations achieved during glucocorticoid therapy in the average subject. Starting from 100 cells, there are 1600 after 4 days growth without suppression, but only 1050 and 655 with 20% and 40% suppression. Thus suppression retards the growth in cell numbers and the effect becomes greater the longer it lasts. When the same dose of glucocorticoid is given, a responsive subject (such as subject L in Figure 6) will, within a few days, show a marked reduction in the number of new lymphocytes available to enter the circulation, compared with an unresponsive subject (such as subject K in Figure 6). This may be the way in which glucocorticoids exert their main immunosuppressive action. The possibility of employing this in vitro test to predict the likely clinical response
of individual patients to glucocorticoid therapy is currently being evaluated in our laboratories. Preliminary studies suggest that a steep dose-response curve is associated with a good response to treatment, but many more patients must be studied prospectively before any claims can be made for the test as a prognostic indicator. In subjects showing little or no inhibition of lymphocyte growth by glucocorticoids in vitro, it would seem logical to employ alternative therapy if immunosuppression is required.
Conclusion Glucocorticoids, because of their different actions in laboratory animals and man, have proved to be difficult drugs to study and, as yet, their exact mode of action in suppressing the immune response is not fully understood, although some aspects have now been elucidated as a result of recent in vitro studies. Despite our lack of understanding of the mode of action of glucocorticoids these drugs remain valuable therapeutic agents, but unwanted and sometimes damaging side effects affecting mineral, carbohydrate, protein and fat metabolism have restricted their use in clinical practice. It is to be hoped that the identification of patients who are unlikely to benefit from glucocorticoid immunosuppressive therapy will prevent these patients being given large doses of glucocorticoids, with the consequent development of iatrogenic disease: there would then be less inihibition in the prescription of glucocorticoids for those patients who are most likely to benefit from their administration.
Acknowledgments: We are grateful to Mrs H Fawkes for drawing the diagrams, and to Mr PR S Fawkes for their preparation for publication. Our current investigations are being supported by a grant from Messrs Glaxo Research Ltd and we have been ably supported in the laboratory by Miss C Roberts and Mr R C Potts.
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Journal of the Royal Society of Medicine Volume 76 June 1983 479
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