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(Journal of Leukocyte Biology. 2000;68:538-544.)
© 2000 by Society for Leukocyte Biology

A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice

Helen Briscoe*,{dagger}, Daniel R. Roach*,{dagger}, Natalie Meadows*, Deborah Rathjen{ddagger} and Warwick J. Britton*,{dagger}

* Department of Medicine, University of Sydney, NSW 2006, Australia;
{dagger} Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No. 6, Newtown, NSW 2042, Australia; and
{ddagger} Peptech Ltd. North Ryde, New South Wales 2113, Australia

Correspondence: Dr. Helen Briscoe, Department of Medicine, Blackburn Building DO6, University of Sydney, NSW 2006, Australia. E-mail: hbriscoe{at}med.usyd.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tumor necrosis factor (TNF) is required to control mycobacterial infections, but its therapeutic value is limited by its in vivo instability and toxicity. The efficacy of a nontoxic TNF-mimetic peptide (TNF70–80) was tested in mice infected with Mycobacterium bovis bacillus Calmette-Guerin (BCG). In vitro TNF70–80 and recombinant human TNF (hTNF) acted with interferon gamma (IFN-{gamma}) to reduce bacterial replication and to induce synthesis of bactericidal nitric oxide (NO) in BCG-infected, bone marrow-derived murine macrophages. The dose-dependent inhibitory effect on bacterial replication was blocked by neutralizing anti-IFN-{gamma} and anti-hTNF mAbs. Further, n-monomethyl-L-arginine (n-MMA) and a soluble TNF-receptor I (TNFRI-IgG) blocked bacterial growth and NO synthesis. Therefore, the peptide acted with IFN-{gamma} via induction of NO synthase and signaled through TNFRI receptors. Concomitant in vivo treatment with TNF70–80 or hTNF prevented reactivation of chronic BCG infection in mice depleted of CD4+ T cells by injecting anti-CD4 antibodies. Granuloma number and bacterial load were comparable in treated, T cell-depleted mice and in chronically infected, intact animals. Thus, TNF70–80 and hTNF can modulate recrudescent BCG infection in CD4+ T cell-deficient mice.

Key Words: mycobacteria • cytokine • nitric oxide • granuloma • macrophages


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Tuberculosis (TB) remains a pressing health problem throughout the world, causing 3 million deaths each year and infecting 50–60 million, mainly children and young adults in developing countries. The failure to control TB in developing countries has multiple causes [1 ], but coexistent human immunodeficiency virus (HIV) infection adds a significant risk factor and is clearly associated with the marked increase in the prevalence of TB in Africa and the resurgence of the disease in the United States and other developed countries [2 ]. The emergence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis compounds the problem and has been associated with outbreaks of TB in hospitals and other institutions [3 ]. Development of novel or improved treatments for M. tuberculosis infection is urgently needed. Infection with mycobacteria induces granulomas, which consist largely of infected macrophages surrounded by CD4+ T cells and a mantle of CD8+ T cells, at the site of infection [4 ]. These serve to contain and eliminate the pathogen. Inhibition of CD4+ T cell function with anti-CD4 antibodies resulted in exacerbated primary mycobacterial infections [5 , 6 ] and recrudescence of chronic infections in mice [7 ]. Moreover, resurgence of M. tuberculosis and M. avium infection occurs in HIV-infected patients as CD4+ T cell numbers decline [8 , 9 ].

T cells function through a complex pattern of cytokine production and induction of bacterial killing mechanisms in macrophages. Interferon (IFN)-{gamma} produced by CD4+ T cells, CD8+ T cells, and natural killer (NK) cells stimulates effective phagocytosis and synthesis of bactericidal reactive nitrogen intermediates (RNI). Mice deficient in IFN-{gamma} fail to control M. tuberculosis [10 , 11 ], and administration of neutralizing anti-IFN-{gamma} antibodies exacerbated the infection [12 ]. Administration of recombinant IFN-{gamma} protected mice against lethal M. tuberculosis infection in some, but not all, animal models [10 , 12 ] and was a useful adjunct to chemotherapy of MDR-TB in humans [13 ]. IFN-{gamma} alone, however, is insufficient to fully activate bactericidal mechanisms in human and murine macrophages [4 ]. Other cytokines, including interleukin (IL)-4 [14 ], IL-6 [15 ], granulocyte-macrophage colony-stimulating factor (GM-CSF) [16 ], 1,25 dihydroxy-vitamin D3 in humans [17 ], and notably tumor necrosis factor (TNF) [14 , 16 ], synergize with IFN-{gamma} to enhance mycobacterial killing.

TNF is produced by macrophages after stimulation with IFN-{gamma} and in direct response to mycobacterial products. Murine bone marrow-derived macrophages (BMM) activated by a combination of IFN-{gamma} and TNF inhibited intracellular growth of M. bovis bacillus Calmette-Guerin (BCG) in vitro through RNI-dependent processes [14 ]. Mice that had disrupted genes for TNF [18 ] or TNFp55 receptor [19 ] were profoundly susceptible to infection with M. tuberculosis, and neutralization of TNF decreased resistance to M. tuberculosis [19 , 20 ] and M. bovis [21 ] in vivo. Defective TNF production may contribute to disseminated M. avium infection in humans [22 ]. Exogenous TNF treatment increased clearance in experimental mycobacterial infections [12 , 23 ], but systemic therapy is complicated by the short half-life of TNF in vivo and its propensity to induce cachexia [24 ]. To overcome this, synthetic peptide analogues of human TNF with prolonged half-life and in vivo activity have been developed [25 , 26 ]. Amino acids 70–80 were identified as essential for activity of human TNF molecules, and the stability of this peptide in vitro was enhanced by substitution of isoleucine for leucine at position 76 (unpublished results). This peptide, TNF70–80, activated human and murine neutrophils in vitro, and in a murine model, systemic treatment with TNF70–80 increased the rate of recovery and clearance of Plasmodium chabaudi [27 ]. Here, we report the efficacy of TNF70–80 in inducing bacteriostasis and preventing recrudescence in a murine model of mycobacterial infection.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacteria
M. bovis (BCG) (CSL, Melbourne, Australia) was grown in Middlebrook 7H9 broth supplemented with ACD (Difco, Detroit, MI) and 0.5% Tween-80 (Sigma, St. Louis, MO) and stored in 30% glycerol at -70°C. After thawing, the number of viable bacteria was determined by culture of serial dilutions on 7H11 agar containing OACD and glycerol for three weeks. Prior to use, the BCG were washed, suspended in RPMI 1640 (Flow, Sydney, Australia) containing 10% fetal calf serum (FCS; CSL) and 2 mM L-glutamine (experimental medium), and sonicated briefly before infection of cultured BMM or intravenous inoculation of mice. The course of bacterial infection in mice was determined by culturing serial dilutions of spleen homogenates on OACD-enriched agar.

Cytokines, peptide, and antibodies
The sequence of TNF70–80 peptide is H-Pro-Ser-Thr-His-Val-Leu-Ileu-Thr-His-Thr-Ileu-OH. The peptide was synthesized by the F-moc-polyamine method [28 ] of solid-phase peptide synthesis using PepSyn KA solid resin [26 ] and purified by high-pressure liquid chromatography (HPLC). Murine IFN-{gamma} (106 U/ml) was purchased from Genzyme (Cambridge, MA). Recombinant human TNF (hTNF; 1.7x107 U/ml) was provided by Peptech Ltd. (Sydney, Australia). Fresh dilutions of peptide and cytokines were prepared in experimental medium. The relative potencies of hTNF and TNF70–80 were compared for stimulation of nitric oxide (NO) release. TNF70–80 (5.0 µg/ml) had activity corresponding to 1000 U/ml of hTNF. Hamster anti-IFN-{gamma} monoclonal antibody (mAb) was purchased from Genzyme. Anti-hTNF mAb 054 was purified from ascites fluid by ammonium sulphate precipitation [25 ]. Isotype control mAb L5 binds to the M. leprae 18 kDa protein. A soluble form of the hTNF-receptor I (RI) composed of recombinant 55 kDa TNFRI fused to human immunoglobulin G (IgG) heavy-chain domain (TNFRI-IgG, designated p55-sf2) [29 ] was kindly provided by Dr. B. Scallon (Centocor, Malvern, PA), along with control Ig. mAb to murine CD4+ [GK1.5, American Type Culture Collection (ATCC), Rockville, MD] was purified from culture supernatant by affinity chromatography on protein-G (Pharmacia Biotech, Uppsala, Sweden).

BCG culture in BMM
Bone marrow cells were flushed from the long bones of C57Bl/6 mice into experimental medium supplemented with 5% horse serum (Trace Biosciences, Sydney, Australia) and 20% macrophage growth factors (supernatant from cultured L929 fibroblasts) and cultured for 7 days in suspension in six-well plates (Interpath Services, Sydney, Australia). BMM were harvested into experimental medium at 106 cells/ml. Aliquots (100 µl, 105 cells) were distributed into microtitre wells (Nunc, Roskilde, Denmark) and incubated for 6 h in 5% CO2 at 37°C. BMM were cultured with varying concentrations of cytokines or the peptide for 16 h. Cytokines were removed by washing in warm RPMI, and cells were infected with 106 BCG organisms and cultured for 3 days. Culture supernatants were tested immediately or stored at -70°C for nitrite measurement. In some experiments, the soluble form of TNFRI (p55-sf2) or normal IgG1 (control) was added to cultures together with cytokines at concentrations of 0.5, 5.0, or 50.0 µg/ml. In other experiments n-monomethyl-L-arginine (n-MMA; Calbiochem, San Diego, CA) was added to the culture medium together with cytokines at concentrations of 0.01–10 mM.

Assay for in vitro BCG growth
After 3 days of culture, supernatants were aspirated, and infected BMM were washed twice with warm medium, then lysed in 100 µl of 0.1% saponin (Sigma), and 1 µCi of 3H-uracil (Amersham, Amersham, UK) was added in 100 µl experimental medium. Further incubation for 24 h allowed incorporation of 3H-uracil into the RNA of viable mycobacteria. 3H-uracil incorporation was determined by liquid scintillation spectroscopy. The percent inhibition of 3H-uracil into BCG was calculated as follows: (mean incorporation of triplicate cultures without cytokines minus mean of triplicate cultures with cytokines) divided by (mean of triplicate cultures without cytokines).

NO measurements
Nitrite levels were measured with the Greiss reagent [30 ]. Briefly, Greiss reagent was freshly prepared by mixing 3.0% phosphoric acid, 1% sulphanilamide, and 0.1% n-(1-naphthyl)ethylenediamine (Sigma) in distilled water. Culture supernatants, 100 µl, were incubated with 100 µl of Greiss reagent in microtitre trays in triplicate for 10 min at room temperature, and the optical density was measured at 540 nm. Nitrite levels were calculated from a standard curve prepared with serial dilutions of sodium nitrite (Sigma) in distilled water.

Cytokine and antibody treatment of mice
SPF C57Bl/6 female mice aged 6–10 weeks were purchased from The University of Sydney Animal Facility. To modulate the course of recrudescent infection, mice were infected intravenously with 106 cfu of BCG organisms 9 weeks prior to CD4+ T cell depletion and cytokine treatment: intraperitoneal injection of 300 µg anti-CD4 mAb daily for 3 days, then 4 days later, and thenceforth, once weekly; and 0.5 µg TNF or 100 µg TNF70–80 administered intraperitoneally every second day, for 28 days.

Histological analysis
Liver tissues fixed in 10% neutral formalin, were set in paraffin blocks and stained using haematoxylin and eosin. The average number of granulomas in 10 randomly chosen high-powered (x400) fields was determined. Sections of liver, frozen in OCT (Tissue-Tek, Sakura, Torrance, CA), were incubated with rat mAb to mouse major histocompatibility complex (MHC) class II (P7/7), then biotin-conjugated goat anti-rat Ig (Caltag, San Francisco, CA), streptavidin alkaline phosphatase (Amersham), and New Fuchsine (Sigma). The average number of foci of MHC class II-positive tissue in 10 high-power fields (x400) was determined by image analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
hTNF and TNF70–80 synergize with IFN-{gamma} to inhibit growth of BCG
BMM were cultured from mouse bone marrow in medium supplemented with macrophage-growth factors. Cells harvested after 7 days in culture (83%) were CD11b (MAC-1)-positive BMM, determined by flow cytometry (unpublished results). Cultured BMM supported the growth of BCG in vitro with an optimal MOI of 10:1 and 105 cells per well. After 3 days, infected BMM were lysed, and replication of BCG was quantified by incorporation of 3H-uracil into viable organisms. Pretreatment of BCG-infected BMM with IFN-{gamma} alone resulted in partial reduction of 3H-uracil uptake (Fig. 1 A and C ). Addition of increasing doses of recombinant hTNF or TNF70–80 enhanced bacteriostasis (decreased 3H-uracil uptake; Fig. 1A and 1C ). This was associated with dose-dependent production of NO, measured as nitrite in culture supernatant 3 days after infection (Fig. 1B and 1D) . hTNF alone or TNF70–80 alone had minimal effect on BCG replication and NO production: The maximum nitrite release was <1.0 µg/ml at 1000 U/ml hTNF or 5.0 µg/ml TNF70–80, with 22% and 27% inhibition of BCG growth, respectively. Therefore, TNF70–80 mimics hTNF function in vitro and acts with IFN-{gamma} to induce NO synthesis and BCG growth inhibition. Murine TNF had similar effects to hTNF and peptide TNF70–80, and pretreatment with control peptide from another region of hTNF (TNF6–18) had no synergistic effect with IFN-{gamma} on BMM activation (unpublished results). Addition of neutralizing antibodies to IFN-{gamma} or hTNF during the activation phase blocked BCG growth inhibition, further emphasizing that the inhibitory effects of macrophage activation were dependent on coactivation with IFN-{gamma} and hTNF or TNF70–80 (Table 1 ).



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  		Figure 1. Activation of BMM by IFN-{gamma} and recombinant hTNF (A, B), and IFN-{gamma} and TNF70–80 (C, D). Activation was determined by measuring the inhibition of BCG growth as reflected by 3H-uracil incorporation (A, C) and by nitrite release (B, D) at 3 days post-infection. The cells were incubated with IFN-{gamma} alone ({square}) or IFN-{gamma} with increasing concentrations of hTNF (A, B) ({blacklozenge} 64 U/ml, • 250 U/ml, or {blacktriangleup} 1000 U/ml) or TNF70–80 (C, D) ({blacklozenge} 0.31 µg/ml, • 1.25 µ/ml, or {blacktriangleup} 5.00 µ/ml). Results are mean values ± SEM in triplicate cultures from one of four similar experiments. The maximum uptake of 3H-uracil into replicating BCG in the absence of cytokines ± standard deviation was 38,826 ± 58.

 

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  		Table 1. The Effect of Antimurine IFN-{gamma} Antibodies and Antihuman TNF Antibodies on the Activation of Antimycobacterial Activity of Bone Marrow-Derived Macrophages by the Combination of IFN-{gamma} with TNF or Peptide TNF70–80 or by IFN-{gamma} Alonea

 
TNF70–80 activities depend on signaling through TNFRI and NO synthase induction
A soluble form of the human TNFRI receptor, p55-sf2 (composed of recombinant 55 kDa TNFRI fused to human IgG heavy-chain domain), inhibits binding of human TNF to the 55 kDa TNFRI [29 ]. Incubating BMM with hTNF and IFN-{gamma} and increasing concentrations of p55-sf2 blocked macrophage activation in a dose-dependent manner. Reduced NO production and concomitant increase in uptake of 3H-uracil into viable BCG organisms were observed (Fig. 2 ). Soluble TNFRI also blocked BMM activation by TNF70–80 (Fig. 2) , consistent with peptide signaling occurring through the TNFRI. Control protein (irrelevant IgG) did not block BMM activation (unpublished results). The modest effects of IFN-{gamma} alone (Fig. 1) were not affected by addition of soluble TNFRI. n-MMA blocks production of NO from L-arginine via induction of NO synthase (iNOS). Addition of n-MMA (1 µM) to cultures blocked the induction of NO by IFN-{gamma} and hTNF or TNF70–80, and prevented the inhibition of BCG growth (Table 2 ). Similar effects also occurred with the addition of a second iNOS inhibitor, aminoguanidine (unpublished results). Therefore, the inhibition of BCG growth by TNF70–80 is dependent on NO production via the L-arginine pathway.



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  		Figure 2. Inhibitory effects of a soluble TNFRI-IgG fusion protein on the activation of BMM by the combination of hTNF or peptide TNF70–80 and IFN-{gamma}. The inhibitory effect was determined by measuring the suppression of antimycobacterial activity as reflected by 3H-uracil incorporation into BCG (A) and by nitrite release at 3 days post-infection (B). Results were calculated from triplicate cultures and are representative of three similar experiments. The cells were incubated with IFN-{gamma} (100 U/ml) and hTNF (250 U/ml; shaded bars) or IFN-{gamma} (100 U/ml) and peptide TNF70–80 (1.25 µ/ml; open bars) with increasing concentrations of soluble TNFRI-IgG fusion protein (0.5, 5.0, or 50.0 µg/ml) for 24 h prior to BCG infection.

 

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  		Table 2. The Inhibitory Effect of Monomethyl-L-Argigine (n-MMA) on the Activation of Anti-Mycobacterial Activity of Bone Marrow-Derived Macrophages by the Combination of IFN-{gamma} with TNF or Peptide TNF70–80a

 
TNF70–80 prevents recrudescence of BCG infection in CD4+ T cell-depleted mice
The observed association between recrudescent M. tuberculosis and M. avium infection and declining numbers of CD4+ T helper cells in HIV-infected patients [8 , 9 ] led us to investigate the in vivo properties of TNF70–80 using a murine model of BCG recrudescence. Intravenous inoculation of mice with 106 BCG led to infection, predominantly of the liver and spleen, which peaked at 3–4 weeks and then resolved over 8–10 weeks to a level of ~1000 organisms per spleen [31 ]. These organisms were contained in small, discrete granulomas. Depleting CD4+ T helper cells by antibody or corticosteroid hormone treatment causes reactivation of infection in the liver and spleen [7 ]. To determine if treatment with hTNF or TNF70–80 can modulate this relapse, mice infected with 106 BCG organisms intravenously were rested for 9 weeks, then treated with anti-CD4 mAb or saline (control) on days 0, 1, and 2 to deplete CD4+ T cells, then on days 7, 14, and 21, to maintain depletion. Concomitant second daily treatment with hTNF (0.5 µg), TNF70–80 (100 µg), or saline commenced at day 0 and continued for the duration of the experiment. This regime was shown by flow cytometric analysis to cause a 97% reduction in the number of CD4+ T cells and no reduction in CD8+ T cells or B cells in the spleens of antibody-treated mice tested before antibody was injected on days 7, 14, and 21 (unpublished results). Figure 3 shows recrudescent BCG infection in CD4+ T cell-depleted mice. Treatment with hTNF or TNF70–80 prevented relapse. Treatment with TNF70–80 or hTNF decreased the bacterial load in the spleen (Fig. 3C) and reduced the numbers of liver granulomas, identified as accumulations of >=10 macrophages and lymphocytes (Fig. 3A) and as foci of activated MHC class II-positive macrophages (Fig. 3B) . Cytokine treatment did not affect the low-level chronic infection in intact mice (Fig. 3) . Administration of control peptide (TNF6–18) in vivo did not modulate the course of BCG infection (unpublished results). Treatment with TNF70–80 or hTNF also resulted in a decrease in the size of liver granulomas in CD4+ T cell-depleted mice measured on stained histological sections (Fig. 4 ). These data suggest that treatment with TNF70–80 or hTNF activated macrophages at sites of chronic infection to inhibit BCG growth secondary to deficiency of CD4+ T cells. This prevented resurgence in the bacterial load and the consequent increase in granulomas.



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  		Figure 3. Reactivation of chronic BCG infection in CD4+ T cell-depleted mice was prevented by in vivo treatment with hTNF and TNF70–80. CD4+ T cell-depleted mice (•) were compared to intact, control mice ({circ}). Reactivation was determined by measuring the number of liver granulomas (A), the number of foci of activated, MHC class II-positive macrophages in the liver (B), and the bacterial load in the spleen (C). Each symbol represents the value in an individual mouse. The columns represent the median value for each treatment group in one of four similar experiments. The number of liver granulomas in hTNF- and TNF70–80-treated, CD4+ T cell-depleted mice was significantly less than that in the CD4+ T cell-depleted, (PBS) phosphate-buffered saline-treated, control group: p < 0.02 (hTNF), and p < 0.05 (TNF70–80) using the nonparametric Mann-Whitney U test. The number of foci in hTNF- and TNF70–80-treated, CD4+ T cell-depleted mice was significantly less than that in the CD4+ T cell-depleted, PBS-treated, control group: p < 0.01 (hTNF), and p < 0.05 (TNF70–80) using the same statistical analysis. The number of BCG cfus in hTNF- and TNF70–80-treated, CD4+ T cell-depleted mice was significantly less than that in the CD4+ T cell-depleted, PBS-treated, control group: p < 0.02 (hTNF), and p < 0.02 (TNF70–80) using the same statistical analysis. No significant difference was observed with any treatment in intact mice.

 


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  		Figure 4. Histology of liver tissue showing treatment with hTNF and TNF70-80 reduced the pathology associated with recrudescent BCG infection. Compact granulomas, consisting of small accumulations of lymphocytes and activated macrophages, were observed in CD4+ T cell-depleted mice treated with hTNF (A) or TNF70-80 (B). Similar lesions were seen in chronic BCG infection (untreated, intact mice, 9 weeks post-infection, D). Larger liver lesions containing higher numbers of lymphocytes and activated macrophages were observed in reactivated infection (sections from saline-treated, CD4+ T cell-depleted mice, C). Tissue was fixed in buffered formalin, sectioned, and stained with haematoxylin and eosin. Original magnification x 200.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study shows that TNF70–80, a peptide with an amino acid sequence derived from that of the TNFRI receptor-binding region of human TNF [32 ], functions similarly to soluble human TNF in conferring protection in mice. Recrudescence of chronic BCG infection following depletion of CD4+ T cells was prevented by peptide TNF70–80 treatment. Peptide treatment prevented resurgence in the number and size of liver granulomas, with apparent reduction in the pathology usually associated with recrudescent infection. A similar effect occurred in a model of primary BCG infection in which mice were infected, and then treated seven days later with TNF70–80 or hTNF for 10 days [31 ]. In this model, the phenotype of cells in the granulomas suggested that treatment expedited granuloma formation and subsequent bacteriostasis.

Our in vitro studies suggest mechanisms for the functional activity of TNF70–80. Peptide TNF70–80, like hTNF, synergized with IFN-{gamma} to activate murine BMM and inhibit the replication of M. bovis (BCG). BCG growth inhibition was abrogated by addition of neutralizing anti-IFN-{gamma} and anti-hTNF. Similar observations were made in a murine macrophage cell line infected with BCG and treated with peptide or soluble human or murine TNF [33 ]. TNF engages two receptors, the p55 TNFRI and p75 TNFRII, on the surface of leukocytes, and the resulting signals induce a wide range of biological effects, including apoptosis, macrophage activation, and cell proliferation [34 ]. A soluble form of TNFRI blocks TNF-induced pathology in experimental allergic encephalitis [35 ] and protects mice against endotoxic shock [36 ]. This TNFRI-IgG fusion protein abrogated the activity of TNF and peptide TNF70–80 on macrophage activation, consistent with the signaling induced by TNF70–80 occurring through the TNFRI. TNF is considered to bind TNF receptors as a trimer [34 ], and the mechanism by which the 11-mer peptide TNF70–80 binds and signals through the TNFRI is currently under investigation. The inhibitory effect of TNF70–80 on BCG replication was dependent on synthesis of RNI via induction of iNOS. hTNF and TNF70–80 synergized with IFN-{gamma} to stimulate NO release. Addition of a competitive inhibitor of iNOS function, n-MMA, to BCG-infected macrophage cultures abrogated the mycobacterial inhibitory effects of peptide TNF70–80 and hTNF.

From these in vitro data, we infer that, as the BCG infection starts to resurge following CD4+ T cell depletion, early therapy with TNF70–80 activates macrophages to contain the bacterial replication through synthesis of RNI. The stimulation of RNI production is also dependent on local levels of other cytokines, notably IFN-{gamma}. Possible sources of IFN-{gamma} include CD8+ T cells [37 ], {gamma}{delta} T cells [38 ], or NK cells [39 ]. Characteristic MHC class II expression on activated macrophages is present in the granulomas in CD4+ T cell-depleted, TNF70–80-treated mice (Fig. 3B) . This is associated with increased iNOS expression in liver granulomas in mice treated with TNF70–80 during primary BCG infection (unpublished results). Therefore, TNF70–80 functions in vivo by stimulating bactericidal RNI in infected macrophages. The low bacterial load in intact mice was not further reduced by treatment with hTNF or TNF70–80, perhaps because a balance has been established between cytokine-induced macrophage activation by IFN-{gamma} and TNF and down-regulatory cytokines, for example TGF-ß and IL-10, that restrict IFN-{gamma} synthesis and macrophage antimycobacterial functions [40 , 41 ]. The effect of TNF and TNF70–80 was apparent when this balance was perturbed.

This TNF70–80 peptide is effective in vivo in other models of infection. Significant reduction in parasitaemia occurred following TNF70–80 treatment of mice infected with P. chabaudi [27 ]. In chronic Pseudomonas aeruginosa infection of mice, TNF70–80 therapy resulted in reduced weight loss and enhanced clearance of organisms (unpublished results). Intrapulmonary administration of TNF70–80 in the lungs of mice prior to infection with Klebsiella pneumoniae or Aspergillus conidia resulted in improved survival and clearance of the microorganisms [42 , 43 ]. TNF70–80 stimulated and primed cultured human neutrophils for increased respiratory burst, release of their granular contents, and consequent enhanced killing of P. falciparum in vitro [27 ]. In this respect TNF70–80 closely resembles the previously described properties of TNF [44 ]. Interestingly, unlike intact TNF, the peptide did not promote expression of adhesion molecules [intercellular adhesion molecule 1 (ICAM-1), endothelial leukocyte adhesion molecule 1 (ELAM-1), and vascular cell adhesion molecule 1 (VCAM-1)] on endothelial cell membranes [27 ]. Also, in contrast to intact TNF, exogenous TNF70–80 was shown to be free of toxicity in P. chabaudi-infected mice in concentrations up to 400 mg/Kg [27 ].

In summary TNF70–80 stimulated macrophage activation and modulated recrudescent mycobacterial infection, leading to a reduction in pathology and bacterial load. Thus, the peptide has clinical therapeutic potential as an agent to modify pathology caused by intracellular pathogens and as an immunostimulatory adjunct to antimicrobial therapy, for example, in the treatment of chronic, drug-resistant M. avium complex infection in advanced HIV/AIDS or treatment of multidrug resistant tuberculosis.


   ACKNOWLEDGEMENTS
 
This work was supported by the Australian Research Council, the National Health and Medical Research Council of Australia, and the Community Health and Anti-Tuberculosis Association. We thank Danielle Avery and John Kamaras for invaluable technical assistance.

Received December 30, 1999; revised April 25, 2000; accepted April 26, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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