INTRODUCTION

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Organisms belonging to the Mycobacterium avium complex (MAC) are rarely pathogenic for healthy individuals but may become a major cause of disseminated bacterial infection in human immunodeficiency virus-infected patients (18). MAC can survive within macrophages (M) and affect various physiological functions, including the production of tumor necrosis factor alpha (TNF-), a cytokine of great importance for anti-MAC resistance in ex vivo and in vivo models (3, 4, 13).

It is generally accepted that the final outcome of TNF- expression in different infection models may depend on its site of action, its local concentration, and the duration of exposure. An excess of TNF- released into the blood can be detrimental for humans, as suggested by the observation that many symptoms of tuberculosis and chronic MAC infections related to TNF-, such as fever and weight loss, are improved by thalidomide, a drug that selectively destabilizes TNF- mRNA (15, 28). TNF- is involved in the development of granulomas, since administration of neutralizing anti-TNF- antibodies leads to granuloma regression and mycobacterial dissemination (3, 17, 19). At the cellular level, TNF- activity is downregulated by MAC in cultured human and mouse M within the first 24 to 48 h of infection, thus preventing a local antimicrobial effect of this cytokine (9, 11, 14).

The wide range of TNF- activities can be in part explained by the presence on almost all nucleated cells of one or two distinct TNF- receptors (TNF-Rs), namely, TNF-RI (p55) and TNF-RII (p75). Shedding of soluble forms from both receptors (sTNF-RI and sTNF-RII, respectively) can modulate the biological effects of TNF- by inhibition of its bioactivity (9, 29) or by stabilization of its quaternary structure (2). Most of the information on the role of TNF- and TNF-Rs in murine mycobacterial infections has been obtained by using neutralizing antibodies (3, 12) and sTNF-Rs (1, 6, 25). Although these studies have provided evidence that TNF- and TNF-RI play a role in mycobacterial infections in ex vivo and in vivo models, they gave little or no information on the site and temporal effects of TNF- and TNF-R activation in MAC-infected mice. An attempt to demonstrate a role for TNF-RI and TNF-RII in the control of MAC infection, based on double-TNF-RII-knockout mice, did not support a role for TNF-Rs in modulating the bacterial load but, rather, pointed to an important role in promoting chronic pathologic changes (8).

To contribute to the dissection of these events and to further investigate the roles of TNF-Rs, we monitored the production of TNF-Rs in the spleen and blood of BALB/c mice throughout a 70-day period of infection. In addition, we used an antagonist anti-TNF-RII antibody to assess whether membrane TNF-RII or sTNF-RII is critically involved in the control of infection with MAC.

	MATERIALS AND METHODS

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Mice. Male BALB/c mice aged 6 to 7 weeks were obtained from Charles River (Calco, Como, Italy). They were bred and maintained under standard conditions, receiving sterilized chow and acidified water ad libitum.

Organism and mouse infection. A clinical isolate of MAC 485 (11) was used throughout this study. Transparent colonies grown on Middlebrook 7H10 agar plates (Difco Laboratories, Detroit, Mich.) were suspended in phosphate-buffered saline (PBS) and sonicated for 10 s to disperse clumps. A suspension adjusted to an optical density of 0.2 at 500 nm (corresponding to approximately 6 × 10⁸ CFU/ml) was prepared. Each mouse was intraperitoneally (i.p.) inoculated with 10⁷ CFU in 0.2 ml of PBS. At different time points, mice were sacrificed and their spleens were aseptically collected and suspended in Middlebrook 7H9 medium (Difco), ground in homogenizers, and briefly sonicated. Each suspension was 10-fold diluted with distilled water and plated onto 7H10 agar medium; after incubation for 10 to 14 days at 37°C in a humidified 5% CO₂ atmosphere, the colonies were counted. Blood was drawn from the retro-orbital sinus, diluted with distilled water, and analyzed for bacterial load as above. Serum samples were stored in aliquots at 80°C for determination of TNF- and sTNF-R levels.

Measurement of TNF- and TNF-R mRNA expression. MAC-infected and PBS-injected (control) mice were sacrificed at the indicated times; their spleens were removed and immediately frozen in liquid nitrogen.

(i) RNA extraction. Frozen tissues were ground in a mortar, and guanidine thiocyanate solution (4 M guanidinium thiocyanate, 0.025 M sodium citrate [pH 7], 0.5% Sarkosyl) was added. The homogenates were mixed sequentially with sodium acetate, water-saturated phenol, and chloroform-isoamyl alcohol (49:1), vortexed thoroughly, and incubated on ice for 15 min. After centrifugation at 12,900 × g for 20 min at 4°C, the RNA in the upper (aqueous) phase was precipitated with isopropanol. Each sample was then incubated for 30 min at 20°C and further centrifuged for 10 min. The RNA pellet was resuspended in cold 75% ethanol, centrifuged, air dried, dissolved in distilled water, and stored frozen (7). RNA gel electrophoresis was performed to confirm that the RNA was intact and that the concentration had been correctly determined.

(ii) Reverse transcription (RT) of RNA. Eppendorf tubes containing 40 µl of total RNA (0.1 µg/µl) and 4 µl of oligo(dT) (27-7858; Pharmacia Biotech, Uppsala, Sweden) were incubated for 10 min at 65°C and chilled on ice (7). Each tube was incubated for 60 min at 37°C after addition of 36 µl of a solution containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, a 0.5 mM concentration of each deoxynucleoside triphosphate (Pharmacia), 10 mM dithiothreitol, and 10 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Grand Island, N.Y.) per µl. The reaction mixture was heated at 95°C for 5 min to denature the reverse transcriptase and cooled on ice. cDNA was diluted 1:10 in distilled water and stored frozen at 20°C until use.

(iii) Semiquantitative PCR. Synthesized cDNAs were amplified by PCR with primers for murine TNF- and TNF-Rs designed to span introns and to enable differential detection of cDNA from genomic DNA. The following sequences were used: 5'-GATCTCAAAGACAACCAACTAGTG-3' (5' primer for TNF-) (33), 5'-CTCCAGCTGGAAGACTCCTCCCAG-3' (3' primer for TNF-) (33), 5'-CTGGCCTGCTGCTGTCACTG-3' (5' primer for TNF-RI), 5'-GTCAGCTTGGCAAGGAGAGATC-3' (3' primer for TNF-RI), 5'-GTCGCGCTGGTCTTCGAACTG-3' (5' primer for TNF-RII), and 5'-GGTATACATGCTTGCCTCACAGTC-3' (3' primer for TNF-RII).

Production of recombinant sTNF-RI and sTNF-RII. The cDNAs coding for the extracellular domains of TNF-RI and TNF-RII were amplified by PCR on plasmids containing the entire cDNA sequences of murine receptors (kindly provided by R. Goodwin, Immunex, Seattle, Wash.), using the following primers: 5'-CATGGAAACATACATCCATCAGGGGTCACTGGA-3' (5' primer for sTNF-RI); 5'-ATGGGATCCTTACGCAGTACCTGAGTCCTGGGG-3' (3' primer for sTNF-RI); 5'-CATGGATCCGTGCCCGCCCAGGTTGTC-3' (5' primer for sTNF-RII); and 5'-ATGGGATCCTTACCTGGTACTTTGTTCAATAATGGG-3' (3' primer for sTNF-RII). sTNF-RI and sTNF-RII were produced by cloning the fragments in the pET-11a system (Novagen, Madison, Wis.) and expressing them in the BL21(DE3) Escherichia coli strain (Novagen). Both receptors were expressed as insoluble forms (40 mg of sTNF-RI and 80 mg of sTNF-RII per liter of culture). Solubilization, denaturation, and partial refolding were performed essentially as described for human sTNF- receptors (20). Soluble receptors were purified by reverse-phase chromatography with a Source 15 RPC column (Pharmacia), with a final yield of 5 to 10%. Recombinant sTNF-RI and sTNF-RII were quantified by commercial enzyme-linked immunosorbent assay (ELISA) kits (HyCult Biotechnologies). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed bands corresponding to the expected molecular mass for monomeric sTNF-RI and sTNF-RII (24 and 29 kDa, respectively).

Production of polyclonal and monoclonal anti-TNF-R antibodies. sTNF-RI and sTNF-RII were used to raise polyclonal anti-sTNF-RI and anti-sTNF-RII in rabbits by standard techniques; the immunoglobulin G (IgG) fraction of each serum sample (named RAM-RI and RAM-RII, respectively) was purified by affinity chromatography on protein A-Sepharose (Pharmacia). Monoclonal antibody (MAb) 6G1 (rat anti-murine TNF-RII IgG2a) was prepared as follows. One rat (Wistar) was immunized by injecting 20 µg of sTNF-RII in 250 µl of a 1:1 emulsion with complete Freund's adjuvant into the base of the tail and into the hind footpad. The animal was boosted with the same amount of sTNF-RII in incomplete Freund's adjuvant and four times with sTNF-RII in PBS, subcutaneously, at the base of the tail, all at 15-day intervals. Three days after the last boost, the rat was killed. The spleen cells were isolated and fused with P3-X63 Ag8-NS1 myeloma cells by standard procedures to generate hybridomas. Hybridomas secreting anti-TNF-RII antibodies were screened by ELISA, using microplates coated with sTNF-RII. MAb 6G1 was purified from cell supernatants by ammonium sulfate precipitation, ion-exchange chromatography (three cycles), and ultrafiltration through a YM 10 membrane (Amicon, Danvers, Mass.). The final product was >90% pure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MAb 19E12 (IgG2a), used as irrelevant antibody in in vivo studies, was purified by ammonium sulfate precipitation and affinity chromatography on protein A-Sepharose (21). This antibody is directed to Thy.1.1 and does not cross-react with the Thy.1.2 antigen expressed by BALB/c mice.

Thymocyte proliferation assay. Thymocytes from C57BL/6 mice (Charles River) were obtained by thymus excision and teasing. The thymocytes were washed twice with RPMI 1640 medium and plated in 96-well flat-bottom plates (Costar, Cambridge, Mass.) (10⁶ cells/well) in a final volume of 100 µl of RPMI 1640 (Life Technologies) supplemented with 20% heat-inactivated (56°C for 30 min) fetal calf serum, 1% glutamine, 100 U of penicillin per ml, and 100 µg of streptomycin per ml in the presence of 50 U of interleukin-2 per ml. After 48 h, the cells were pulsed with 1 µCi of [³H]thymidine (Amersham, Milan, Italy) per well for 20 h, harvested onto a fiberglass filter, and lysed with distilled water. The radioactivity bound to the filter was measured with a liquid scintillation counter.

Measurement of TNF- bioactivity and sTNF-Rs. (i) Determination of TNF- bioactivity levels by a cytolytic assay. TNF- bioactivity was measured by a cytolytic assay based on mouse WEHI 164 clone 13 cells (10, 21); the detection limit of this assay was 0.21 pg/ml.

(ii) Determination of sTNF-RI and sTNF-RII levels by ELISA. sTNF-RI and sTNF-RII were quantified by sandwich ELISA with rabbit anti-sTNF-RI or anti-sTNF-RII polyclonal IgG, prepared as described above, in the capturing step and biotinylated anti-sTNF-RI or anti-sTNF-RII MAbs (HyCult Biotechnologies, Uden, The Netherlands) as the second antibody. Streptavidin-peroxidase dissolved in PBS containing 0.5% bovine serum albumin (BSA) and 1% normal goat serum was used as the detection reagent (24). The detection limits of the assays were 0.32 ng/ml for sTNF-RI and 0.1 ng/ml for sTNF-RII.

Preparation and stimulation of splenocytes from mice infected once or twice with MAC. Mice were infected intravenously by injecting 5 × 10³ CFU of MAC into the tail vein. After 60 days, some mice were sacrificed and their spleens were aseptically collected and processed for CFU counts as described above. Infected mice were reinfected i.p. with 10⁵ CFU of MAC on day 60; uninfected mice of the same age were infected in parallel and used as once-infected control mice. All the mice were sacrificed after 3 months and processed for determination of CFU, bioactive TNF-, sTNF-RI, and sTNF-RII in spleen cell supernatants. Spleen cells collected from individual mice of each group were incubated (in 24-well plates at 3 × 10⁶ cells/well) in culture medium (RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 25 mM HEPES, and 2 mM glutamine) with or without 10 µg of MAC 485 sonicate per ml. After 48 h, the levels of bioactive TNF-, sTNF-RI, and sTNF-RII in the cell supernatants were measured as described above. The sonicate was prepared by lysing MAC cells at 4°C with an ultrasonic disintegrator (VCX 400 W; Sonics & Materials Inc., Danbury, Conn.) for 20 min (1-min cycles with 1-min cooling intervals); after centrifugation, the protein content in the supernatant was determined by the method of Bradford (5).

	RESULTS

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Kinetics of MAC infection in mice. MAC-infected BALB/c mice developed a chronic infection which persisted for more than 70 days (Fig. 1). In the spleen, after a 1.3 log₁₀ unit increase in the number of CFU from day 2 to day 21, the bacterial burden remained fairly constant throughout the remaining period. About 10² CFU/ml was found in the blood between days 2 and 14, and then mycobacteremia dropped to undetectable levels. No animal deaths were recorded during the whole experimental period.

View larger version (10K): [in this window] [in a new window]   FIG. 1.   Time course of MAC infection in the spleens and blood of BALB/c mice inoculated i.p. with 107 CFU. Data are shown as CFU per spleen and CFU per milliliter of blood. Each point represents the mean value for five mice and the standard deviation.

Kinetics of TNF- and TNF-R production. The production of TNF-, TNF-RI, and TNF-RII mRNAs in the spleens of infected mice was monitored by reverse transcription-PCR throughout the 70-day experimentation period (Fig. 2). The TNF- mRNA level increased fourfold (P = 0.002) on day 21 in comparison with the level in control mice and then slowly decreased (Fig. 2A). The level of TNF-RI mRNA did not significantly increase at any time (Fig. 2B); in contrast, TNF-RII mRNA was significantly overexpressed in the spleen on day 21 (15-fold increase [P = 0.009]) (Fig. 2C).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   Semiquantitative analysis of TNF-, TNF-RI, and sTNF-RII gene expression by RT-PCR in the spleens of MAC-infected and PBS-treated BALB/c mice. Data are presented as arbitrary units; each point represents the mean value for three mice and the standard deviation. At each time point, a statistically significant increase in expression compared to PBS-injected mice is labelled with an asterisk, indicating P < 0.05 (Student's t test).

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Kinetics of release of sTNF-RII in the blood of MAC-infected and PBS-treated BALB/c mice. Each point represents the mean value and the standard deviation for three mice. At each time point, a statistically significant increase compared to PBS-treated mice is labelled with an asterisk indicating P < 0.05.

Production and characterization of an antagonist anti-TNF-RII MAb. To investigate the role of TNF-RII in MAC infection, we have generated a new rat anti-murine TNF-RII MAb (MAb 6G1). Polyclonal anti-TNF-RI and anti-TNF-RII IgGs (termed RAM-RI and RAM-RII, respectively) were also prepared. No cross-reactivity of polyclonal anti-TNF-R antibodies or MAbs with irrelevant sTNF-RI or sTNF-RII was observed in the ELISA (results not shown).

View larger version (13K): [in this window] [in a new window]   FIG. 4.   Effect of MAb 6G1 on the TNF--induced proliferation of mouse thymocytes (left) and on the binding of TNF- to sTNF-RII (right). The thymocyte proliferation assay was carried out in the absence and in the presence of murine TNF-, without or with 2 µg of the indicated antibody per ml (left). The effect of MAb 6G1 on the binding of 3 µg of TNF- per ml to sTNF-RII (right) was evaluated by a competitive ELISA with microplates coated with sTNF-RII (10 µg/ml in PBS, 1 h at room temperature) and blocked with 3% BSA for 2 h. After a 1-h incubation with biotin-TNF- in PBS containing 1% BSA and MAb 6G1 at various doses, the plates were washed with PBS and further incubated with the detecting reagents (anti-TNF- rabbit IgGs, followed by goat anti-rabbit IgG-peroxidase conjugate and chromogenic substrate) by standard techniques.

Effect of anti-TNF-RII antibodies on MAC growth in mice. To assess whether TNF-RII and/or its soluble form is involved in the regulation of TNF- activity during MAC infection, we carried out an experiment in which MAb 6G1 was administered to animals. This treatment significantly inhibited MAC growth in the spleens of mice after both 21 days (P = 0.0002) and 42 days (P = 0.0005) of infection (Fig. 5). Treatment with an irrelevant antibody (MAb 19E12) of the same isotype (IgG2a) did not cause significant changes in the number of CFU in comparison with the control (saline-treated) animals. This observation indicates that TNF-RII and/or its soluble form is critically involved in the control of MAC infection in mice.

View larger version (12K): [in this window] [in a new window]   FIG. 5.   Effect of anti-TNF-RII treatment on the time course of MAC growth in the spleens of BALB/c mice infected with 107 CFU i.p. The mice were treated i.p. with 100 µg of anti-TNF-RII antibody (MAb 6G1) or an irrelevant antibody (MAb 19E12) of the same isotype (IgG2a) as MAb 6G1, administered 1 day before infection and every 5 days up to day 34. Control (infected) mice were treated with saline. Each point represents the mean value and the standard deviation of two independent experiments, each with four mice per group. At each time point, a statistically significant decrease compared to control mice is labelled with two asterisks, indicating P < 0.005.

Production of TNF- and sTNF-Rs in mice infected once or twice with MAC. After 60 days from the initial infection, the number of CFU per spleen in untreated mice was (2.6 ± 0.5) × 10⁶. To explore the response to a further mycobacterial stimulation, infected mice were reinfected with MAC on day 60. After 3 months, CFU, TNF-, and sTNF-R levels were determined and compared with those for the once-infected control mice. No significant difference in viable counts between twice-and once-infected mice was observed; the CFU were (0.71 ± 0.46) × 10⁶ and (1 ± 0.46) × 10⁶, respectively.

View larger version (9K): [in this window] [in a new window]   FIG. 6.   Release of bioactive TNF- (A), sTNF-RI (B), and sTNF-RII (C) in the supernatants of spleen cells of once- or twice-infected mice, either unstimulated (solid bars) or stimulated with 10 µg of MAC sonicate per ml (open bars). Each point represents the mean value and the standard deviation for six mice. Detection limits are indicated by dotted lines.

	DISCUSSION

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Anti-MAC resistance in BALB/c mice developed in two phases: the first, up to day 21, was associated with bacterial multiplication in the spleen, a drop in mycobacteremia, and transient activation of the TNF-/TNF-RII system; the second was associated with containment of infection in the spleen and blood.

The marked increase of TNF- and TNF-RII mRNA levels on day 21 coincided with or immediately preceded the highest protective activity against infection and indicated that the control of MAC growth in the spleen closely correlates with the local activation of the TNF-/TNF-RII system. This is in keeping with results of previous studies (3) in which early neutralization of TNF- with antibodies led to an enhanced bacterial load in tissues. TNF-RII appeared to be regulated not only at the transcriptional level but also posttranscriptionally, as shown by a concomitant increase in the level of sTNF-RII in the blood. The correlation between activation of TNF-RII in the spleen and blood and the capability of animals to efficiently control MAC growth strongly suggests that this receptor and its soluble form play a role in modulating TNF- activity locally and systemically during the immune response to MAC.

Interestingly, in contrast to TNF-RII, transcription of TNF-RI mRNA in the spleen did not change appreciably during infection. Previous reports showed that TNF-RI is essential for protection against Mycobacterium tuberculosis (12) and Mycobacterium bovis BCG (25) infections in mice. Although TNF-RI may also be involved in signalling in a constitutive mode, our data suggest that the fine modulation of TNF- activity in MAC infection during the early phase is mainly due to TNF-RII. It has been shown that TNF-RII can play an accessory role by increasing the local concentration of TNF- and by rapid "ligand passing" to TNF-RI for signalling (26), while TNF-RI can mediate most of the effects of TNF- (29, 31). Moreover, TNF-RII can also enhance TNF-RI-mediated activities by cooperative signalling (32). Besides these supportive or modulating effects, TNF-RII can also directly contribute to local restriction of the responses induced by the membrane-bound precursor of TNF- (16) and to the mediation of other TNF- activities, such as thymocyte and T-cell proliferation and granulocyte-macrophage colony-stimulating factor production (27, 30). Thus, the modulation of TNF-RII during MAC infection not only may regulate the signals triggered by soluble TNF- through TNF-RI but also could markedly affect the overall activity of soluble and membrane-bound TNF-. Shedding of sTNF-RII can additionally contribute to the downregulation of the receptors and to the release of inhibitors that locally or systemically impair interaction with membrane receptors. Recent ex vivo studies have shown that the supernatant of MAC-infected M contains a significant amount of TNF- antigen, mostly inactive (9); shedding of soluble TNF-RII has also been suggested as one of the mechanisms of TNF- inhibition in cell culture supernatants. These studies suggest that upregulation of soluble TNF- receptors may play a role in the ability of some virulent MAC strains to survive in M (9). Our findings that shedding of TNF-RII indeed occurs in vivo during infection support the hypothesis of a functional importance of TNF-RII and its soluble form in the regulation of TNF- activity during MAC infection.

This hypothesis is strengthened by the finding that the antagonist anti-TNF-RII MAb 6G1 can affect bacterial growth in the spleens of infected mice. Interestingly, this antibody was able to significantly reduce bacterial growth after 21 or 42 days of infection, suggesting that the antigen accessible to this antibody acts as a negative modulator of the antibacterial activity exerted by TNF- in the early phase of infection (3). Circulating sTNF-RII is probably accessible to systemically administered antibodies. Since MAb 6G1 can prevent the binding of TNF- to sTNF-RII, one possibility is that the MAb 6G1 blocked the inhibitory activity of this soluble receptor, leaving TNF- free to interact with membrane receptors (at least TNF-RI) and to trigger antibacterial effects. However, since MAb 6G1 can also prevent the binding of TNF- to membrane-bound TNF-RII, the inhibition of MAC growth may be related to inhibition of TNF- binding to both forms of this receptor. Although it is difficult to draw conclusions on this point, these data suggest that the TNF-RII system is indeed functionally involved in the modulation of TNF- during infection.

The above results indicate that upon a single challenge with MAC the TNF-/TNF-R system is transiently activated during the early phase of infection. Since mycobacterial lesions are acutely sensitive to further stimulation with mycobacterial products or whole organisms (22, 23), we explored whether restimulation of infected mice with live MAC could induce a chronic activation of the TNF-/TNF-R system in the spleens of twice-infected or once-infected mice. Although CFU numbers in twice-infected and once-infected mice were not significantly different 3 months after reinfection, the TNF-/TNF-RII system was markedly activated in the spleens of twice-infected mice, as suggested by a consistent upregulation of the release of bioactive TNF- and sTNF-RII in spleen cell supernatants. These data suggest that under conditions in which reinfection was efficiently controlled, a stronger chronic activation of the TNF-/TNF-RII system than the one observed in mice infected with a single MAC dose occurred. The observation that MAC antigens can induce a vigorous increase (>15-fold) in the production of bioactive TNF- by in vitro stimulation of spleen cells from once-infected mice and only a 2-fold increase in spleen cells from twice-infected mice can be explained in part by an increased production of sTNF-RII.

Overall, our data indicate that TNF-RII can be activated to modulate TNF- activity in infected mice either transiently, in the first month of infection, or chronically, in reinfected mice. The time-related modulation of TNF- activity by TNF-RII may be an important mechanism by which immunocompetent hosts efficiently control natural infections by this environmental organism.