Granulysin is a cytolytic granule protein with a broad range of antimicrobial and tumoricidal activities. Recently, two short synthetic peptides derived from granulysin, have been shown to inhibit the in vitro
growth of clinical isolates of both multidrug resistant and drug susceptible strains of M. tuberculosis
Relevant to M. tuberculosis
infection, most T cell subsets with anti-mycobacterial activity express granulysin2-6
and, at least for some of them, requirement for granulysin has been found as a requisite to kill both intracellular and extracellular M. tuberculosis
inferring an important role of granulysin in the protective immune response against M. tuberculosis
. Accordingly, recent in vivo
mouse studies have demonstrated that delivery of a recombinant plasmid containing full-length cDNA of granulysin significantly reduced the numbers of viable bacilli in the lung lesions indicating that granulysin has a therapeutic effect against M. tuberculosis
In another study, a viable therapeutic vaccine of recombinant M. smegmatis
containing the IL-12 and the granulysin genes induced efficient protective immune response against M. tuberculosis
Because of the importance of correlates of protective immune responses as potential tools to evaluate the efficacy of TB interventions, including therapy or vaccination, we decided to measure serum granulysin levels in children affected by TB and healthy controls, in order to assess whether serum granulysin levels correlated with disease activity and the clinical outcome after therapy.
Results here reported show that serum granulysin levels before the initiation of TB therapy were significantly lower in children with TB compared to controls, with the lowest levels being found in TB patients who were PPD skin test negative at the time of diagnosis. Interestingly, we did not find any significant differences between serum granulysin levels and clinical severity (mild/moderate or advanced) of pulmonary TB or the clinical form (pulmonary or extrapulmonary) of TB. At four months after completion of therapy, serum granulysin levels were increased in children with TB, not significantly different to values observed in control children.
In this study, we were unable to determine granulysin levels in relation to treatment failure, due to the low number of failure cases in our cohort (one case). However, after TB therapy, granulysin levels decreased in three patients and remained unchanged in additional six patients. Thus, a total of 9 out of 72 (12.5%) did not show any increase in serum granulysin. In the accompanying paper by Sahiratmadja et al., granulysin remained low in 17.5% of patients with mild/moderate TB and 23.7% of patients with advanced TB. Therefore, additional longer follow-up studies are necessary to obtain better insight into the dynamics of serum granulysin levels after anti-TB therapy.
The finding of increased serum levels of granulysin at four months after completion of therapy was paralleled by the increased in vitro
mycobactericidal activity of sera from TB patients, which correlates with granulysin concentration. These results are worth of additional comments. Granulysin has been shown to kill M. tuberculosis
at higher concentrations than those found in sera.10
Therefore, it is possible that other components in sera contribute to the killing of M. tuberculosis
. Antibacterial activity of sera does not appear to be attributable to administration of antituberculous drugs on the grounds that (a) sera were collected four months after completion of therapy to allow washout of drugs from serum and (b) we did not find any difference in serum granulysin in 40 healthy PPD+
children before, and four months after completion of a nine-month isoniazide prophylaxis (our unpublished observations). Thus, it is likely that the increased serum anti-tuberculous activity post therapy could be attributable to increased concentration of peptides with anti-mycobacterial activity, other than granulysin. Further studies with neutralization of granulysin activity by specific antibodies are required to confirm this hypothesis.
Previous studies have correlated granulysin levels with other pathological states. These include the analysis of granulysin expression in leprosy model as a relevant marker for effective host defence.6
T cells expressing granulysin, especially CD4 cells, were found more abundant (8–15%), in the skin lesions of patients with the localized tuberculoid form as compared to those with the disseminated lepromatous form of disease, suggesting a correlation between the clinical form of disease and the frequency of T cell expressing granulysin: localized disease is associated with increased granulysin expression. Similarly, children affected by TB have lower levels of serum granulysin than healthy contacts, i.e. those subjects who control M. tuberculosis
In addition, high granulysin serum levels were found in primary viral infections, such as those with parvovirus B19 and Epstein-Barr virus,12
especially during the acute phase of disease and thereafter granulysin levels rapidly decrease to normal range during the convalescent phase.12
A transient granulysin increase was also found in both lung19
of transplant rejection and measurement of serum granulysin concentration has been suggested as a useful marker of graft-versus-host reaction in patients with hematopoietic stem-cell transplantation.21
Also in preeclamptic patients, serum levels of granulysin were significantly elevated when compared to those of normal pregnancy subjects.10
By contrast, very low granulysin serum levels have been detected in patients with severe immunodeficiencies12
and a reduced expression of this protein in different cell types or even in serum is observed in carcinoma patients, which could be correlated with tumor progression.14,22,23
The reason for the reduction of serum granulysin concentration during active TB and its recovery after therapy is unknown. One possibility is that granulysin is rapidly consumed during active disease, because of the ongoing effector immune response. Another possibility is that serum granulysin is reduced during active disease because of a reduction of the T cell subset dedicated to its production. In agreement with this possibility, we previously reported that γδ
T cells expressing granulysin are decreased during active TB, but they recover after therapy.15
Similarly, effector CD8 T cells expressing perforin (and presumably also granulysin) specific for the mycobacterial antigen 85A, are also decreased during active TB, but recover after therapy.24
Thus the decrease, or even the loss, of many different T cell subsets which secrete granulysin during active disease, might well explain the low granulysin concentration observed in such patients. Alternatively, we must also consider the possibility that the increased serum granulysin levels after therapy may represent increased production of granulysin from the same number of cells, rather than an increase in numbers of granulysin-producing cells.
However, further analysis of granulysin-producing cells during active TB and after completion of therapy are required to define the underlying mechanisms.
In conclusion, results reported in this paper clearly indicate that granulysin is not a marker of TB severity but rather a marker of TB disease activity. The potential use of serum granulysin levels to monitor TB disease activity and response to treatment merits further investigation.