Clin. exp. Immunol. (1991) 83, 466-471
ADONIS
000991049100085N
Modulation of Mycobacterium avium growth in vivo by cytokines: involvement of tumour necrosis factor in resistance to atypical mycobacteria M. DENIS Research Unit, Centre de Pneumologie, Laval Hospital, Ste-Foy, Quebec, Canada
(Acceptedfor publication 4 October 1990)
SUMMARY The protective mechanisms associated with resistance to atypical mycobacteria infections are not clear. In an effort to broaden our understanding of the mechanisms involved, susceptible mice were infected with a virulent strain of M. avium and various treatments were applied so as to modify the course of the disease. Treatment with an antiserum against tumour necrosis factor-alpha (TNF-a) significantly enhanced the experimental infection, as judged by enumeration of colony-forming units (CFU) in the spleens and livers of infected mice, suggesting a role for TNF-a in resistance to M. avium. In other sets of experiments, recombinant cytokines were directly infused into infected mice. Infusion of recombinant interferon-gamma (IFN-y) did not modify the experimental infection significantly, and infusion of interleukin-2 was also without effect. Injection of TNF-a enhanced resistance in susceptible animals, as seen by a reduction in the viable bacilli recovered from the spleens and livers. In a final set of experiments, we demonstrate that combinations of cytokines may induce strong resistance against M. avium, namely injection of 1 ug of interleukin- 1 a and 1 yg of TNF-a at 5-day intervals which was seen to eradicate M. avium in both spleens and livers of susceptible BALB/c mice. Overall, our results suggest that induction of protection against M. avium by treatment with cytokines may be feasible, and that TNF-cx may be a pivotal molecule in resistance to M. avium.
Keywords Mycobacterium avium cytokines immunomodulation
INTRODUCTION Infections with M. avium intracellulare constitute an important health problem in both immunosuppressed (O'Brien, Geiter & Snyder, 1987) and otherwise normal individuals (Prince et al., 1989). These life-threatening infections are problematic because most strains of M. avium intracellulare are resistant to antituberculous drugs (Rastogi et al., 1981). These bacteria are facultative intracellular pathogens and will invade and multiply quickly within normal human and murine macrophages (Crowle et al., 1986). Under intense scrutiny at this point in the field of infectious diseases is the ability of cytokines to endow resistance against a variety of infectious diseases (Rook, 1987; Shellenkens, 1989). It is not known how cytokines may influence M. avium infections. In vitro evidence suggests that human macrophages are not activated by recombinant interferon-gamma (rIFN-y) to inhibit the intracellular growth of M. avium (Squires et al., 1989; Toba, Crawford & Ellner, 1989), whereas tumour necrosis factor-alpha (TNF-a) may trigger the killing of relatively avirulent strains of M. avium in human and murine macrophages (Bermudez, Kolonoski & Young, 1990; Bermudez & Young, 1988). This last
set of experiments was specifically followed up by showing that administration of TNF-a with interleukin-2 (IL-2) could endow mice with significant resistance against infections with these strains of M. avium (Bermudez et al., 1989). Moreover exogenous IL-2 may increase the resistance of mice to a variety of bacterial pathogens (Sharma, Hoffin & Remington, 1985; Weyland et al., 1987; Jeevan & Asherson, 1988; Haak-Frendscho, Young & Czuprynski, 1989). Other in vivo experiments have suggested that rIFN-y may not protect mice or humans against virulent M. avium infections (Squires et al., 1989). The administration of cytokines in vivo to modify the course of an infectious disease may lead to data that are difficult to interpret inasmuch as many cytokines will induce the synthesis of other soluble factors (Granelli-Piperno, Andrus & Steinman, 1986). Nevertheless, such experiments may prove useful in terms of dissecting the host response to this class of pathogens. We describe here a simple experimental model, growth of a virulent M. avium in the organs of susceptible mice where we attempt to modify the growth of the pathogen with recombinant cytokines or neutralizing antibodies against cytokines.
Correspondence: Michel Denis, Unite de Recherche, Centre de Pneumologie, H6pital Laval, 2725 chemin Ste-Foy, Ste-Foy, Quebec GI V 4G5, Canada.
Animals BALB/c mice, weighing 20-25 g were used throughout. Those mice were fed with Purina Chow and tap water ad tibitum.
MATERIALS AND METHODS
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Cytokines in M. avium infections Mycobacterium Mycobacterium avium (Trudeau mycobacterial collection 702) was used throughout. It was grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) for 21 days. Aliquots were then frozen at - 70'C. For infection, bacteria were thawed and sonicated briefly to obtain a dispersed suspension, bacterial counts were adjusted by diluting in phosphate-buffered saline (PBS).
Infections Mice were injected intravenously via the caudal vein with the appropriate numbers of M. avium. Mice were divided in seven experimental groups of about 30 mice, as follows: (I), mice infected with 104 colony-forming units (CFU) of M. avium and injected intravenously with I mg of normal rabbit globulin at day 0 and day 20 of infection; (II) mice infected with I04 CFU of M. avium and injected intravenously with I mg of rabbit antiserum against TNF-a at day 0 and day 20 of infection; (III) mice injected with 104 CFU of M. avium and injected intraperitoneally with 1 pg of IFN-y every week; (IV) mice infected with 104 CFU of M. avium and injected intraperitoneally with 5000 U of IL-2 every second day; (V) mice infected with 04 CFU of M. avium and injected intraperitoneally with 1 pg of TNF-a every week; (VI) mice infected with 103 CFU of M. avium intraperitoneally with 1 pg of TNFx and I pg of IL- I every week; and (VII) infected mice treated with the saline used to reconstitute the cytokines. At predetermined intervals, mice were killed by exposure to CO2. The degree of infection in the spleens and livers was quantified by plating serial dilutions of organ homogenates as described in detail elsewhere (Denis et al., 1986). Immunomodulators Human IL-la was obtained from Hoffman-Laroche (Nutley, NW), specific activity 2-5 x 107 U/mg; there was less than 0-125 EU/ml ofendotoxin contamination in this preparation. Recombinant murine TNF-a (Genzyme, Boston, MA) had a specific activity of 4 x 107 U/mg, the preparation had 0 12 EU/ml of endotoxin contamination. Recombinant human IFN-y was obtained from Amgen Biochemicals (Thousand Oaks, CA). The original solution was diluted in pyrogen-free saline. Recombinant murine IL-2 (Genzyme) had a specific activity of 1 5 x 106 U/mg; the preparation was endotoxin-free (less than 0-01 ng of endotoxin/105 U). All cytokines were diluted in pyrogen-free saline prior to in vivo administration. Rat IgGI monoclonal antibody (MoAb) to murine IFN-y, produced by Lee Biomolecular Research, and purified rat IgG (Sigma, St Louis, MO) as controls were used. Antibodies were resuspended according to the supplier's instructions. An immunoglobulin fraction, which was prepared and purified by ammonium sulphate precipitation from the serum of rabbits hyperimmunized with purified murine rTNF-x was used as the anti-TNF-a antiserum. The antiserum (I mg) neutralized 6 x 106 U of the cytolytic activity of mouse rTNF-a but did not affect the activity of mouse rTNF-# or human rTNF-a in an L929 killing assay (Titus, Sherry & Ceram, 1989), it did not interfere with the antiviral activities of mouse IFN-y and mouse IFN-fl, and did not affect the co-stimulatory activity of mouse IL-I (Oppenheim, Sheyour & Cook, 1976) or IL-2 (Gillis et al., 1978; and unpublished results). Normal rabbit globulin (NRG)
used as a control was also prepared by ammonium sulphate fractionation from the serum of pre-immune rabbits. Isolation of resident peritoneal macrophages and assessment of H202 release Resident peritoneal macrophages were isolated from mice as described in detail elsewhere (Denis & Chadee, 1989). (PMAtriggered release of H202 of peritoneal macrophages was assessed as previously described in detail (Denis & Chadee, 1989).
Statistical analysis Differences between means were analysed by Student's t-test. RESULTS Administration of an antiserum against TNF-a decreases resistance against M. avium As shown in Fig. 1, following injection with 5 x 104 CFU, M. avium grew progressively in the spleens of susceptible BALB/c, with little sign of any decrease in the CFU recovered. Counts remained static in the livers during the observation period. There was no significant difference between NRGtreated and untreated animals in their susceptibility to M. avium. Mice were injected intravenously with 1 mg of rabbit anti-mouse TNF-a immunoglobulin or normal rabbit globulin as controls, 2 h before infection and 20 days after infection and
LL 0 0 -J
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Fig. 1. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 1 mg of rabbit anti-TNF-cx (0) or normal rabbit globulin (0) 2 h before infection and 20 days after infection. Vertical bars represent the standard deviations of five mice. Representative results of one experiment repeated three times with similar results.
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Fig. 2. Growth of 104 of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 1 pg of IFN-y (0) or PBS (0) every week. Results expressed as in Fig. 1.
the splenic and liver bacterial load assessed. As suggested by Fig. 1, elimination of endogenous TNF-a led to significant increase in the number of CFU recovered from both organs at all times, with a 1-log increase being apparent at day 100 in the liver and a 1-2-log increase in the spleen. No deaths were observed in either groups (NRG- and anti-TNF-x-treated) although CFU counts were shown to increase up to 5 months following infection in anti-TNF-a-treated mice (data not shown).
Administration of a MoAb against IFN-y does not modify the growth pattern of M. avium Mice injected intravenously with 2 x 103 U of anti-IFN-y MoAb 2 h before injection of M. avium were not more susceptible to infection than control groups given rat IgG (data not shown). In a separate set of experiments, mice injected with 2 x 103 U of anti-IFN-y MoAb at 5-day intervals during infection with M. avium were no more susceptible to infection (data not shown).
Infusion ofIFN-y or IL-2 in infected animals does not significantly modify growth of M. avium As shown in Fig. 2. IFN-y treatment did not modify the course of the disease significantly. Our rIFN-y was fully active at priming mouse peritoneal macrophages for an enhanced respiratory burst following PMA triggering (data not shown). Moreover, as Fig. 3 suggests, there was no significant difference between IL-2-treated mice and PBS controls in terms of the CFU recovered from the spleens and the livers. This suggests that within the limits of our experimental model, IL-2 may not afford protection against virulent M. avium. Our IL-2 was fully bioactive as a T cell growth factor, using a standard assay (Gillis et
al., 1978) (data not shown).
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Fig. 3. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 5000 U of IL-2 (0) or PBS (0) every second day. Results expressed as in Fig. 1.
TNF-c. increases resistance to M. avium Infusion of TNF-a (I pg/mouse at the time of infection and I pg/mouse at 5-day intervals following infection) led to substantial protection (Fig. 4) as measured by recovery of M. avium from the spleen and liver at all times measured. Counts in the liver of TNF-cx-treated mice declined steadily during the course of the experiment whereas the CFU remained stable in the spleens, in contrast to control animals, where progressive growth was apparent in the spleens. In this system boiled rTNF-a was unable to induce any protection, suggesting that residual lipopolysaccharide (LPS) was not the active component (data not shown). Combination of cytokines may endow mice with strong resistance against M. avium Having determined a role for TNF-x in resistance to M. avium, we set out to investigate the ability of TNF-a and IL-I to endow mice with resistance against M. avium. In preliminary experiments it was determined that infusion of IL-I (up to 5 pg/day) had no effect on mouse resistance to M. avium (data not shown). In three other separate experiments (Fig. 5), it was observed that administration of 1 pg of IL-1 and 1 pg of TNF-a per mouse enhanced resistance against mycobacterial growth significantly. This protection was significant starting at day 20 post-infection, and remained significant for the remainder of the infection. Protection by TNF-a/IL-la was significantly superior to that seen with TNF-a alone at day 60, 80 and 100 (P'
4
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:>
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,, 5 - b co r3
2 0
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40 60 80 100 Time (days) Fig. 5. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice which were treated with I pg of TNF-a and 1 jpg of IL-la (0) or PBS (-) at 5-day intervals. Results expressed as in Fig. 1.
This study was aimed at understanding the importance of several cytokines in M. avium infections. One observation was the apparent lack of involvement of IFN-y in M. avium TMC 702. This was shown both by administration of anti-IFN-y and by in vivo infusion of IFN-y; both treatments did not affect the course of M. avium infection in susceptible BALB/c mice. Our experimental protocol for depletion of endogenous IFN-y has been used successfully by other investigators working on intracellular parasites (Rose, Wakelin & Hesketh, 1989). Similarly, the dose of IFN-y we infused in the infected mice is within the range used by other investigators in related systems where protection against an infectious agent was sought (Edwards et al., 1986; Reed, 1988; Shellenkens, 1989). The role of IFN-y in mycobacterial infections is equivocal, with most studies suggesting a positive effect of IFN-y on the anti-mycobacterial properties of mouse macrophages whereas they are rather inactive on human monocytes/macrophages (Douvas et al., 1985; Mor, Goren & Crowle, 1989; Rook et al., 1985). Infusion of IFN-y in BCG-infected BALB/c nu/nu mice led to an increased susceptibility in these mice, as seen by an increase in the viable counts in the spleens whereas their normal counterparts were marginally protected by IFN-y infusion (Banerjee, Sharp & Lowrie, 1986). However, the involvement of IFN-y in resistance to atypical mycobacteria is quite unclear. A particular study has shown that in vivo infusion of IFN-y drastically reduced the bacterial burden in the spleens of mice infected with a virulent strain of M. intracellulare (Edwards et al., 1986), whereas another recent study has shown that in vivo infusion of IFN-y in mice infected with a virulent M. avium strain was ineffective at regulating bacterial growth (Squires et al., 1989). Other studies have shown that crude lymphokines may endow macrophages from susceptible C57BL/6 strain but not from resistant A/J mice with significant anti-mycobacterial activity against the same strain of M. avium used here (Stokes & Collins, 1988). Our results also suggest that TNF-ot may play a role in M. avium infection in this murine model. Depletion of endogenous TNF-ax by treatment with antibodies against TNF-a enhanced susceptibility against M. avium significantly; in addition, treatment of M. avium infection with recombinant TNF-a resulted in enhanced resistance. Recent results have suggested an important role for TNF-a in anti-microbial resistance (Titus et al., 1989; Bermudez et al., 1990; Liew et al., 1990; Roll et al., 1990; Chang, Grau & Pechere, 1990). Depletion of endogenous TNF-a led to a much increased susceptibility ofmice to infection with Listeria monocytogenes (Havell, 1989; Nakane, Minagawa & Kato, 1988) and M. bovis BCG (Kindler et al., 1989). Kindler et al. (1989) suggested that anti-TNF-a was blocking the recruitment of monocytes to the infectious foci. It has been shown that TNF-a may activate murine and human macrophages to kill M. avium, although the actual virulence of the particular strain used was rather obscure (Bermudez & Young, 1988). TNF-a may also act via the lysis of infected cells, as it has
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been shown that TNF-a may lyse viral-infected targets (Koff & Fann, 1986). This would allow release of the bacteria into an hostile environment, with freshly emigrated monocytes arriving at the lesion site (Kaufmann & Flesch, 1990). In this regard, macrophages heavily infected with mycobacteria have been shown to become unresponsive to IFN-y, this unresponsiveness is likely to be caused by release of prostaglandins (Sibley & Krahenbuhl, 1988). This may explain in part our data with IFN-y in vivo. IL-2, a pivotal molecule in immune regulation (Gillis et al., 1978), has been used recently in modulation of neoplasia in humans (Donahue & Rosenburg, 1983). Other recent evidence suggests that IL-2 may protect mice against infections with a variety of bacterial pathogens (Haak-Frendscho et al., 1989; Weyland et al., 1987). In most cases, the actual mechanism responsible for the protection was not clear. IL-2 may stimulate in vivo the release of IFN-y, which may in turn activate macrophages and/or neutrophils to an enhanced antibacterial activity (Granelli-Piperno et al., 1986). IL-2 may also activate macrophages directly for enhanced tumour cytotoxicity (Malkovsky et al., 1987). In our hands, IL-2 was inactive at modifying the growth of M. avium in vivo. This finding is somewhat surprising, in that IL-2 is a strong inducer of TNF-a in vivo and in vitro (Granelli-Piperno et al., 1986). The use of a cocktail of cytokines that may synergize and offer maximal protection against microbial challenge is a strategy which is being increasingly recognized in in vivo and in vitro systems (Chang et al., 1990; Roll et al., 1990). In our system, TNF-a and IL-la infusion led to a maximal protective phenomenon. Similar results have been reported recently regarding the increase in resistance to another intracellular parasite, L. monocytogenes (Roll et al., 1990). Our findings reinforce the notion that optimal therapeutic action ofcytokines may be obtained via the use of combinations of soluble factors. As for the mechanisms(s) involved in the increased resistance to M. avium infections in IL-la- and TNF-y-treated animals, we have no real indication of the component involved. Peritoneal or splenic macrophages of cytokine-treated animals did not show a superior ability to restrict in vivo the growth of M. avium, as compared with cells from control animals (unpublished data). This would seem to parallel findings in the Listeria system where the protection sponsored by IL-1/TNF infusion did not correlate with macrophage anti/listerial activity. Nevertheless, our results with IL-I/TNF used together are impressive, inasmuch as eradication of the bacilli is achieved in the organs of treated mice. In the Listeria model, there was also a lack of correlation between protection and the production of colony-stimulating activity in the animals infused with IL-I and TNF-a (Roll et al., 1990). The present work has underscored a major role for TNF-a in protection against virulent M. avium infections, whereas IFN-y and IL-2 are not seemingly involved in resistance. Our results also indicate that IL-la may potentiate TNF-a induced resistance.
ACKNOWLEDGMENTS I wish to thank E. Shavers, Franqoise Maher and Louise Gendron for their help in the preparation of the manuscript.
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Cytokines in M. avium infections mediates host protection against cutaneous leishmaniasis. Immunology, 69, 570. MALKOVSKY, M., LOVELAND, B., NORTH, M., ASHERSON, G.L., GAO, L., WARD, P. & FIERS, W. (1987) Recombinant interleukin-2 directly augments the cytotoxicity of human monocytes. Nature, 325, 262. MOR, N., GOREN, M.B. & CROWLE, A.J. (1989) Enhancement of growth of Mycobacterium lepraemurium in macrophages by gamma interferon. Infect. Immun. 57, 2586. NAKANE, A., MINAGAWA, P. & KATO, K. (1988) Endogenous tumour necrosis factor (cachectin) is essential to hose resistance against Listeria monocytogenes infection. Infect. Immun. 56, 2563. O'BRIEN, R.J., GEITER, L.J. & SNYDER, D.E. (1987) The epidemiology of non-tuberculous mycobacterial diseases in the United States: results from a national survey. Am. Rev. respir. Dis. 135, 1007. OPPENHEIM, J.J., SHEYOUR, A. & COOK, A.I. (1976) Enhancement of DNA synthesis and cAMP content in mouse thymocytes by mediator(s) derived from adherent cells. J. Immunol. 116, 1466. PRINCE, D.S., PETERSON, D.D., STEINER, R.M., GOTTLIEB, J.E., SCOTT, R., ISRAEL, H.L., FIGUEROA, W.J. & FISH, J.E. (1989) Infection with Mycobacterium avium complex in patients without predisposing conditions. N. Engl. J. Med. 321, 863. RASTOGI, N., FREHEL, C., RYTER, A., OHAYON, H., LESOURD, M. & DAVID, H.L. (1981) Multiple drug resistance in Mycobacterium avium: is the wall architecture responsible for the exclusion of antimicrobial agents? Antimicrob. Agents Chemother. 20, 666. REED, S.G. (1988) In vivo administration of recombinant IFN-y induces macrophage activation and prevents acute disease, immune suppression, and death in experimental Trypanosoma cruzi infections. J. Immunol. 140, 4342. Roll, J.T., Young, K.M., Kurtz, R.S. & Czuprynski, C.J. (1990) Human rTNF-a augments antibacterial resistance in mice; potentiation of its effects by recombinant human rIL- Io. Immunology, 69, 316. ROOK, G.A.W. (1987) Progress in the immunology of the mycobacterioses. Clin. exp. Immunol. 69, 1. ROOK, G.A.W., CHAMPION, B.R., STEELE, J., YOUNG, A.M. & STANFORD,
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J.L. (1985) I-A restricted activation by T cell lines of anti-tuberculosis activity in murine macrophages. Clin. exp. Immunol. 59, 414. ROOK, G.A.W., STEELE, J., AINSWORTH, M. & CHAMPION, B.R. (1986) Activation of macrophages to inhibit proliferation of Mycobacterium tuberculosis: comparison of the effects of recombinant gammainterferon on human monocytes and murine peritoneal macrophages. Immunology, 59, 333. ROSE, M.E., WAKELIN, D. & HESKETH, P. (1989) Gamma interferon controls Eimeria vermiformis primary infection in BALB/c mice. Infect. Immun. 57, 1599. SHARMA, S.D., HOFFIN, J.M. & REMINGTON, J.S. (1985) In vivo recombinant interleukin-2 administration enhances survival against a lethal challenge with Toxoplasma gondii. J. Immunol. 135, 4160. SHELLENKENS, H. (1989) Animal models in inteferon research: some current trends. Experientia, 45, 558. SIBLEY, D.L. & KRAHENBUHL, J.L. (1988) Induction of unresponsiveness to gamma interferon in macrophages infected with Mycobacterium leprae. Infect. Immun. 56, 1912. SQuIREs, K.E., MURPHY, W.F., MADOFF, L.C. & MURRAY, H.W. (1989) Interferon-y and Mycobacterium avium-intracellulare infection. J. infect. Dis. 159, 599. STOKES, R.W. & COLLINS, F.M. (1988) Growth of Mycobacterium avium in activated macrophages harvested from inbred mice with differing innate susceptibilities to mycobacterial infection. Infect. Immun. 56, 2250. TITUS, R.G., SHERRY, B. & CERAMI, A. (1989) Tumour necrosis factor plays a protective role in experimental murine cutaneous leishmaniasis. J. erp. Med. 170, 2097. TOBA, H., CRAWFORD, J.T. & ELLNER, J.J. (1989) Pathogenicity of Mycobacterium avium for human monocytes: absence of macrophage-activating factor activity of gamma interferon. Infect. Immun. 57, 239. WEYLAND, C., GORONZY, J., DALLMAN, M.J. & HANLEY, P.O. (1987) Administration in vivo of recombinant interleukin-2 protects against septic death. J. clin. Invest. 79, 1956.
ADONIS
000991049100085N
Modulation of Mycobacterium avium growth in vivo by cytokines: involvement of tumour necrosis factor in resistance to atypical mycobacteria M. DENIS Research Unit, Centre de Pneumologie, Laval Hospital, Ste-Foy, Quebec, Canada
(Acceptedfor publication 4 October 1990)
SUMMARY The protective mechanisms associated with resistance to atypical mycobacteria infections are not clear. In an effort to broaden our understanding of the mechanisms involved, susceptible mice were infected with a virulent strain of M. avium and various treatments were applied so as to modify the course of the disease. Treatment with an antiserum against tumour necrosis factor-alpha (TNF-a) significantly enhanced the experimental infection, as judged by enumeration of colony-forming units (CFU) in the spleens and livers of infected mice, suggesting a role for TNF-a in resistance to M. avium. In other sets of experiments, recombinant cytokines were directly infused into infected mice. Infusion of recombinant interferon-gamma (IFN-y) did not modify the experimental infection significantly, and infusion of interleukin-2 was also without effect. Injection of TNF-a enhanced resistance in susceptible animals, as seen by a reduction in the viable bacilli recovered from the spleens and livers. In a final set of experiments, we demonstrate that combinations of cytokines may induce strong resistance against M. avium, namely injection of 1 ug of interleukin- 1 a and 1 yg of TNF-a at 5-day intervals which was seen to eradicate M. avium in both spleens and livers of susceptible BALB/c mice. Overall, our results suggest that induction of protection against M. avium by treatment with cytokines may be feasible, and that TNF-cx may be a pivotal molecule in resistance to M. avium.
Keywords Mycobacterium avium cytokines immunomodulation
INTRODUCTION Infections with M. avium intracellulare constitute an important health problem in both immunosuppressed (O'Brien, Geiter & Snyder, 1987) and otherwise normal individuals (Prince et al., 1989). These life-threatening infections are problematic because most strains of M. avium intracellulare are resistant to antituberculous drugs (Rastogi et al., 1981). These bacteria are facultative intracellular pathogens and will invade and multiply quickly within normal human and murine macrophages (Crowle et al., 1986). Under intense scrutiny at this point in the field of infectious diseases is the ability of cytokines to endow resistance against a variety of infectious diseases (Rook, 1987; Shellenkens, 1989). It is not known how cytokines may influence M. avium infections. In vitro evidence suggests that human macrophages are not activated by recombinant interferon-gamma (rIFN-y) to inhibit the intracellular growth of M. avium (Squires et al., 1989; Toba, Crawford & Ellner, 1989), whereas tumour necrosis factor-alpha (TNF-a) may trigger the killing of relatively avirulent strains of M. avium in human and murine macrophages (Bermudez, Kolonoski & Young, 1990; Bermudez & Young, 1988). This last
set of experiments was specifically followed up by showing that administration of TNF-a with interleukin-2 (IL-2) could endow mice with significant resistance against infections with these strains of M. avium (Bermudez et al., 1989). Moreover exogenous IL-2 may increase the resistance of mice to a variety of bacterial pathogens (Sharma, Hoffin & Remington, 1985; Weyland et al., 1987; Jeevan & Asherson, 1988; Haak-Frendscho, Young & Czuprynski, 1989). Other in vivo experiments have suggested that rIFN-y may not protect mice or humans against virulent M. avium infections (Squires et al., 1989). The administration of cytokines in vivo to modify the course of an infectious disease may lead to data that are difficult to interpret inasmuch as many cytokines will induce the synthesis of other soluble factors (Granelli-Piperno, Andrus & Steinman, 1986). Nevertheless, such experiments may prove useful in terms of dissecting the host response to this class of pathogens. We describe here a simple experimental model, growth of a virulent M. avium in the organs of susceptible mice where we attempt to modify the growth of the pathogen with recombinant cytokines or neutralizing antibodies against cytokines.
Correspondence: Michel Denis, Unite de Recherche, Centre de Pneumologie, H6pital Laval, 2725 chemin Ste-Foy, Ste-Foy, Quebec GI V 4G5, Canada.
Animals BALB/c mice, weighing 20-25 g were used throughout. Those mice were fed with Purina Chow and tap water ad tibitum.
MATERIALS AND METHODS
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Cytokines in M. avium infections Mycobacterium Mycobacterium avium (Trudeau mycobacterial collection 702) was used throughout. It was grown in Middlebrook 7H9 broth (Difco Laboratories, Detroit, MI) for 21 days. Aliquots were then frozen at - 70'C. For infection, bacteria were thawed and sonicated briefly to obtain a dispersed suspension, bacterial counts were adjusted by diluting in phosphate-buffered saline (PBS).
Infections Mice were injected intravenously via the caudal vein with the appropriate numbers of M. avium. Mice were divided in seven experimental groups of about 30 mice, as follows: (I), mice infected with 104 colony-forming units (CFU) of M. avium and injected intravenously with I mg of normal rabbit globulin at day 0 and day 20 of infection; (II) mice infected with I04 CFU of M. avium and injected intravenously with I mg of rabbit antiserum against TNF-a at day 0 and day 20 of infection; (III) mice injected with 104 CFU of M. avium and injected intraperitoneally with 1 pg of IFN-y every week; (IV) mice infected with 104 CFU of M. avium and injected intraperitoneally with 5000 U of IL-2 every second day; (V) mice infected with 04 CFU of M. avium and injected intraperitoneally with 1 pg of TNF-a every week; (VI) mice infected with 103 CFU of M. avium intraperitoneally with 1 pg of TNFx and I pg of IL- I every week; and (VII) infected mice treated with the saline used to reconstitute the cytokines. At predetermined intervals, mice were killed by exposure to CO2. The degree of infection in the spleens and livers was quantified by plating serial dilutions of organ homogenates as described in detail elsewhere (Denis et al., 1986). Immunomodulators Human IL-la was obtained from Hoffman-Laroche (Nutley, NW), specific activity 2-5 x 107 U/mg; there was less than 0-125 EU/ml ofendotoxin contamination in this preparation. Recombinant murine TNF-a (Genzyme, Boston, MA) had a specific activity of 4 x 107 U/mg, the preparation had 0 12 EU/ml of endotoxin contamination. Recombinant human IFN-y was obtained from Amgen Biochemicals (Thousand Oaks, CA). The original solution was diluted in pyrogen-free saline. Recombinant murine IL-2 (Genzyme) had a specific activity of 1 5 x 106 U/mg; the preparation was endotoxin-free (less than 0-01 ng of endotoxin/105 U). All cytokines were diluted in pyrogen-free saline prior to in vivo administration. Rat IgGI monoclonal antibody (MoAb) to murine IFN-y, produced by Lee Biomolecular Research, and purified rat IgG (Sigma, St Louis, MO) as controls were used. Antibodies were resuspended according to the supplier's instructions. An immunoglobulin fraction, which was prepared and purified by ammonium sulphate precipitation from the serum of rabbits hyperimmunized with purified murine rTNF-x was used as the anti-TNF-a antiserum. The antiserum (I mg) neutralized 6 x 106 U of the cytolytic activity of mouse rTNF-a but did not affect the activity of mouse rTNF-# or human rTNF-a in an L929 killing assay (Titus, Sherry & Ceram, 1989), it did not interfere with the antiviral activities of mouse IFN-y and mouse IFN-fl, and did not affect the co-stimulatory activity of mouse IL-I (Oppenheim, Sheyour & Cook, 1976) or IL-2 (Gillis et al., 1978; and unpublished results). Normal rabbit globulin (NRG)
used as a control was also prepared by ammonium sulphate fractionation from the serum of pre-immune rabbits. Isolation of resident peritoneal macrophages and assessment of H202 release Resident peritoneal macrophages were isolated from mice as described in detail elsewhere (Denis & Chadee, 1989). (PMAtriggered release of H202 of peritoneal macrophages was assessed as previously described in detail (Denis & Chadee, 1989).
Statistical analysis Differences between means were analysed by Student's t-test. RESULTS Administration of an antiserum against TNF-a decreases resistance against M. avium As shown in Fig. 1, following injection with 5 x 104 CFU, M. avium grew progressively in the spleens of susceptible BALB/c, with little sign of any decrease in the CFU recovered. Counts remained static in the livers during the observation period. There was no significant difference between NRGtreated and untreated animals in their susceptibility to M. avium. Mice were injected intravenously with 1 mg of rabbit anti-mouse TNF-a immunoglobulin or normal rabbit globulin as controls, 2 h before infection and 20 days after infection and
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Fig. 1. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 1 mg of rabbit anti-TNF-cx (0) or normal rabbit globulin (0) 2 h before infection and 20 days after infection. Vertical bars represent the standard deviations of five mice. Representative results of one experiment repeated three times with similar results.
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Fig. 2. Growth of 104 of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 1 pg of IFN-y (0) or PBS (0) every week. Results expressed as in Fig. 1.
the splenic and liver bacterial load assessed. As suggested by Fig. 1, elimination of endogenous TNF-a led to significant increase in the number of CFU recovered from both organs at all times, with a 1-log increase being apparent at day 100 in the liver and a 1-2-log increase in the spleen. No deaths were observed in either groups (NRG- and anti-TNF-x-treated) although CFU counts were shown to increase up to 5 months following infection in anti-TNF-a-treated mice (data not shown).
Administration of a MoAb against IFN-y does not modify the growth pattern of M. avium Mice injected intravenously with 2 x 103 U of anti-IFN-y MoAb 2 h before injection of M. avium were not more susceptible to infection than control groups given rat IgG (data not shown). In a separate set of experiments, mice injected with 2 x 103 U of anti-IFN-y MoAb at 5-day intervals during infection with M. avium were no more susceptible to infection (data not shown).
Infusion ofIFN-y or IL-2 in infected animals does not significantly modify growth of M. avium As shown in Fig. 2. IFN-y treatment did not modify the course of the disease significantly. Our rIFN-y was fully active at priming mouse peritoneal macrophages for an enhanced respiratory burst following PMA triggering (data not shown). Moreover, as Fig. 3 suggests, there was no significant difference between IL-2-treated mice and PBS controls in terms of the CFU recovered from the spleens and the livers. This suggests that within the limits of our experimental model, IL-2 may not afford protection against virulent M. avium. Our IL-2 was fully bioactive as a T cell growth factor, using a standard assay (Gillis et
al., 1978) (data not shown).
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Fig. 3. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice treated with 5000 U of IL-2 (0) or PBS (0) every second day. Results expressed as in Fig. 1.
TNF-c. increases resistance to M. avium Infusion of TNF-a (I pg/mouse at the time of infection and I pg/mouse at 5-day intervals following infection) led to substantial protection (Fig. 4) as measured by recovery of M. avium from the spleen and liver at all times measured. Counts in the liver of TNF-cx-treated mice declined steadily during the course of the experiment whereas the CFU remained stable in the spleens, in contrast to control animals, where progressive growth was apparent in the spleens. In this system boiled rTNF-a was unable to induce any protection, suggesting that residual lipopolysaccharide (LPS) was not the active component (data not shown). Combination of cytokines may endow mice with strong resistance against M. avium Having determined a role for TNF-x in resistance to M. avium, we set out to investigate the ability of TNF-a and IL-I to endow mice with resistance against M. avium. In preliminary experiments it was determined that infusion of IL-I (up to 5 pg/day) had no effect on mouse resistance to M. avium (data not shown). In three other separate experiments (Fig. 5), it was observed that administration of 1 pg of IL-1 and 1 pg of TNF-a per mouse enhanced resistance against mycobacterial growth significantly. This protection was significant starting at day 20 post-infection, and remained significant for the remainder of the infection. Protection by TNF-a/IL-la was significantly superior to that seen with TNF-a alone at day 60, 80 and 100 (P'
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40 60 80 100 Time (days) Fig. 5. Growth of 104 CFU of M. avium in (a) spleen and (b) liver of BALB/c mice which were treated with I pg of TNF-a and 1 jpg of IL-la (0) or PBS (-) at 5-day intervals. Results expressed as in Fig. 1.
This study was aimed at understanding the importance of several cytokines in M. avium infections. One observation was the apparent lack of involvement of IFN-y in M. avium TMC 702. This was shown both by administration of anti-IFN-y and by in vivo infusion of IFN-y; both treatments did not affect the course of M. avium infection in susceptible BALB/c mice. Our experimental protocol for depletion of endogenous IFN-y has been used successfully by other investigators working on intracellular parasites (Rose, Wakelin & Hesketh, 1989). Similarly, the dose of IFN-y we infused in the infected mice is within the range used by other investigators in related systems where protection against an infectious agent was sought (Edwards et al., 1986; Reed, 1988; Shellenkens, 1989). The role of IFN-y in mycobacterial infections is equivocal, with most studies suggesting a positive effect of IFN-y on the anti-mycobacterial properties of mouse macrophages whereas they are rather inactive on human monocytes/macrophages (Douvas et al., 1985; Mor, Goren & Crowle, 1989; Rook et al., 1985). Infusion of IFN-y in BCG-infected BALB/c nu/nu mice led to an increased susceptibility in these mice, as seen by an increase in the viable counts in the spleens whereas their normal counterparts were marginally protected by IFN-y infusion (Banerjee, Sharp & Lowrie, 1986). However, the involvement of IFN-y in resistance to atypical mycobacteria is quite unclear. A particular study has shown that in vivo infusion of IFN-y drastically reduced the bacterial burden in the spleens of mice infected with a virulent strain of M. intracellulare (Edwards et al., 1986), whereas another recent study has shown that in vivo infusion of IFN-y in mice infected with a virulent M. avium strain was ineffective at regulating bacterial growth (Squires et al., 1989). Other studies have shown that crude lymphokines may endow macrophages from susceptible C57BL/6 strain but not from resistant A/J mice with significant anti-mycobacterial activity against the same strain of M. avium used here (Stokes & Collins, 1988). Our results also suggest that TNF-ot may play a role in M. avium infection in this murine model. Depletion of endogenous TNF-ax by treatment with antibodies against TNF-a enhanced susceptibility against M. avium significantly; in addition, treatment of M. avium infection with recombinant TNF-a resulted in enhanced resistance. Recent results have suggested an important role for TNF-a in anti-microbial resistance (Titus et al., 1989; Bermudez et al., 1990; Liew et al., 1990; Roll et al., 1990; Chang, Grau & Pechere, 1990). Depletion of endogenous TNF-a led to a much increased susceptibility ofmice to infection with Listeria monocytogenes (Havell, 1989; Nakane, Minagawa & Kato, 1988) and M. bovis BCG (Kindler et al., 1989). Kindler et al. (1989) suggested that anti-TNF-a was blocking the recruitment of monocytes to the infectious foci. It has been shown that TNF-a may activate murine and human macrophages to kill M. avium, although the actual virulence of the particular strain used was rather obscure (Bermudez & Young, 1988). TNF-a may also act via the lysis of infected cells, as it has
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been shown that TNF-a may lyse viral-infected targets (Koff & Fann, 1986). This would allow release of the bacteria into an hostile environment, with freshly emigrated monocytes arriving at the lesion site (Kaufmann & Flesch, 1990). In this regard, macrophages heavily infected with mycobacteria have been shown to become unresponsive to IFN-y, this unresponsiveness is likely to be caused by release of prostaglandins (Sibley & Krahenbuhl, 1988). This may explain in part our data with IFN-y in vivo. IL-2, a pivotal molecule in immune regulation (Gillis et al., 1978), has been used recently in modulation of neoplasia in humans (Donahue & Rosenburg, 1983). Other recent evidence suggests that IL-2 may protect mice against infections with a variety of bacterial pathogens (Haak-Frendscho et al., 1989; Weyland et al., 1987). In most cases, the actual mechanism responsible for the protection was not clear. IL-2 may stimulate in vivo the release of IFN-y, which may in turn activate macrophages and/or neutrophils to an enhanced antibacterial activity (Granelli-Piperno et al., 1986). IL-2 may also activate macrophages directly for enhanced tumour cytotoxicity (Malkovsky et al., 1987). In our hands, IL-2 was inactive at modifying the growth of M. avium in vivo. This finding is somewhat surprising, in that IL-2 is a strong inducer of TNF-a in vivo and in vitro (Granelli-Piperno et al., 1986). The use of a cocktail of cytokines that may synergize and offer maximal protection against microbial challenge is a strategy which is being increasingly recognized in in vivo and in vitro systems (Chang et al., 1990; Roll et al., 1990). In our system, TNF-a and IL-la infusion led to a maximal protective phenomenon. Similar results have been reported recently regarding the increase in resistance to another intracellular parasite, L. monocytogenes (Roll et al., 1990). Our findings reinforce the notion that optimal therapeutic action ofcytokines may be obtained via the use of combinations of soluble factors. As for the mechanisms(s) involved in the increased resistance to M. avium infections in IL-la- and TNF-y-treated animals, we have no real indication of the component involved. Peritoneal or splenic macrophages of cytokine-treated animals did not show a superior ability to restrict in vivo the growth of M. avium, as compared with cells from control animals (unpublished data). This would seem to parallel findings in the Listeria system where the protection sponsored by IL-1/TNF infusion did not correlate with macrophage anti/listerial activity. Nevertheless, our results with IL-I/TNF used together are impressive, inasmuch as eradication of the bacilli is achieved in the organs of treated mice. In the Listeria model, there was also a lack of correlation between protection and the production of colony-stimulating activity in the animals infused with IL-I and TNF-a (Roll et al., 1990). The present work has underscored a major role for TNF-a in protection against virulent M. avium infections, whereas IFN-y and IL-2 are not seemingly involved in resistance. Our results also indicate that IL-la may potentiate TNF-a induced resistance.
ACKNOWLEDGMENTS I wish to thank E. Shavers, Franqoise Maher and Louise Gendron for their help in the preparation of the manuscript.
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