4. Material y m´ etodos
4.8. Evaluaci´ on y verificaci´ on de los planes de tratamiento de IMRT y VMAT
4.1 Introduction
4.2 Experimental protocol 4.3 Results and Discussion
4.3.1 Thl-Th2 cytokines in protective / pathogenic immune responses 4.3.2 Interactions between T hl and Th2 cytokines
4.1 INTRODUCTION
Mycobacterial antigens (Ags) selectively induce T h l cytokines (IFN-y and TNF-p) in humans lymphocytes in vitro (Haanen et al, 1991; Mutis
et al, 1991). Human PBMCs in vitro were found to secrete T h l cytokines on day 6-8 and Th2 cytokines on day 12 in response to live BCG (Sander et al, 1995). The early T h l response is thought to be followed by Th2 cytokine production in order to down-regulate the T h l inflammatory response. This suggests that superimposition of a Th2 response on a pre-existing T hl response occurs in tuberculosis. The regulation of T hl-T h2 balance is thought to be critical in mounting an appropriate immune response against mycobacteria.
The main aim of this chapter is to observe the production of T h l and Th2 cytokines (IFN-y and IL-4) in healthy subjects and tuberculosis patients in response to sonicated antigens of M.vaccae and
M.tuberculosis. This was followed by studying the interactions between T h l and Th2 cytokines in both groups, by testing the effect of exogenous IL-4 on their IFN-y production. The investigation involved
analysis of cytokines in the supernatants using Enzyme-linked Immunoassay (ELISA). Cytokines in the supernatant can be readsorbed or degraded. Therefore, the ELISA method measures the net amount of cytokine that remains in the supernatant after they have been utilised or removed by various cells.
4.2 EXPERIMENTAL PROTOCOL
The dose-response production of cytokines was measured by removing supernatant from the whole blood cultures set up for the lymphocyte proliferation assay. Supernatant (100 pi) was removed on the sixth day for the measurement of IFN-y and IL-4 using ELISA before the addition of radio-active thymidine for labelling the proliferating cells.
Initially, healthy people were tested by adding exogenous IL-4 at the beginning of the culture and on days 2 and 4. The addition of IL-4 from the beginning of the culture and on day 2, completely abolished the production of IFN-y production. Therefore, IL-4 was added on day 4 of culture. IL-4 (10 pi) was added to each well to give the final concentrations of 1000 Units/ml. Supernatant (100 pi) was removed on day 6 for the measurement of IFN-y.
4.3 RESULTS AND DISCUSSION
4.3.1 T H l AND TH2 CYTOKINES IN PROTECTIVE AND PATHOGENIC IMMUNE RESPONSES
Healthy people and TB patients produced similar amounts of IFN-y in response to vaccin and tuberculin (Figs. 4.1 and 4.3). Therefore, the simple presence or absence of IFN-y alone is unlikely to make the immune response beneficial or harmful. IFN-y could be involved in both protective or pathogenic immune processes. As IFN-y acts in concert with other cytokines, its protective or pathogenic effects could be modulated by the effects of other cytokines.
From the in vitro results, it seems likely that mycobacterial antigens can also be used to stimulate IFN-y in healthy individuals or TB patients. The T h l induction pattern of mycobacterium-reactive lymphocytes has been found to be independent of health or disease status (Mutis et al,
1993). Lepromatous leprosy (LL) was found to be charaterised by immune non-responsiveness to M. leprae antigens. However, multibacillary patients showed only a slightly decreased response to mycobacterial antigens from other mycobacteria eg. M.bovis BCG, and PPD from M.tuberculosis. In spite of this non-responsiveness to
M.leprae, multibacillary patients produced high levels of T h l cytokines
X.0 M .tuberculosis M.bovis (Myrvang a/, 1973).
Tuberculosis patients have lymphocytes capable of producing similar amounts of IFN-y compared to healthy controls but they can produce higher amounts of IL-4 when cultured with mycobacterial antigens (Surcel et al, 1995). Other researchers have proposed that the impairment of T hl cytokine production occurs in tuberculosis and not the over-production of Th2 cytokines (Zhang et al, 1995). My results
Figure 4.1 IFN-gamma production to vaccin in TB patients and healthy people (±SD)
700 Healthy (n=15) 600 - TB (n=14) 5 0 0 - 4 0 0 - 3 0 0 - 20 0- 1 0 0- 5 10 20 50 0.05 0.5 o Vaccin (jig/ml)
Figure 4.2 IL-4 production to vaccin in TB patients and healthy people (±SD)
150 Healthy TB D — — 50 0.5 5 10 20 0.05 o
Vaccin conc (|ig/ml)
Figure 4.1 Healthy people and TB patients produced IFN-gammain réponse to vaccin in a dose related manner. Healthy people produced a maximum level of IFN-gamma at 10|ig/ml of vaccin whereas TB patients required 20|ig/ml of vaccin. At lower doses of vaccin, healthy people produced more IFN-gamma than TB patients. In fact, the graph of the TB patients seems to have been moved to the right of the healthy people rather than downwards ie. TB patients required higher dose of vaccin to produce a similar level of IFN-gamma as healthy people.
Figure 4.2 TB patients showed slight IL-4 production in réponse to vaccin whereas healthy people showed no production of IL-4.
I
I
Figure 4.3 IFN-gamma production in response to tuberculin in TB patients and healthy people
700 600 - 5 0 0 - 400 - 3 0 0 - 2 0 0- 1 0 0- 0 Healthy (n=15) TB (n=14) Tuberculin (p.g/ml)
Figure 4.4 IL-4 production to tuberculin in healthy people and TB patients (±SD) 150 Healthy TB p<0.006 p<0.005 5 0 - X 0.5 5 10 20 50 0.05 o
Tuberculin conc. (|ig/ml)
Figure 4.3 Both healthy people and TB patients produced a similar amount of IFN- Y in response to tuberculin.
The level of IFN-y produced by tuberculin was higher than vaccin (fig.4.1) in healthy people and TB patients.
Figure 4.4 Healthy people produced no IL-4 in response to tuberculin. TB patients produced significantly higher levels of IL-4 for 5jig/ml or higher concentrations of tuberculin (Mann-Whitney test). TB patients also produced higher levels of IL-4 to tuberculin than vaccin.
show that tuberculosis patients have an undiminished T h l response but an elevated Th2 response, especially to tuberculin. The superimposition of Th2 cytokines over a T hl state leads to a mixed T hl+ T h2 response. The suppressive effects of IL-4 and IL-10 on the T h l response is believed to result in the chronicity of infection (Yamamura et al, 1992).
It is notable that the ratio of T hl:T h2 cytokine production depends on the disease status of the subject as well as the mycobacterial antigen. Both vaccin and tuberculin induced a higher T hl:T h2 cytokine response in healthy people compared to TB patients. Although vaccin produced lower levels of IFN-y than tuberculin, it gave a higher T hl:T h2 response in TB patients. Therefore, vaccin is likely to be better suited for the immunogenic property of stimulating a T hl response.
TB patients produced IL-4 mainly in response to tuberculin (Fig. 4.4). Healthy people failed to produce IL-4 in response to vaccin o r tuberculin (Figs. 4.1 and 4.3). This suggests that IL-4 production is a marker of the disease process in TB and it also appears to be linked with a response to antigens derived from M.tuberculosis rather than to
M.vaccae. Generally, IFN-y and IL-4 are known to be inhibit each other’s production. However, TB patients showed a higher level of IFN-y in response to tuberculin than vaccin in spite of TB patients producing higher levels of IL-4 in response to tuberculin than vaccin. This suggests that vaccin and tuberculin stimulate IFN-y production differently. Their differences might be accounted by other cytokines apart from IFN-y which are induced by vaccin and tuberculin.
4.3.2 INTERACTIONS BETWEEN T H l AND TH2 CYTOKINES
T hl and Th2 cells cross-regulate each other via their cytokines. Thus, T hl cells inhibit the production of Th2 cytokines whilst potentiating the production of T hl cytokines. This should lead to the establishment of dominant T hl or Th2 cell populations. There are likely to be homeostatic regulatory mechanisms to dampen any excessive march in one direction. For example, the early wave of the T h l response following infection by intracellular parasites is thought to be followed by a physiological feedback response consisting of Th2 cytokine production in order to down-regulate the T hl inflammatory response. There might also be critical switches responsible for the shift from one state to another during pathogenesis.
This study found that the interactions between T h l and Th2 cytokines were different in healthy people and TB patients. IL-4 suppressed mycobacterial Ag-stimulated IFN-y production of healthy people but not TB patients (Fig 4.5). IL-4 actually enhanced IFN-y production of some TB patients in response to tuberculin (Fig.4.6). Different effects of exogenous IL-4 on IFN-y production of healthy people and TB patients suggest that the functions, as well as the amounts of cytokines produced are altered in disease. The sequence in which different T h l and Th2 cytokines are produced determines the eventual composition of cytokines. Other Th2 cytokines apart from IL-4 such IL-10 could be involved in regulating the IFN-y production. Absence of these cytokines might be responsible for the excessive production of IL-4 in TB patients.
Figure 4.5 The effect of exogenous IL-4 on IFNgamma production in response to vaccin (±SD) 800 □ n o IL-4 □ + IL -4 Q 0 1 6 0 0 - p<0.003 T3 2 H ealthy TB R esp o n se to va ccin ± IL -4
Figure 4.6 The effect of exogenous IL-4 on IFNgamma production in response to tuberculin (±SD) Q d 8 0 0 p<0.007 no IL-4 6 0 0 - 4- IL-4 p<0.002 4 0 0 - 200 - H ealthy TB R esp o n se to tuberculin ± IL -4
Figures 4.5-4.6: IFN-gamma production of healthy people (n=10) to IQiig/ml of vaccin (fig.4.5) and tuberculin (fig.4.6) was suppressed by exogenous IL-4. In contrast, TB patients (n=10) showed no difference in their IFNgamma production in response to vaccin following exposure to IL-4 (fig.4.5). IFNgamma production of TB patients to tuberculin was actually enhanced after the addition of IL-4 (fig.4.6). The p values in the figures were derived from the Wilcoxon signed rank test.
Although T cells are generally believed to be the main regulatory cells, cytokines produced by other cells such as macrophages can also modulate the T cells in the early stages of the immune response. These monokines can be divided in to pro-inflammatory cytokines (T N F -a, IL-1, IL-6 and IL-12) which promote the T hl response and anti inflammatory cytokines (IL-4, IL-10 and TGF-P) which select the Th2 response. However, macrophages and T cells in humans are found to produce mixtures of these cytokines. B cells are also recruited as antigen-presenting cells (APC) when the antigen load is high. Macrophages usually produce IL-12 which help select the T h l cytokines whereas the B cells are thought to select the Th2 response. Therefore, the fine tuning of the cytokine response might be achieved by a combination of cytokines from different cells acting at different stages of the immune response.
T hl and Th2 cells are believed to be derived from common precursors which are uncommited or ThO cells. They might be preferentially activated to produce particular cytokines by certain antigens. Environmental mycobacteria responsible for protective priming might be able to “lock in” T hl mode while unfavourable sensitisation might give a Th2 response. Different doses of mycobacterial antigens from
M.vaccae were found to produce different cytokine responses in mice (Hemandez-Pando and Rook, 1994). A lower dose consisting of 10^ killed M.vaccae yielded a T hl response whereas 10^ killed M.vaccae
resulted in a mixed Thl+Th2 response.
The properties of common antigens from fast growing environmental mycobacteria to give small, non-necrotic skin-test responses and also to reduce skin-test to antigens from some slow growing species (Nye et al,
cytokines may dampen the excessive T hl inflammatory response, and indirectly also inhibit the pathogenic Th2 response. The early Th2 response might even be protective. Different Th2 signals may be operating at low-dose and high dose induced responses. Large amounts of a late suppressive signal such as IL-4 may be produced only if the early Th2 factors are not sufficient to dampen the early T h l response. In fact IL-10 was found to be raised along with IFN-y in tubercular pleurisy compared to non-tubercular pleurisy (Barnes et al, 1993) and has been correlated with the protective immune response linked with this condition. Thus, IL-10 might serve as the early protective Th2 signal.
Continuous exposure to mycobacterial Ags might select immune ceUs which repond differently. A favourable sensitisation process may involve a ‘locked-in’ response to common Ags. Early Th2 cytokines might be responsible for preventing excessive lymphocyte proliferation (LP) and IFN-y production. This might also account for the generally lower LP and IFN-y secretion in response to the M.vaccae antigens (even at high doses) compared with M.tuberculosis antigens. An unfavourable sensitisation process may result in bypassing the early protective Th2 response.
This expanded hypothesis explains how the moderate T h l response with ‘early non-pathogenic Th2 signals’ might be responsible for the non- necrotic small skin-test responses given by common antigens; and the higher T h l response combined with a ‘late and large pathogenic Th2 signal’ which might be indicative of necrotic skin test responses to tuberculin existing in the tuberculosis patients. Indeed, the TB patients produced IL-4 mainly in response to tuberculin rather than to vaccin. Unfortunately, the TB patients were not skin-tested to check if the IL-4 production was associated with a necrotic skin-test response.
The main requirement for developing a suitable immunotherapeutic agent appears to be able to induce suitable conditions for IFN -y to exert its beneficial effects. The low dose of mycobacterial antigens is predicted to correlate with a protective T h l state whereas the high dose of mycobacterial antigens may induce an inflammatory T h l or mixed T hl+T h2 state. A suitable immunotherapeutic strategy should utilise an immunisation protocol geared towards administering the optimum dose. Individuals can also show variable responsiveness to a particular dose of immunotherapy owing to their genetic variations. For example, genetic polymorphism on cytokine genes or receptor genes could affect the amount of cytokine produced as well as receptor affinity on binding to their receptors.
An ideal immunotherapeutic agent should also inhibit the Th2 response to prevent the mixed T hl+T h2 state which may mediate the immunopathogenic process. The common antigens, by inducing early Th2 factors, may prevent an excessive increase in the T h l response and indirectly, also avoid the late Th2 response. The property of M.vaccae
antigens to give a lower proliferation response (chapter 3; figure 3.2-3) and lower IFN-y than with M.tuberculosis antigens (Fig. 4.1-3) from human lymphocytes could be due to protective anti-inflammatory Th2 factors which might indirectly suppress the pathogenic Th2 response as well. This property of M.vaccae might be useful as a T h l adjuvant. However, an immunotherapeutic agent should actually reduce any on going excessive inflammatory T h l response as well as the pathogenic Th2 response which is likely to be present in mycobacterial patients. These two seperate goals of immunotherapy have to be clearly delineated since they might require two separate mechanisms.