Recombinant human interleukin 2 adjunctive therapy

It has long been noted that cell-mediated immunity may be impaired in patients with tuberculosis. This abnormality is associated with a deficiency in IL-2-induced T cell proliferation, reduced numbers of IL-2-responsive cells in the circulation and decreased IL-2 receptor expression on blood leukocytes (Toossi et al 1986, Schauf et al 1993, Wallis & Ellner 1994). Our previous studies using recombinant human (rhu) IL-2 adjunctive therapy in patients with leprosy and with Leishmania infections suggest that IL-2 may be effective in enhancing cell-mediated immunity (Hancock et al 1989, 1991, Kaplan et al 1989, 1991, Converse et al 1991, Akuffo et al 1990). When low dose rhuIL-2 was administered to leprosy patients, there was an increased dermal cell-mediated immune response, as well as an increased production of IFN-y, resulting in a more rapid clearance of Mycobacterium leprae from the skin as compared to multidrug therapy alone (Kaplan et al 1989, 1991). Since IFN-y is an important inducer of the Th1 protective immune response, the observation that rhuIL-2 treatment of patients induces the production of this cytokine suggests that IL-2 may co-ordinate the Th1 type protective response in patients.

Recombinant human interleukin 2 therapy in tuberculosis patients

We conducted a patient-based investigation using rhuIL-2 as an adjunct to drug therapy in tuberculosis to evaluate the safety of this approach and to determine whether IL-2 can affect the response and outcome in these patients. Our results showed that rhuIL-2 administration in combination with multidrug therapy is safe (Johnson et al 1995). Daily rhuIL-2 administered at a low dose (12.5 ßg twice daily) for 30 days to patients was associated with immune activation, as manifested by increased numbers of CD25+ and CD56+ leukocytes in the peripheral blood. The proliferative response of lymphocytes to purified protein derivative of tuberculin

(PPD) and the percentage of IL-2-responsive cells in the circulation of the treated patients was estimated by assaying the [3H] thymidine incorporation into peripheral blood mononuclear cells (PBMC) in vitro following exposure to exogenous PPD and/or IL-2. At the pre-study time point, the per cent of responsive cells was the same as that seen in PBMC from PPD+ normal controls. However, following rhuIL-2 therapy for 30 days, the PBMC of treated patients showed a statistically significant increase in the frequency of cells capable of proliferation in response to PPD (p < 0.006) and IL-2 (p < 0.005) (Johnson et al 1995).

Elevated soluble IL-2 receptor (sIL-2R) levels have been associated with immune activation (Deehan et al 1995) and may correlate with the patient clinical response to rhuIL-2 immunotherapy (Deehan et al 1994, Gooding et al 1995, Mangge et al 1995). We therefore assayed sIL-2R levels in multidrug-resistant tuberculosis patient plasma at pre-, mid- and post-study time points. There was a significant increase in plasma sIL-2R of treated patients at the mid-study time point, which decreased to baseline after the last injection of rhuIL-2 (johnson et al 1998a).

Effect of recombinant human interleukin 2 therapy on cytokine mRNA levels in patient leukocytes

To investigate the effect of exogenous rhuIL-2 administration on the expression of cytokine mRNA in patient leukocytes, we used semi-quantitative reverse transcriptase (RT)-PCR to assay cytokine mRNA levels. IFN-y mRNA was observed in PBMC obtained from all tuberculosis patients tested, while IL-2 mRNA levels were lower (Fig. 3). Following rhuIL-2 therapy, IFN-y mRNA levels in patient PBMC showed decreases at both the mid - and post -study time points when compared to the pre-ILy-2 treatment time point (Johnson et al 1995, 1998b). When the ratio of IFN-y mRNA to CD3^ mRNA was calculated, a decrease in the level of IFN-y mRNA expression per T cell during and immediately following rhuIL-2 therapy was observed (Johnson et al 1998b). No change was observed in control untreated patients. Thus, rhuIL-2 therapy was associated with a reduction of IFN-y levels in blood leukocytes. Other cytokine mRNAs, including TNF-a, IL-4, IL-10 and IL-12, did not show obvious changes in PBMC in response to rhuIL-2 therapy.

mRNA expression ofIFN-y and IL-2 was higher in the PPD skin test site than in cells of the peripheral blood of the same patients (Fig. 3). IFN-y mRNA expression was also higher than IL-2 mRNA expression in the cells at this site. The effect of rhuIL-2 administration on cytokine gene expression at the PPD site was studied. In

FIG. 3. A representative Southern blot of serially diluted RNA isolated from peripheral blood mononuclear cells and purified protein derivative of tuberculin (PPD) biopsy sites of multidrug-resistant tuberculosis patients. Total RNA from the samples was amplified by reverse transcriptase-PCR and hybridized to radiolabelled probes specific for y-interferon (IFN-y) and interleukin (IL)-2 mRNA.

FIG. 3. A representative Southern blot of serially diluted RNA isolated from peripheral blood mononuclear cells and purified protein derivative of tuberculin (PPD) biopsy sites of multidrug-resistant tuberculosis patients. Total RNA from the samples was amplified by reverse transcriptase-PCR and hybridized to radiolabelled probes specific for y-interferon (IFN-y) and interleukin (IL)-2 mRNA.

contrast to the results obtained with PBMC we observed that the mean mRNA expression levels for the T cell cytokines IL-2 and IFN-y were increased during rhuIL-2 therapy relative to the pre-study time point.

'Differential regulation of other genes in response to recombinant human interleukin 2 treatment of multidrug-resistant tuberculosis patients

We have recently begun to utilize differential display RT-PCR to facilitate the identification of changes in expression of unselected genes as a result of rhuIL-2 treatment. PCR amplifications of RNA from biopsies of PPD skin tests placed before and during rhuIL-2 administration to multidrug-resistant tuberculosis patients (or the corresponding time points in control patients not treated with rhuIL-2) were performed. Comparison of cDNA displays generated from the RNA isolated from rhuIL-2-treated patient biopsies and from control patient biopsies were carried out (Liang et al 1993, 1995). A number of bands were found to be consistently differentially expressed (Johnson et al 1998b). These genes included components of endocytic vacuoles, enzymes of the respiratory pathway and other regulators of cellular function. The physiological importance of the differential expression of these genes is under investigation to determine their roles in leukocyte activation and in the development of an anti-mycobacterial response.

Effect of recombinant human interleukin 2 on disease manifestations

Multidrug-resistant tuberculosis presents serious problems in that patients remain infectious despite treatment. Thus, any therapy that would enhance the rate of clearance of sputum bacterial load and lead to clinical improvement would have great potential in patient management. We therefore evaluated sputum bacterial load and changes in chest radiographs during the 30 days of daily adjunctive rhulL-2 treatment. Although this study consisted of a small number of patients, a trend toward sputum bacterial clearance was seen (Johnson et al 1998a). We observed that five out of the eight multidrug-resistant tuberculosis patients who entered the study sputum acid-fast bacilli positive and received 30 days of daily rhuIL-2 injections showed reduction or clearance of bacterial burden by the end of the study. In contrast, only three of nine placebo control patients showed decrease in bacterial load. A similar trend toward improvement was also noted in the chest radiographs. Seven of 12 patients treated with rhuIL-2 adjunctive therapy demonstrated definite improvement in the chest radiographs; four patients showed highly significant improvement.

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