- Email: [email protected]
Aerosol infection of mice with recombinant BCG secreting murine IFN-γ partially reconstitutes local protective immunity
Article available online at http://www.idealibrary.com on
Microbial Pathogenesis 2000; 29: 175–185 doi:10.1006/mpat.2000.0382
MICROBIAL PATHOGENESIS
Aerosol infection of mice with recombinant BCG secreting murine IFN- partially reconstitutes local protective immunity Andre´ L. Moreiraa,b, Liana Tsenovaa, Peter J. Murrayc, Sherry Freemana, Amy Bergtolda, Luis Chiribogad & Gilla Kaplan∗a a
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, U.S.A.; b New York University School of Medicine, Dept. of Pathology, 550 First Ave., New York, NY 10016, U.S.A.; c Dept. Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, U.S.A.; d New York University School of Medicine, Kaplan Comprehensive Cancer Center, New York, NY 10016, U.S.A. (Received January 28, 2000; accepted in revised form May 28, 2000)
To better understand the contribution of interferon-gamma (IFN-) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN- gene disrupted (IFN--/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN- (BCG-IFN-) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN--/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN- resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN- by the recombinant BCG did not protect IFN--/- mice from subsequent challenge with M. tuberculosis. Infection of IFN--/- peritoneal macrophages in vitro with BCG-IFN- led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN- induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth. 2000 Academic Press Key words: Interferon-gamma, Mycobacterium bovis BCG, inducible nitric oxide synthase, macrophages.
∗ Author for correspondence. E-mail: kaplang@rockvax. rockefeller.edu This study was supported in part by US Public Health Service grants AI-22616 and AI-43526 to G.K. and by a grant from Pott’s Memorial Foundation to A.L.M.
0882–4010/00/090175+11 $35.00/0
2000 Academic Press
176
A. L. Moreira et al. 5
Introduction
(a)
3 2 cfu in lungs (log10)
BCG infection of mice has long been used as a model for studying the host immune response to mycobacterial infection. Infection of immunocompetent mice with BCG leads to initial growth or persistence of the organisms predominantly in the lungs of the animal. Subsequently, the growth of BCG is contained by the developing cellular immune response [1, 2]. The profile of cytokines induced in alveolar macrophages and other cells in the lungs following infection plays an important role in establishment and/or control of the infection [2–6]. IFN- is an important regulatory molecule in the protective immune response. In adoptive transfer experiments in mice, T-cells that confer protection against M. tuberculosis produce IFN when stimulated in vitro [7]. The local production of IFN- in infected tissues is associated with a protective host response to mycobacterial infection [8]. This cytokine has been shown to activate macrophages, rendering the cells capable of killing or controlling the growth of intracellular pathogens, including mycobacteria [9, 10]. The antibacterial activity of activated macrophages involves at least two pathways: the generation of reactive nitrogen intermediates (RNI) and the production of reactive oxygen intermediates (ROI) [9, 11–13]. Components of both pathways have been shown to be upregulated by IFN- in vitro [9, 13]. IFN- also downregulates the production of cytokines that negatively regulate macrophage function, such as interleukin-10 (IL-10) [14]. In experiments using IFN- gene disrupted mice (knock-out; IFN--/-) [15–17] or IFN- receptor (IFN--R) knock-out mice, the role of the cytokine was further explored [18]. These mice were unable to mount an effective cell mediated immune response to M. bovis BCG or to M. tuberculosis, resulting in unrestricted growth of the bacilli in the organs of infected animals. The animals succumbed to the infection earlier than wildtype control animals [16–18]. Thus, IFN- and its receptor are essential for controlling mycobacterial infection. To date it has been difficult to reconstitute the host response in IFN- deficient mice [16, 19]. Such reconstitution is necessary to conclusively prove the role of IFN- as a regulatory cytokine and to define the mechanism of its effects in the host protective response to mycobacterial infection. In the present study, we used a strain
*
*
4
1 0 5
(b) *
4 3 2 1 0
0
10
50 20 30 40 Time post-infection (days)
60
Figure 1. Bacillary load in the lungs of BCG-infected IFN--/- (a) and BALB/c (b) mice. Results are the means of three experiments (with quadruplicate samples) expressed as log10 cfu±SD. Closed symbols represent mice infected with BCG-IFN-. Open symbols represent mice infected with BCG-vector. Statistically significant differences (indicated by ∗) in the numbers of BCG-IFN- vs BCG-vector were observed (P<0.05).
of recombinant BCG that secretes murine IFN (BCG-IFN-) to attempt to reconstitute the host response to BCG infection and to an aerosol challenge with M. tuberculosis.
Results Bacillary load in the organs of BCG infected mice Two recombinant BCG strains were used to infect mice: BCG-vector and BCG-IFN- both grew similarly in liquid medium with generation times of 38.4 and 37.2 h, respectively. BCG-IFN used for infection were shown to secrete IFN (by ELISA) and the IFN- was biologically active (by IRF-1 induction in J774 cells). When IFN--/- mice were infected with BCG-vector, the bacillary load increased rapidly for the first 35 days of the infection [Fig.1(a)]. After 35 days,
Reconstitution of anti-mycobacterial immunity
numbers of bacilli in the lungs stabilized. Hematogenous spread of BCG-vector to the spleens was observed at 60 days (cfu log 3.2±1.6). When IFN--/- mice were infected with the recombinant BCG-IFN-, the number of organisms in the lungs did not increase, resulting in a significant difference between the numbers of BCG-vector compared to BCG-IFN- in the lungs at day 35 (P=0.04). Hematogeneous spread of BCG-IFN- to the spleens was observed by day 60 of infection (cfu log 2.4±0.5). Infection of control BALB/c mice with BCGvector resulted in slow bacillary growth in the lungs of aerosol infected mice for the first 60 days [Fig. 1(b)] confirming the attenuated nature of BCG Montreal in mice. Hematogenous spread of the organisms from the lungs to the spleen was first observed at day 60 post-infection (cfu log 2.2±0.5). When BALB/c mice were infected with BCG-IFN-, bacterial numbers in the lung were significantly reduced throughout the infection compared to baseline (35 days P=0.03; 60 days P=0.02). This resulted in a significant difference in the numbers of BCG-vector vs the numbers of BCG-IFN- in the lungs of infected mice at 60 days (P<0.05) [Fig. 1(b)]. In addition, no hematogenous spread of BCG-IFN- to the spleens was observed. These results indicate that IFN- production at the site of infection partially controls the growth of BCG in immunocompromised IFN--/- mice. In the immune competent mice the growth of BCG is more efficiently restricted in the presence of the additional IFN-.
177
in cuffs most often around blood vessels and some neutrophils were observed within the inflammatory infiltrate. However, in contrast to the BCG-vector-infected mice, few AFB were seen in association with the epithelioid or foamy macrophages in BCG-IFN- infected mice, thus morphologically confirming the cfu results (Fig. 1). Aerosol infection with either BCG-vector or BCG-IFN- induced similar small granulomas in the lungs of control BALB/c mice (not shown).
Expression of iNOS in the lungs of BCGinfected mice To better characterize the local host response to the infecting bacilli, immunohistochemical staining for iNOS, an IFN- induced protein, was carried out on the lung sections. In the lungs of IFN--/- mice infected with BCG-vector, there were large collections of macrophages which were negative for the expression of iNOS [Fig. 3(a)]. In contrast, when mice were infected with BCG-IFN-, some of the epithelioid macrophages within the granulomas were positive for the expression of iNOS [Fig. 3(b)]. Therefore, the local production of IFN- by the recombinant mycobacteria induced local macrophage differentiation and iNOS expression. iNOS expression was observed in the lungs of BALB/c control mice infected with either BCG-vector or BCG-IFN- (not shown).
Cytokine expression in the lungs of mice infected with recombinant BCG The granulomatous response in the lungs of BCG infected mice In the IFN--/- mice, aerosol infection with BCGvector induced a vigorous cellular response in the lungs. By day 60 post-infection, the lungs were filled with many loose aggregates of undifferentiated foamy macrophages [Fig. 2(a) and (b)] and cuffs of lymphocytes [Fig. 2(b)] with scattered neutrophils. Interestingly, most of the lymphoid infiltrate in the lungs was restricted to perivascular areas [Fig. 2(a)]. Acid-fast bacilli (AFB) were seen in the large macrophages [Fig. 2(c)]. In contrast, when IFN--/- mice were infected with BCG-IFN-, the mononuclear cell infiltrate was more focal [Fig. 2(d) and (e)]. Differentiation of the macrophages into epithelioid cells was more pronounced [Fig. 2(e) and (f)]. Here too, lymphocytes were organized
The expression of IL-10, TNF- and IFN- mRNA in the lungs of infected mice was studied. Infection of IFN--/- mice with BCG-IFN- resulted in significantly decreased expression of IL10 mRNA at day 60 post-infection when compared to the levels obtained in the lungs of IFN-/- mice infected with the BCG-vector (P<0.05). Comparable low levels of TNF- mRNA were observed in the lungs of IFN--/- mice infected with either recombinant strain [Fig. 4(a)]. As expected, no IFN- mRNA was detected in these mice. In contrast to the IFN--/- mice, IFN- mRNA was expressed in the lungs of control BALB/c mice. In the latter, infection with BCGIFN- resulted in a significant up-regulation of this cytokine compared with infection with BCGvector. No difference in the low level of expression of TNF- mRNA and IL-10 mRNA was
178
Figure 2. Legend on facing page.
Figure 3. Legend on facing page.
A. L. Moreira et al.
Reconstitution of anti-mycobacterial immunity 100
(a)
Survival of mice (%)
Cytokine mRNA in infected lungs (fold increase)
2
179
1
50
0 5
(b)
4
0
10
20
30
40
Time post-challenge (days)
3
Figure 5. Survival of IFN--/- mice (12/group) preinfected by aerosol with: BCG-vector (Β), BCG-IFN- (Χ) followed by infection with 1.6 log10 M. tuberculosis CDC1551 24 h later. Control mice infected only with M. tuberculosis CDC1551 (Α).
2 1 0
IL-10
TNF-α
IFN-γ
Figure 4. Cytokine mRNA expression in the lungs of BCG-infected IFN--/- (a) and BALB/c (b) mice. RTPCR results were first normalized to the amount of -actin mRNA in each lane and then expressed as fold increase over the amount of cytokine mRNA in the lungs of mice infected with BCG-vector at day 60 post-infection (100%=1). The results are means±SD (two experiments with quadruplicate samples). Levels in mice infected with BCG-vector (∆); levels in mice infected with BCG-IFN- (Ε).
noted [Fig. 4(b)]. Neither IFN-, IL-10 or TNF- was detected by ELISA in the serum of IFN--/or BALB/c mice infected with either strain of recombinant BCG at any time during the infection.
Effect of infection with BCG-IFN- on response to M. tuberculosis challenge To determine whether the local production of IFN- in the lungs of IFN--/- mice would protect these animals from tuberculosis, IFN--/mice already infected with BCG-IFN- were challenged with a small inoculum of M. tuberculosis strain CDC1551 [20]. When uninfected IFN--/- mice were infected by aerosol with M. tuberculosis CDC1551, mice began to die on day 35 post-infection. By day 38, all mice had succumbed to the infection, thus confirming the immunodeficiency of IFN--/- mice. When mice were first infected with either of the recombinant BCG strains and 24 h later challenged with aerosolized M. tuberculosis, similar survival patterns were observed (Fig. 5). These results suggest
Figure 2. Histologic sections of lungs of IFN--/- mice infected with BCG-vector (a–c) or BCG-IFN- (d–f) at day 60 post-infection. (a), (b), (d) and (e): hematoxylin and eosin, (c) and (f): Ziehl-Nielsen stains. Arrow heads indicate mycobacteria in large undifferentiated macrophages. Ly-lymphocyte cuff. Magnification: (a), (d) ×25; (b), (e) ×100; (c), (f) ×250. Figure 3. Immunohistology for iNOS expression in the sections of lungs of IFN--/- mice 60 days after infection with either BCG-vector (a) or BCG-IFN- (b). Ly-lymphocyte cuff; asterisks indicate alveolar space. Arrows indicate large undifferentiated iNOS-negative macrophages in BCG-vector-infected mice (a), and iNOS-positive (dark pink stain) as well as iNOS-negative epithelioid macrophages in BCG-IFN- infected mice (b). Magnification ×250.
180
A. L. Moreira et al. 20
240
(a)
200 TNF-α (pg/ml)
15
5
120 80 40
0 20
0
(b)
100
15
80 IL-10 (pg/ml)
cfu in macrophages (fold increase)
10
160
10
5
60 40 20
0
1
2 3 4 5 Time post-infection (days)
6
Figure 6. Growth of BCG-vector (Β) and BCG-IFN (Χ) in peritoneal macrophages of IFN--/- (a) and BALB/c (b) mice. Results are expressed as means±SE of the mean for four experiments each carried out in duplicate.
that the focal presence of intracellular IFN- in the lungs of IFN--/- mice could not control the growth of M. tuberculosis in other cells.
Growth of recombinant BCG in peritoneal macrophages in vitro To investigate whether the secretion of IFN- by the recombinant BCG would impact on the intracellular growth of the organisms in vitro, 4 day old peritoneal macrophages from uninfected IFN--/- or BALB/c mice were infected with either recombinant BCG-vector or BCG-IFN-. Both BCG recombinants grew exponentially in cultured macrophages obtained from IFN--/mice with generation times of 26.8 h (BCG-vector), 30.3 h (BCG-IFN-); generation times in control BALB/c mice were 31.6 h (BCG-vector) and 33.7 h (BCG-IFN-) (Fig. 6). Thus, despite the presence of IFN- in the culture supernatant (confirmed by ELISA), no difference in the growth of BCG-IFN- and BCG-vector was noted (Fig. 6). Growth of extracellular BCG was not
0
1
2 3 4 Time post-infection (days)
6
Figure 7. Cytokine levels in culture supernatants of peritoneal macrophages from IFN--/- mice infected in vitro with BCG-vector (∆) or BCG-IFN- (Ε). Results are expressed as mean pg/ml+SD for three experiments carried out in duplicate.
observed in the cell culture medium. These results suggest that the presence of murine IFN was not sufficient to activate macrophages from IFN--/- mice to control growth of the intracellular mycobacteria.
Cytokine production by peritoneal macrophages infected in vitro with recombinant BCG When peritoneal macrophages of IFN--/- mice were infected with BCG-vector, low levels of IL10 and TNF- were produced. The production of TNF- was highest at day 1 and much lower on days 2–6 of the experiment. The levels of IL10 were maximal at day 2 post-infection and slowly decreased (Fig. 7). The infection with the BCG-IFN- induced markedly reduced levels of IL-10 compared to the levels observed in cultures infected with BCG-vector. No difference was
BCG-IFNγ
BCG-Vector
BCG-IFNγ
BCG-Vector
Reconstitution of anti-mycobacterial immunity
αiNOS
αIRF-1
αGrb-2
8h
24 h
Figure 8. iNOS induction in peritoneal macrophages from IFN--/- mice infected with either BCG-vector or BCG-IFN--in vitro. Immunoblots were carried out on cell lysates prepared 8 and 24 h after infection. Grb-2 was used as a control.
observed in the levels of TNF- induced by infection with BCG-IFN- compared with BCGvector (Fig. 7).
Expression of iNOS by peritoneal macrophages infected with recombinant BCG In order to determine whether the infection of macrophages with the recombinant BCG could induce the expression of iNOS, cell homogenates were prepared 8 h, 1, 3 and 6 days post-infection. Specific iNOS protein immunoprecipitates were observed in IFN--/- peritoneal macrophages infected with BCG-IFN- at the 8 and 24 h time points but not in cells infected with BCG-vector. IRF-1 was similarly induced (Fig. 8).
Discussion and Conclusions IFN- is a central regulatory cytokine of the protective immune response to mycobacterial infection. The absence of the gene for IFN- or IFN- receptor leads to loss of this protection. In a previous study of IFN--/- mice infected
181
with M. tuberculosis, Flynn et al. [16] delivered IFN- by either intramuscular injection or with an implanted osmotic pump. However, this treatment was unable to fully reconstitute the protective immune response and prevent the death of infected IFN--/- animals. The authors suggested that an insufficient concentration of IFN- at the infected site (lung granulomas) might account for their findings. In the present study we infected IFN--/- mice with recombinant BCG-IFN- thereby ensuring the continuous presence of the cytokine at the site of infection. We show here that the effect of IFN- is limited to the site of cytokine production. The local production of IFN- is insufficient for complete systemic immune reconstitution. The aerosol infection of IFN--/- mice or control BALB/c mice with a recombinant BCG-IFN accelerates the rate of bacterial clearance early in the course of infection. This observation directly demonstrates the importance of this cytokine in restriction of mycobacterial growth and/or survival within the infected tissues [16, 18]. This early IFN- mediated effect on mycobacterial growth/survival mimics the effects of the specific cellular immune response that usually occurs in immune competent mice after T cell mediated immunity develops (>28 days). Our observations would therefore suggest that the earlier the T cell response is activated leading to IFN- production, the sooner mycobacterial growth can be controlled. In a recent study of clinical isolates of M. tuberculosis, we noted that early (7 days) production of cytokines including IFN- in the infected lung, is associated with earlier control of mycobacterial growth and prolonged survival of the mice [20]. Infection of IFN--/- mice with BCG-IFN- also induced a marked reduction in the expression of mRNA for the inhibitory cytokine IL-10. IL-10 is produced by T cells and by macrophages in response to mycobacteria or mycobacterial antigens. IL-10 inhibits IFN- production by Th1 T cell clones [21]. In addition IL-10 is a potent inhibitor of macrophage function, thereby favoring the survival of mycobacteria within the macrophage [14]. Our results demonstrate that local IFN- levels in the lungs reduce the production of IL-10 mRNA thus presumably promoting macrophage activation and control of mycobacterial growth. In our study the presence of IFN- in the lungs was associated with increased differentiation of macrophages into epithelioid cells and the development of smaller granulomas. IFN- also
182
induced iNOS protein expression in the macrophages in the lungs of IFN--/- infected mice. iNOS induction is dependent on at least two signals provided by two distinct cytokines: IFN (in the present study produced by the recombinant BCG) and TNF- (produced in low levels by macrophages in response to the infection). IFN- induces the specific activation of the nuclear factors STAT-1 and IRF-1, and TNF induces the activation of NF-B [11, 22]. Together, IRF-1 and NF-B regulate the transcription of nitric oxide synthase 2 (NOS2) [23]. However, despite the presence of both cytokine signals and iNOS protein production, peritoneal macrophages in vitro failed to restrict the growth of BCG. Hanano et al. [24] have shown that macrophages must be first activated with IFN prior to exposure to mycobacteria for adequate killing of the organisms. If macrophages were infected first and later stimulated with IFN-, no BCG killing was observed, despite adequate iNOS expression. Our results suggest that in vitro this may also be the case. Alternatively, additional stimuli may be required to activate macrophages for adequate control of BCG growth. Hematogenous spread is an indicator of the failure of the host immune response to completely contain the infection in the lungs, the primary site of infection. In the control BALB/ c mice, hematogenous spread of the BCG-IFN was not observed, either because the infection was fully contained in the lungs or because the immune response in the spleen was efficient at eliminating any organisms that seeded to that site. However, in the IFN--/- mice infected with BCG-IFN-, the cellular immune response in the lungs was not adequate to control the infection, thereby allowing hematogenous spread. The presence of cfu in the spleen indicated that the immune response in the spleen was also not adequate. Histology of the lung of IFN--/- mice revealed lymphoid cells aggregated around the vasculature and unevenly distributed throughout the infected tissue (Fig. 2). This sequestration of the lymphoid cells together with the absence of IFN- production by the T cells of IFN--/mice may explain the lack of expression of a systemic immune response against the M. tuberculosis challenge. IFN--/- mice were unable to control the growth of M. tuberculosis despite the presence of IFN- in the lungs due to BCG-IFN. This result suggests that the reconstitution of immunity may be restricted to the cells containing the BCG-IFN-. The same level of acti-
A. L. Moreira et al.
vation, including iNOS induction, may not be available in the cells containing M. tuberculosis. A systemic protective response to M. tuberculosis may be achieved only by the presence of specific cytokine producing and/or cytotoxic T lymphocytes. In vivo, T-cells including CD8+ T cells, may contribute to the control of infection by the production of cytokines and/or cytotoxic activity [25–27]. Recently, cytotoxic T cells have been shown to recognize and kill macrophages infected with mycobacteria and other intracellular parasites by either Fas–FasL interaction or by a Fas-independent granule-dependent mechanism. The latter pathway has been shown to lead to death of the host cell and also killing of the intracellular mycobacteria [28, 29]. In conclusion we have directly shown that although IFN--/- mice can be partially reconstituted by delivery of IFN- directly to the site of infection, the local reconstitution does not confer systemic anti-mycobacterial immunity. We have also shown that although iNOS protein expression and diminished IL-10 mRNA levels are associated with control of mycobacterial infection in the lung, these are insufficient to induce killing of intracellular mycobacteria in vitro. The potential contribution of additional IFN driven leukocyte responses to mycobacterial killing is under investigation.
Materials and Methods Mice Female BALB/c control and BALB/c IFN- gene disrupted (IFN--/-) mice [15] (6–7 weeks old) were obtained from the Jackson Laboratories (Bar Harbor, ME, U.S.A.). Mice were housed in group cages at the Rockefeller University Laboratory Animal Research Center.
Mycobacteria M. Bovis Bacillus Calmette-Gue´rin (BCG) strain Montreal (a strain of low virulence in mice) [2] was used to generate the recombinant mycobacteria. BCG strain Montreal expressing murine interferon-gamma (BCG-IFN-) and control BCG carrying the plasmid vector only (BCGvector) were grown to mid log phase in Middlebrook 7H9 medium supplemented with 20 g/ ml of kanamycin (Sigma Corp., St Louis, MO,
Reconstitution of anti-mycobacterial immunity
U.S.A.) as described [30, 31], and kept frozen in aliquots until use.
Aerosol infection Mice were inoculated via the respiratory route by exposure to an aerosolized suspension of mycobacteria generated by a Lovelace nebulizer using a nose-only exposure apparatus (In-Tox Products, Albuquerque, NM, U.S.A.) [32]. Stocks of 1.5×107 bacilli/ml were used to implant approximately 300 organisms into the lungs of mice, confirmed by plating lung homogenates 3 h after infection. Group of infected animals were killed at different time points as indicated. A group of BCG-infected mice was challenged 24 h later by an aerosol exposure to M. tuberculosis, strain CDC1551, at a dose of 50 mycobacteria/mouse. Survival of mice was recorded over time. Blood was collected by cardiac puncture from mice anesthetized with a solution containing 44 mg/kg of ketamine (Aveco Co., Inc. Fort Dodge, IO, U.S.A.) and 5 mg/kg of xylazine (Rompum, Mobay Corp., Shawnee, KS, U.S.A.). Serum was prepared and kept frozen at −80°C until assay. Lungs and spleens were collected aseptically immediately after cardiac puncture and used for further study. Serum and lungs from uninfected mice were used for determination of baseline cytokine and mRNA levels. This protocol was approved by the Rockefeller University Animal Care and Use Committee.
Histology Lungs were fixed in 10% buffered formalin, paraffin embedded and processed. Sections were stained with haematoxylin-eosin for histologic evaluation and with Ziehl-Nielsen for evaluation of acid fast bacilli (AFB) by light microscopy.
Immunohistology Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated through graded alcohols. For antigen retrieval the slides were boiled in 10 mmol/l citrate buffer pH 6.0 for 20 min. Staining was performed in an automated immunostainer (Ventana, Tucson, AZ,
183
U.S.A.) using a polyclonal rabbit anti-mouse iNOS (1:300) (Calbiochem, La Jolla, CA, U.S.A.).
Infection in vitro Peritoneal macrophages were collected from the peritoneal cavity of naı¨ve control BALB/c and IFN--/- mice, washed in RPMI (GIBCO Laboratories, Grand Island, NY, U.S.A.) and cultured in RPMI supplemented with 10% fetal calf serum (Gemini, Calabasas, CA, U.S.A.), 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (GIBCO) (complete medium) in 24 well plates (Falcon, Becton Dickinson, Lincoln Park, NJ, U.S.A.) for 4 days. Cells in complete medium without antibiotics were infected with recombinant BCG-vector or BCG-IFN- at a MOI of five mycobacteria/cell (5:1). Culture supernatant and cell lysates for cytokine analysis and cfu assay, respectively, were harvested daily for 6 days post-infection.
ELISA assays for cytokines TNF- and IL-10 levels were measured in the serum of infected and uninfected control animals, and in culture supernatants of peritoneal macrophages infected with recombinant BCG. Commercial ELISA kits (Endogen Inc., Boston, MA, U.S.A.) were used according to the manufacturer’s specification.
Polymerase chain reaction (PCR) for cytokine mRNA Total cellular RNA was obtained from lungs of mice infected with either BCG-vector or BCGIFN- at 60 days following aerosol exposure to the mycobacteria. Tissues were homogenized in 3 ml RNAzolB (Cinna/Biotecx Lab. Inc., Houston, TX, U.S.A.) and RNA was extracted according to the manufacturer’s instructions. The RT-PCR reaction using primers for TNF-, IL-10, IFN- and -actin was carried out as described [32]. Densitometry of the amplified bands was carried out using a Phosphorimager (Molecular Dynamics, CA, U.S.A.). Results were normalized to the density of -actin.
Colony forming units (cfu) assay Bacterial load in the lungs and spleens of infected mice was evaluated by plating 10-fold serial
184
dilutions of organ homogenates. cfu in infected cultured macrophages were evaluated by disrupting the cells in PBS containing 0.016% digitonin (Sigma) and 0.25% Tween 80 (Sigma) for 10 min at 37°C followed by probe sonication using 0.5 s pulses at low output. Organ homogenates and cell lysates were plated onto Middlebrook 7H10 agar plates (Difco Laboratories, Detroit, MI, U.S.A.) and 7H10 agar containing 20 g/ml of kanamycin (Sigma) then incubated at 37°C for 2–3 weeks. Organisms were enumerated as cfu as described [32].
Determination of IFN- levels and biologic activity in the bacterial culture supernatants The ability of the bacilli to secrete murine recombinant IFN- was verified by measuring the concentration of the cytokine by ELISA (Endogen Inc.) in the bacterial culture supernatants. Only mycobacterial suspensions that produced the cytokine were used for infection. Infecting stocks and mycobacteria recovered from the lungs of the infected mice (colonies in the cfu assay) were grown in 7H9 medium supplemented with 20 g/ml of kanamycin for 1 week, and the supernatants tested for IFN- by ELISA. In preliminary experiments it was observed that some of the BCG-IFN- colonies recovered from the lungs of infected mice had lost the ability to secrete IFN- by 90 days postinfection. The instability of the IFN- carrying plasmid in BCG has been described elsewhere [31]. Therefore, experiments in vivo were carried out only up to day 60. There was no difference in growth of recombinant BCG-vector and BCGIFN- in Middlebrook 7H9 medium. In addition, supernatants from BCG-vector and BCG-IFN- were diluted in RPMI (1:6 to 1:150) and added to J774 cells (a murine macrophage cell line) with 100 ng/ml LPS to evaluate the induction of IRF-1 and iNOS as described below.
Immunoblotting for IFN- induced proteins J774 cells were plated at a density of 5×105/ well in six well plates (Fisher, Pittsburgh, PA, U.S.A.) and stimulated with BCG culture supernatants diluted in RPMI as described above. At 48 h, the cells were washed in PBS and lysed in RIPA buffer. Peritoneal macrophage cultures were infected with recombinant BCG and cell
A. L. Moreira et al.
lysates were prepared at 8 h, 1,3 and 6 days post-infection as described. Cell lysates were electrophoresed on a 4–15% SDS PAGE gel and transferred to nitrocellulose. Immunoblots were probed for IRF-1 (48 kDa) and iNOS (130 kDa) using a rabbit anti-IRF-1 antibody at 1:2000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and a monoclonal anti-iNOS antibody at 1:1000 (Transduction Laboratories, Lexingon, KY, U.S.A.). HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Pierce Chemical Co., Rockford, IL, U.S.A.) were used to visualize the bands by chemiluminescence [22].
Statistical analysis Data were analysed using an independent t-test when indicated.
Acknowledgements We would like to thank Dr R. Young for helpful advice and discussion during the project. We acknowledge Dr Victoria H. Freedman for critical review of this manuscript, Judy Adams for preparing the figures and Marguerite Nulty for typing the manuscript. We also thank Drs Ian R. Orme and Andrea Cooper for their help with the early part of these studies. Andre´ L. Moreira was a Villares fellow.
References 1 Bourassa D, Forget A, Pelletier M, Skamene E, Turcotte R. Cellular immune response to Mycobacterium bovis (BCG) in genetically-susceptible and resistant congenic mouse strains. Clin Exp Immunol 1985; 62: 31–8. 2 Orme IM, Collins FM. Demonstration of acquired resistance in Bcgr inbred mouse strains infected with a low dose of BCG Montreal. Clin Exp Immunol 1984; 56: 81–8. 3 Kramnik I, Radzioch D, Skamene E. T-helper 1-like subset selection in Mycobacterium bovis Bacillus Calmette-Guerin-infected resistant and susceptible mice. Immunol 1994; 81: 618–25. 4 Medina E, North RJ. Mice that carry the resistance allele of BCG gene (Bcgr) develop a superior capacity to stabilize Bacilli Calmette-Guerin (BCG) infection in their lungs and spleen over a protracted period in the absence of specific immunity. Clin Exp Immunol 1996; 104: 44–7. 5 North RJ. Mycobacterium tuberculosis is strikingly more virulent for mice when given via the respiratory than the intravenous route. J Infect Dis 1995; 172: 1550–3.
Reconstitution of anti-mycobacterial immunity 6 Schluger NW, Rom WN. The host immune response to tuberculosis. Am J Resp Crit Care Med 1998; 157: 679–91. 7 Orme IM, Miller ES, Roberts AD et al. T Lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. J Immunol 1992; 148: 189–96. 8 Barnes PF, Fong S-J, Brennan PJ, Twomey PE, Mazumder A, Modlin RL. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J Immunol 1990; 145: 149–54. 9 Denis M. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell Immunol 1991; 132: 150–7. 10 Flesch IEA, Kaufmann SHE. Activation of tuberculostatic macrophage functions by gamma interferon, interleukin-4, and tumor necrosis factor. Infect Immun 1990; 58: 2675–7. 11 Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 1996; 84: 443–50. 12 Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 1983; 58: 670–89. 13 Xie QW, Cho HJ, Calaycay J et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256: 225–8. 14 Murray PJ, Wang L, Onufryk C, Tepper RI, Young RA. T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J Immunol 1997; 158: 315–21. 15 Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon- genes. Science 1993; 259: 1739–42. 16 Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon- in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993; 178: 2249–54. 17 Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon gene-disrupted mice. J Exp Med 1993; 178: 2243–7. 18 Kamijo R, Le J, Shapiro D et al. Mice that lack the interferon- receptor have profoundly altered responses to infection with Bacillus Calmette-Guerin and subsequent challenge with lipopolysaccharide. J Exp Med 1993; 178: 1435–40. 19 Flynn JL, Goldstein MM, Triebold KJ, Sypek J, Wolf S, Bloom BR. IL-12 increases resistance of BALB/c mice to
185
20
21 22
23 24
25 26
27
28 29 30
31
32
Mycobacterium tuberculosis infection. J Immunol 1995; 155: 2515–24. Manca C, Tsenova L, Barry III CE et al. Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 1999; 162: 6740–6. Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 1993; 11: 165–90. Meraz MA, White JM, Sheehan KC et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 1996; 84: 431–42. Kamijo R, Harada H, Matsuyama T et al. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 1994; 263: 1612–5. Hanano R, Kaufmann SHE. Nitric oxide production and mycobacterial growth inhibition by murine alveolar macrophages: the sequence of rIFN-gamma stimulation and Mycobacterium bovis BCG infection determines macrophage activation. Immunol Letters 1995; 45: 23. Mazzaccaro RJ, Stenger S, Rock KL et al. Cytotoxic T lymphocytes in resistance to tuberculosis. Adv Exp Med Biol 1998; 452: 85–101. Flynn JL, Goldstein MM, Triebold KJ, Koller B, Bloom BR. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Nat Acad Sci USA 1992; 89: 12013–7. Serbina NV, Flynn JL. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect Immun 1999; 67: 3980–8. Stenger S, Mazzaccaro RJ, Uyemura K et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science 1997; 276: 1684–7. Stenger S, Hanson DA, Teitelbaum R et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998; 282: 121–5. Murray PJ, Aldovini A, Young RA. Manipulation and potentiation of antimycobacterial immunity using recombinant bacilli Calmette-Guerin strains that secrete cytokines. Proc Natl Acad Sci USA 1996; 93: 934–9. Murray PJ, Young RA. Secretion of mammalian proteins from mycobacteria. In: Parish T, Stoker NG, eds. Methods in Molecular Biology; mycobacteria protocols. Totowa: Humana Press Inc., 1998: 275–84. Moreira AL, Tsenova-Berkova L, Wang J et al. Effect of cytokine modulation by thalidomide on the granulomatous response in murine tuberculosis. Tubercle Lung Dis 1997; 78: 47–55.
Microbial Pathogenesis 2000; 29: 175–185 doi:10.1006/mpat.2000.0382
MICROBIAL PATHOGENESIS
Aerosol infection of mice with recombinant BCG secreting murine IFN- partially reconstitutes local protective immunity Andre´ L. Moreiraa,b, Liana Tsenovaa, Peter J. Murrayc, Sherry Freemana, Amy Bergtolda, Luis Chiribogad & Gilla Kaplan∗a a
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021, U.S.A.; b New York University School of Medicine, Dept. of Pathology, 550 First Ave., New York, NY 10016, U.S.A.; c Dept. Infectious Diseases, St. Jude Children’s Research Hospital, 332 North Lauderdale St., Memphis, TN 38105, U.S.A.; d New York University School of Medicine, Kaplan Comprehensive Cancer Center, New York, NY 10016, U.S.A. (Received January 28, 2000; accepted in revised form May 28, 2000)
To better understand the contribution of interferon-gamma (IFN-) to the immune response during the first 60 days of mycobacterial infection in the lungs, IFN- gene disrupted (IFN--/-) mice were infected via aerosol with recombinant Mycobacterium bovis Bacillus Calmette-Guerin (BCG) secreting murine IFN- (BCG-IFN-) and compared to mice infected with recombinant BCG containing the vector only (BCG-vector). When IFN--/- mice were infected with BCG-vector, increasing bacillary loads and large undifferentiated granulomas that did not express inducible nitric oxide synthase (iNOS) were observed in the lungs. In contrast, infection with BCG-IFN- resulted in reduced bacillary load and better differentiated granulomas containing epithelioid macrophages expressing iNOS as well as reduced levels of interleukin 10 (IL-10) mRNA. However, local production of IFN- by the recombinant BCG did not protect IFN--/- mice from subsequent challenge with M. tuberculosis. Infection of IFN--/- peritoneal macrophages in vitro with BCG-IFN- led to induction of iNOS expression and lower IL-10 mRNA levels. Nevertheless, the growth of the intracellular BCG was unaffected. Since IFN- induced-iNOS protein and reduced IL-10 production were insufficient to control mycobacterial growth in vitro, the results suggest that additional mediator(s) present in vivo are required for control of mycobacterial growth. 2000 Academic Press Key words: Interferon-gamma, Mycobacterium bovis BCG, inducible nitric oxide synthase, macrophages.
∗ Author for correspondence. E-mail: kaplang@rockvax. rockefeller.edu This study was supported in part by US Public Health Service grants AI-22616 and AI-43526 to G.K. and by a grant from Pott’s Memorial Foundation to A.L.M.
0882–4010/00/090175+11 $35.00/0
2000 Academic Press
176
A. L. Moreira et al. 5
Introduction
(a)
3 2 cfu in lungs (log10)
BCG infection of mice has long been used as a model for studying the host immune response to mycobacterial infection. Infection of immunocompetent mice with BCG leads to initial growth or persistence of the organisms predominantly in the lungs of the animal. Subsequently, the growth of BCG is contained by the developing cellular immune response [1, 2]. The profile of cytokines induced in alveolar macrophages and other cells in the lungs following infection plays an important role in establishment and/or control of the infection [2–6]. IFN- is an important regulatory molecule in the protective immune response. In adoptive transfer experiments in mice, T-cells that confer protection against M. tuberculosis produce IFN when stimulated in vitro [7]. The local production of IFN- in infected tissues is associated with a protective host response to mycobacterial infection [8]. This cytokine has been shown to activate macrophages, rendering the cells capable of killing or controlling the growth of intracellular pathogens, including mycobacteria [9, 10]. The antibacterial activity of activated macrophages involves at least two pathways: the generation of reactive nitrogen intermediates (RNI) and the production of reactive oxygen intermediates (ROI) [9, 11–13]. Components of both pathways have been shown to be upregulated by IFN- in vitro [9, 13]. IFN- also downregulates the production of cytokines that negatively regulate macrophage function, such as interleukin-10 (IL-10) [14]. In experiments using IFN- gene disrupted mice (knock-out; IFN--/-) [15–17] or IFN- receptor (IFN--R) knock-out mice, the role of the cytokine was further explored [18]. These mice were unable to mount an effective cell mediated immune response to M. bovis BCG or to M. tuberculosis, resulting in unrestricted growth of the bacilli in the organs of infected animals. The animals succumbed to the infection earlier than wildtype control animals [16–18]. Thus, IFN- and its receptor are essential for controlling mycobacterial infection. To date it has been difficult to reconstitute the host response in IFN- deficient mice [16, 19]. Such reconstitution is necessary to conclusively prove the role of IFN- as a regulatory cytokine and to define the mechanism of its effects in the host protective response to mycobacterial infection. In the present study, we used a strain
*
*
4
1 0 5
(b) *
4 3 2 1 0
0
10
50 20 30 40 Time post-infection (days)
60
Figure 1. Bacillary load in the lungs of BCG-infected IFN--/- (a) and BALB/c (b) mice. Results are the means of three experiments (with quadruplicate samples) expressed as log10 cfu±SD. Closed symbols represent mice infected with BCG-IFN-. Open symbols represent mice infected with BCG-vector. Statistically significant differences (indicated by ∗) in the numbers of BCG-IFN- vs BCG-vector were observed (P<0.05).
of recombinant BCG that secretes murine IFN (BCG-IFN-) to attempt to reconstitute the host response to BCG infection and to an aerosol challenge with M. tuberculosis.
Results Bacillary load in the organs of BCG infected mice Two recombinant BCG strains were used to infect mice: BCG-vector and BCG-IFN- both grew similarly in liquid medium with generation times of 38.4 and 37.2 h, respectively. BCG-IFN used for infection were shown to secrete IFN (by ELISA) and the IFN- was biologically active (by IRF-1 induction in J774 cells). When IFN--/- mice were infected with BCG-vector, the bacillary load increased rapidly for the first 35 days of the infection [Fig.1(a)]. After 35 days,
Reconstitution of anti-mycobacterial immunity
numbers of bacilli in the lungs stabilized. Hematogenous spread of BCG-vector to the spleens was observed at 60 days (cfu log 3.2±1.6). When IFN--/- mice were infected with the recombinant BCG-IFN-, the number of organisms in the lungs did not increase, resulting in a significant difference between the numbers of BCG-vector compared to BCG-IFN- in the lungs at day 35 (P=0.04). Hematogeneous spread of BCG-IFN- to the spleens was observed by day 60 of infection (cfu log 2.4±0.5). Infection of control BALB/c mice with BCGvector resulted in slow bacillary growth in the lungs of aerosol infected mice for the first 60 days [Fig. 1(b)] confirming the attenuated nature of BCG Montreal in mice. Hematogenous spread of the organisms from the lungs to the spleen was first observed at day 60 post-infection (cfu log 2.2±0.5). When BALB/c mice were infected with BCG-IFN-, bacterial numbers in the lung were significantly reduced throughout the infection compared to baseline (35 days P=0.03; 60 days P=0.02). This resulted in a significant difference in the numbers of BCG-vector vs the numbers of BCG-IFN- in the lungs of infected mice at 60 days (P<0.05) [Fig. 1(b)]. In addition, no hematogenous spread of BCG-IFN- to the spleens was observed. These results indicate that IFN- production at the site of infection partially controls the growth of BCG in immunocompromised IFN--/- mice. In the immune competent mice the growth of BCG is more efficiently restricted in the presence of the additional IFN-.
177
in cuffs most often around blood vessels and some neutrophils were observed within the inflammatory infiltrate. However, in contrast to the BCG-vector-infected mice, few AFB were seen in association with the epithelioid or foamy macrophages in BCG-IFN- infected mice, thus morphologically confirming the cfu results (Fig. 1). Aerosol infection with either BCG-vector or BCG-IFN- induced similar small granulomas in the lungs of control BALB/c mice (not shown).
Expression of iNOS in the lungs of BCGinfected mice To better characterize the local host response to the infecting bacilli, immunohistochemical staining for iNOS, an IFN- induced protein, was carried out on the lung sections. In the lungs of IFN--/- mice infected with BCG-vector, there were large collections of macrophages which were negative for the expression of iNOS [Fig. 3(a)]. In contrast, when mice were infected with BCG-IFN-, some of the epithelioid macrophages within the granulomas were positive for the expression of iNOS [Fig. 3(b)]. Therefore, the local production of IFN- by the recombinant mycobacteria induced local macrophage differentiation and iNOS expression. iNOS expression was observed in the lungs of BALB/c control mice infected with either BCG-vector or BCG-IFN- (not shown).
Cytokine expression in the lungs of mice infected with recombinant BCG The granulomatous response in the lungs of BCG infected mice In the IFN--/- mice, aerosol infection with BCGvector induced a vigorous cellular response in the lungs. By day 60 post-infection, the lungs were filled with many loose aggregates of undifferentiated foamy macrophages [Fig. 2(a) and (b)] and cuffs of lymphocytes [Fig. 2(b)] with scattered neutrophils. Interestingly, most of the lymphoid infiltrate in the lungs was restricted to perivascular areas [Fig. 2(a)]. Acid-fast bacilli (AFB) were seen in the large macrophages [Fig. 2(c)]. In contrast, when IFN--/- mice were infected with BCG-IFN-, the mononuclear cell infiltrate was more focal [Fig. 2(d) and (e)]. Differentiation of the macrophages into epithelioid cells was more pronounced [Fig. 2(e) and (f)]. Here too, lymphocytes were organized
The expression of IL-10, TNF- and IFN- mRNA in the lungs of infected mice was studied. Infection of IFN--/- mice with BCG-IFN- resulted in significantly decreased expression of IL10 mRNA at day 60 post-infection when compared to the levels obtained in the lungs of IFN-/- mice infected with the BCG-vector (P<0.05). Comparable low levels of TNF- mRNA were observed in the lungs of IFN--/- mice infected with either recombinant strain [Fig. 4(a)]. As expected, no IFN- mRNA was detected in these mice. In contrast to the IFN--/- mice, IFN- mRNA was expressed in the lungs of control BALB/c mice. In the latter, infection with BCGIFN- resulted in a significant up-regulation of this cytokine compared with infection with BCGvector. No difference in the low level of expression of TNF- mRNA and IL-10 mRNA was
178
Figure 2. Legend on facing page.
Figure 3. Legend on facing page.
A. L. Moreira et al.
Reconstitution of anti-mycobacterial immunity 100
(a)
Survival of mice (%)
Cytokine mRNA in infected lungs (fold increase)
2
179
1
50
0 5
(b)
4
0
10
20
30
40
Time post-challenge (days)
3
Figure 5. Survival of IFN--/- mice (12/group) preinfected by aerosol with: BCG-vector (Β), BCG-IFN- (Χ) followed by infection with 1.6 log10 M. tuberculosis CDC1551 24 h later. Control mice infected only with M. tuberculosis CDC1551 (Α).
2 1 0
IL-10
TNF-α
IFN-γ
Figure 4. Cytokine mRNA expression in the lungs of BCG-infected IFN--/- (a) and BALB/c (b) mice. RTPCR results were first normalized to the amount of -actin mRNA in each lane and then expressed as fold increase over the amount of cytokine mRNA in the lungs of mice infected with BCG-vector at day 60 post-infection (100%=1). The results are means±SD (two experiments with quadruplicate samples). Levels in mice infected with BCG-vector (∆); levels in mice infected with BCG-IFN- (Ε).
noted [Fig. 4(b)]. Neither IFN-, IL-10 or TNF- was detected by ELISA in the serum of IFN--/or BALB/c mice infected with either strain of recombinant BCG at any time during the infection.
Effect of infection with BCG-IFN- on response to M. tuberculosis challenge To determine whether the local production of IFN- in the lungs of IFN--/- mice would protect these animals from tuberculosis, IFN--/mice already infected with BCG-IFN- were challenged with a small inoculum of M. tuberculosis strain CDC1551 [20]. When uninfected IFN--/- mice were infected by aerosol with M. tuberculosis CDC1551, mice began to die on day 35 post-infection. By day 38, all mice had succumbed to the infection, thus confirming the immunodeficiency of IFN--/- mice. When mice were first infected with either of the recombinant BCG strains and 24 h later challenged with aerosolized M. tuberculosis, similar survival patterns were observed (Fig. 5). These results suggest
Figure 2. Histologic sections of lungs of IFN--/- mice infected with BCG-vector (a–c) or BCG-IFN- (d–f) at day 60 post-infection. (a), (b), (d) and (e): hematoxylin and eosin, (c) and (f): Ziehl-Nielsen stains. Arrow heads indicate mycobacteria in large undifferentiated macrophages. Ly-lymphocyte cuff. Magnification: (a), (d) ×25; (b), (e) ×100; (c), (f) ×250. Figure 3. Immunohistology for iNOS expression in the sections of lungs of IFN--/- mice 60 days after infection with either BCG-vector (a) or BCG-IFN- (b). Ly-lymphocyte cuff; asterisks indicate alveolar space. Arrows indicate large undifferentiated iNOS-negative macrophages in BCG-vector-infected mice (a), and iNOS-positive (dark pink stain) as well as iNOS-negative epithelioid macrophages in BCG-IFN- infected mice (b). Magnification ×250.
180
A. L. Moreira et al. 20
240
(a)
200 TNF-α (pg/ml)
15
5
120 80 40
0 20
0
(b)
100
15
80 IL-10 (pg/ml)
cfu in macrophages (fold increase)
10
160
10
5
60 40 20
0
1
2 3 4 5 Time post-infection (days)
6
Figure 6. Growth of BCG-vector (Β) and BCG-IFN (Χ) in peritoneal macrophages of IFN--/- (a) and BALB/c (b) mice. Results are expressed as means±SE of the mean for four experiments each carried out in duplicate.
that the focal presence of intracellular IFN- in the lungs of IFN--/- mice could not control the growth of M. tuberculosis in other cells.
Growth of recombinant BCG in peritoneal macrophages in vitro To investigate whether the secretion of IFN- by the recombinant BCG would impact on the intracellular growth of the organisms in vitro, 4 day old peritoneal macrophages from uninfected IFN--/- or BALB/c mice were infected with either recombinant BCG-vector or BCG-IFN-. Both BCG recombinants grew exponentially in cultured macrophages obtained from IFN--/mice with generation times of 26.8 h (BCG-vector), 30.3 h (BCG-IFN-); generation times in control BALB/c mice were 31.6 h (BCG-vector) and 33.7 h (BCG-IFN-) (Fig. 6). Thus, despite the presence of IFN- in the culture supernatant (confirmed by ELISA), no difference in the growth of BCG-IFN- and BCG-vector was noted (Fig. 6). Growth of extracellular BCG was not
0
1
2 3 4 Time post-infection (days)
6
Figure 7. Cytokine levels in culture supernatants of peritoneal macrophages from IFN--/- mice infected in vitro with BCG-vector (∆) or BCG-IFN- (Ε). Results are expressed as mean pg/ml+SD for three experiments carried out in duplicate.
observed in the cell culture medium. These results suggest that the presence of murine IFN was not sufficient to activate macrophages from IFN--/- mice to control growth of the intracellular mycobacteria.
Cytokine production by peritoneal macrophages infected in vitro with recombinant BCG When peritoneal macrophages of IFN--/- mice were infected with BCG-vector, low levels of IL10 and TNF- were produced. The production of TNF- was highest at day 1 and much lower on days 2–6 of the experiment. The levels of IL10 were maximal at day 2 post-infection and slowly decreased (Fig. 7). The infection with the BCG-IFN- induced markedly reduced levels of IL-10 compared to the levels observed in cultures infected with BCG-vector. No difference was
BCG-IFNγ
BCG-Vector
BCG-IFNγ
BCG-Vector
Reconstitution of anti-mycobacterial immunity
αiNOS
αIRF-1
αGrb-2
8h
24 h
Figure 8. iNOS induction in peritoneal macrophages from IFN--/- mice infected with either BCG-vector or BCG-IFN--in vitro. Immunoblots were carried out on cell lysates prepared 8 and 24 h after infection. Grb-2 was used as a control.
observed in the levels of TNF- induced by infection with BCG-IFN- compared with BCGvector (Fig. 7).
Expression of iNOS by peritoneal macrophages infected with recombinant BCG In order to determine whether the infection of macrophages with the recombinant BCG could induce the expression of iNOS, cell homogenates were prepared 8 h, 1, 3 and 6 days post-infection. Specific iNOS protein immunoprecipitates were observed in IFN--/- peritoneal macrophages infected with BCG-IFN- at the 8 and 24 h time points but not in cells infected with BCG-vector. IRF-1 was similarly induced (Fig. 8).
Discussion and Conclusions IFN- is a central regulatory cytokine of the protective immune response to mycobacterial infection. The absence of the gene for IFN- or IFN- receptor leads to loss of this protection. In a previous study of IFN--/- mice infected
181
with M. tuberculosis, Flynn et al. [16] delivered IFN- by either intramuscular injection or with an implanted osmotic pump. However, this treatment was unable to fully reconstitute the protective immune response and prevent the death of infected IFN--/- animals. The authors suggested that an insufficient concentration of IFN- at the infected site (lung granulomas) might account for their findings. In the present study we infected IFN--/- mice with recombinant BCG-IFN- thereby ensuring the continuous presence of the cytokine at the site of infection. We show here that the effect of IFN- is limited to the site of cytokine production. The local production of IFN- is insufficient for complete systemic immune reconstitution. The aerosol infection of IFN--/- mice or control BALB/c mice with a recombinant BCG-IFN accelerates the rate of bacterial clearance early in the course of infection. This observation directly demonstrates the importance of this cytokine in restriction of mycobacterial growth and/or survival within the infected tissues [16, 18]. This early IFN- mediated effect on mycobacterial growth/survival mimics the effects of the specific cellular immune response that usually occurs in immune competent mice after T cell mediated immunity develops (>28 days). Our observations would therefore suggest that the earlier the T cell response is activated leading to IFN- production, the sooner mycobacterial growth can be controlled. In a recent study of clinical isolates of M. tuberculosis, we noted that early (7 days) production of cytokines including IFN- in the infected lung, is associated with earlier control of mycobacterial growth and prolonged survival of the mice [20]. Infection of IFN--/- mice with BCG-IFN- also induced a marked reduction in the expression of mRNA for the inhibitory cytokine IL-10. IL-10 is produced by T cells and by macrophages in response to mycobacteria or mycobacterial antigens. IL-10 inhibits IFN- production by Th1 T cell clones [21]. In addition IL-10 is a potent inhibitor of macrophage function, thereby favoring the survival of mycobacteria within the macrophage [14]. Our results demonstrate that local IFN- levels in the lungs reduce the production of IL-10 mRNA thus presumably promoting macrophage activation and control of mycobacterial growth. In our study the presence of IFN- in the lungs was associated with increased differentiation of macrophages into epithelioid cells and the development of smaller granulomas. IFN- also
182
induced iNOS protein expression in the macrophages in the lungs of IFN--/- infected mice. iNOS induction is dependent on at least two signals provided by two distinct cytokines: IFN (in the present study produced by the recombinant BCG) and TNF- (produced in low levels by macrophages in response to the infection). IFN- induces the specific activation of the nuclear factors STAT-1 and IRF-1, and TNF induces the activation of NF-B [11, 22]. Together, IRF-1 and NF-B regulate the transcription of nitric oxide synthase 2 (NOS2) [23]. However, despite the presence of both cytokine signals and iNOS protein production, peritoneal macrophages in vitro failed to restrict the growth of BCG. Hanano et al. [24] have shown that macrophages must be first activated with IFN prior to exposure to mycobacteria for adequate killing of the organisms. If macrophages were infected first and later stimulated with IFN-, no BCG killing was observed, despite adequate iNOS expression. Our results suggest that in vitro this may also be the case. Alternatively, additional stimuli may be required to activate macrophages for adequate control of BCG growth. Hematogenous spread is an indicator of the failure of the host immune response to completely contain the infection in the lungs, the primary site of infection. In the control BALB/ c mice, hematogenous spread of the BCG-IFN was not observed, either because the infection was fully contained in the lungs or because the immune response in the spleen was efficient at eliminating any organisms that seeded to that site. However, in the IFN--/- mice infected with BCG-IFN-, the cellular immune response in the lungs was not adequate to control the infection, thereby allowing hematogenous spread. The presence of cfu in the spleen indicated that the immune response in the spleen was also not adequate. Histology of the lung of IFN--/- mice revealed lymphoid cells aggregated around the vasculature and unevenly distributed throughout the infected tissue (Fig. 2). This sequestration of the lymphoid cells together with the absence of IFN- production by the T cells of IFN--/mice may explain the lack of expression of a systemic immune response against the M. tuberculosis challenge. IFN--/- mice were unable to control the growth of M. tuberculosis despite the presence of IFN- in the lungs due to BCG-IFN. This result suggests that the reconstitution of immunity may be restricted to the cells containing the BCG-IFN-. The same level of acti-
A. L. Moreira et al.
vation, including iNOS induction, may not be available in the cells containing M. tuberculosis. A systemic protective response to M. tuberculosis may be achieved only by the presence of specific cytokine producing and/or cytotoxic T lymphocytes. In vivo, T-cells including CD8+ T cells, may contribute to the control of infection by the production of cytokines and/or cytotoxic activity [25–27]. Recently, cytotoxic T cells have been shown to recognize and kill macrophages infected with mycobacteria and other intracellular parasites by either Fas–FasL interaction or by a Fas-independent granule-dependent mechanism. The latter pathway has been shown to lead to death of the host cell and also killing of the intracellular mycobacteria [28, 29]. In conclusion we have directly shown that although IFN--/- mice can be partially reconstituted by delivery of IFN- directly to the site of infection, the local reconstitution does not confer systemic anti-mycobacterial immunity. We have also shown that although iNOS protein expression and diminished IL-10 mRNA levels are associated with control of mycobacterial infection in the lung, these are insufficient to induce killing of intracellular mycobacteria in vitro. The potential contribution of additional IFN driven leukocyte responses to mycobacterial killing is under investigation.
Materials and Methods Mice Female BALB/c control and BALB/c IFN- gene disrupted (IFN--/-) mice [15] (6–7 weeks old) were obtained from the Jackson Laboratories (Bar Harbor, ME, U.S.A.). Mice were housed in group cages at the Rockefeller University Laboratory Animal Research Center.
Mycobacteria M. Bovis Bacillus Calmette-Gue´rin (BCG) strain Montreal (a strain of low virulence in mice) [2] was used to generate the recombinant mycobacteria. BCG strain Montreal expressing murine interferon-gamma (BCG-IFN-) and control BCG carrying the plasmid vector only (BCGvector) were grown to mid log phase in Middlebrook 7H9 medium supplemented with 20 g/ ml of kanamycin (Sigma Corp., St Louis, MO,
Reconstitution of anti-mycobacterial immunity
U.S.A.) as described [30, 31], and kept frozen in aliquots until use.
Aerosol infection Mice were inoculated via the respiratory route by exposure to an aerosolized suspension of mycobacteria generated by a Lovelace nebulizer using a nose-only exposure apparatus (In-Tox Products, Albuquerque, NM, U.S.A.) [32]. Stocks of 1.5×107 bacilli/ml were used to implant approximately 300 organisms into the lungs of mice, confirmed by plating lung homogenates 3 h after infection. Group of infected animals were killed at different time points as indicated. A group of BCG-infected mice was challenged 24 h later by an aerosol exposure to M. tuberculosis, strain CDC1551, at a dose of 50 mycobacteria/mouse. Survival of mice was recorded over time. Blood was collected by cardiac puncture from mice anesthetized with a solution containing 44 mg/kg of ketamine (Aveco Co., Inc. Fort Dodge, IO, U.S.A.) and 5 mg/kg of xylazine (Rompum, Mobay Corp., Shawnee, KS, U.S.A.). Serum was prepared and kept frozen at −80°C until assay. Lungs and spleens were collected aseptically immediately after cardiac puncture and used for further study. Serum and lungs from uninfected mice were used for determination of baseline cytokine and mRNA levels. This protocol was approved by the Rockefeller University Animal Care and Use Committee.
Histology Lungs were fixed in 10% buffered formalin, paraffin embedded and processed. Sections were stained with haematoxylin-eosin for histologic evaluation and with Ziehl-Nielsen for evaluation of acid fast bacilli (AFB) by light microscopy.
Immunohistology Formalin-fixed, paraffin-embedded sections were deparaffinized and rehydrated through graded alcohols. For antigen retrieval the slides were boiled in 10 mmol/l citrate buffer pH 6.0 for 20 min. Staining was performed in an automated immunostainer (Ventana, Tucson, AZ,
183
U.S.A.) using a polyclonal rabbit anti-mouse iNOS (1:300) (Calbiochem, La Jolla, CA, U.S.A.).
Infection in vitro Peritoneal macrophages were collected from the peritoneal cavity of naı¨ve control BALB/c and IFN--/- mice, washed in RPMI (GIBCO Laboratories, Grand Island, NY, U.S.A.) and cultured in RPMI supplemented with 10% fetal calf serum (Gemini, Calabasas, CA, U.S.A.), 2 mM L-glutamine, 100 U/ml penicillin and 100 g/ml streptomycin (GIBCO) (complete medium) in 24 well plates (Falcon, Becton Dickinson, Lincoln Park, NJ, U.S.A.) for 4 days. Cells in complete medium without antibiotics were infected with recombinant BCG-vector or BCG-IFN- at a MOI of five mycobacteria/cell (5:1). Culture supernatant and cell lysates for cytokine analysis and cfu assay, respectively, were harvested daily for 6 days post-infection.
ELISA assays for cytokines TNF- and IL-10 levels were measured in the serum of infected and uninfected control animals, and in culture supernatants of peritoneal macrophages infected with recombinant BCG. Commercial ELISA kits (Endogen Inc., Boston, MA, U.S.A.) were used according to the manufacturer’s specification.
Polymerase chain reaction (PCR) for cytokine mRNA Total cellular RNA was obtained from lungs of mice infected with either BCG-vector or BCGIFN- at 60 days following aerosol exposure to the mycobacteria. Tissues were homogenized in 3 ml RNAzolB (Cinna/Biotecx Lab. Inc., Houston, TX, U.S.A.) and RNA was extracted according to the manufacturer’s instructions. The RT-PCR reaction using primers for TNF-, IL-10, IFN- and -actin was carried out as described [32]. Densitometry of the amplified bands was carried out using a Phosphorimager (Molecular Dynamics, CA, U.S.A.). Results were normalized to the density of -actin.
Colony forming units (cfu) assay Bacterial load in the lungs and spleens of infected mice was evaluated by plating 10-fold serial
184
dilutions of organ homogenates. cfu in infected cultured macrophages were evaluated by disrupting the cells in PBS containing 0.016% digitonin (Sigma) and 0.25% Tween 80 (Sigma) for 10 min at 37°C followed by probe sonication using 0.5 s pulses at low output. Organ homogenates and cell lysates were plated onto Middlebrook 7H10 agar plates (Difco Laboratories, Detroit, MI, U.S.A.) and 7H10 agar containing 20 g/ml of kanamycin (Sigma) then incubated at 37°C for 2–3 weeks. Organisms were enumerated as cfu as described [32].
Determination of IFN- levels and biologic activity in the bacterial culture supernatants The ability of the bacilli to secrete murine recombinant IFN- was verified by measuring the concentration of the cytokine by ELISA (Endogen Inc.) in the bacterial culture supernatants. Only mycobacterial suspensions that produced the cytokine were used for infection. Infecting stocks and mycobacteria recovered from the lungs of the infected mice (colonies in the cfu assay) were grown in 7H9 medium supplemented with 20 g/ml of kanamycin for 1 week, and the supernatants tested for IFN- by ELISA. In preliminary experiments it was observed that some of the BCG-IFN- colonies recovered from the lungs of infected mice had lost the ability to secrete IFN- by 90 days postinfection. The instability of the IFN- carrying plasmid in BCG has been described elsewhere [31]. Therefore, experiments in vivo were carried out only up to day 60. There was no difference in growth of recombinant BCG-vector and BCGIFN- in Middlebrook 7H9 medium. In addition, supernatants from BCG-vector and BCG-IFN- were diluted in RPMI (1:6 to 1:150) and added to J774 cells (a murine macrophage cell line) with 100 ng/ml LPS to evaluate the induction of IRF-1 and iNOS as described below.
Immunoblotting for IFN- induced proteins J774 cells were plated at a density of 5×105/ well in six well plates (Fisher, Pittsburgh, PA, U.S.A.) and stimulated with BCG culture supernatants diluted in RPMI as described above. At 48 h, the cells were washed in PBS and lysed in RIPA buffer. Peritoneal macrophage cultures were infected with recombinant BCG and cell
A. L. Moreira et al.
lysates were prepared at 8 h, 1,3 and 6 days post-infection as described. Cell lysates were electrophoresed on a 4–15% SDS PAGE gel and transferred to nitrocellulose. Immunoblots were probed for IRF-1 (48 kDa) and iNOS (130 kDa) using a rabbit anti-IRF-1 antibody at 1:2000 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) and a monoclonal anti-iNOS antibody at 1:1000 (Transduction Laboratories, Lexingon, KY, U.S.A.). HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Pierce Chemical Co., Rockford, IL, U.S.A.) were used to visualize the bands by chemiluminescence [22].
Statistical analysis Data were analysed using an independent t-test when indicated.
Acknowledgements We would like to thank Dr R. Young for helpful advice and discussion during the project. We acknowledge Dr Victoria H. Freedman for critical review of this manuscript, Judy Adams for preparing the figures and Marguerite Nulty for typing the manuscript. We also thank Drs Ian R. Orme and Andrea Cooper for their help with the early part of these studies. Andre´ L. Moreira was a Villares fellow.
References 1 Bourassa D, Forget A, Pelletier M, Skamene E, Turcotte R. Cellular immune response to Mycobacterium bovis (BCG) in genetically-susceptible and resistant congenic mouse strains. Clin Exp Immunol 1985; 62: 31–8. 2 Orme IM, Collins FM. Demonstration of acquired resistance in Bcgr inbred mouse strains infected with a low dose of BCG Montreal. Clin Exp Immunol 1984; 56: 81–8. 3 Kramnik I, Radzioch D, Skamene E. T-helper 1-like subset selection in Mycobacterium bovis Bacillus Calmette-Guerin-infected resistant and susceptible mice. Immunol 1994; 81: 618–25. 4 Medina E, North RJ. Mice that carry the resistance allele of BCG gene (Bcgr) develop a superior capacity to stabilize Bacilli Calmette-Guerin (BCG) infection in their lungs and spleen over a protracted period in the absence of specific immunity. Clin Exp Immunol 1996; 104: 44–7. 5 North RJ. Mycobacterium tuberculosis is strikingly more virulent for mice when given via the respiratory than the intravenous route. J Infect Dis 1995; 172: 1550–3.
Reconstitution of anti-mycobacterial immunity 6 Schluger NW, Rom WN. The host immune response to tuberculosis. Am J Resp Crit Care Med 1998; 157: 679–91. 7 Orme IM, Miller ES, Roberts AD et al. T Lymphocytes mediating protection and cellular cytolysis during the course of Mycobacterium tuberculosis infection. J Immunol 1992; 148: 189–96. 8 Barnes PF, Fong S-J, Brennan PJ, Twomey PE, Mazumder A, Modlin RL. Local production of tumor necrosis factor and IFN-gamma in tuberculous pleuritis. J Immunol 1990; 145: 149–54. 9 Denis M. Interferon-gamma-treated murine macrophages inhibit growth of tubercle bacilli via the generation of reactive nitrogen intermediates. Cell Immunol 1991; 132: 150–7. 10 Flesch IEA, Kaufmann SHE. Activation of tuberculostatic macrophage functions by gamma interferon, interleukin-4, and tumor necrosis factor. Infect Immun 1990; 58: 2675–7. 11 Durbin JE, Hackenmiller R, Simon MC, Levy DE. Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 1996; 84: 443–50. 12 Nathan CF, Murray HW, Wiebe ME, Rubin BY. Identification of interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity. J Exp Med 1983; 58: 670–89. 13 Xie QW, Cho HJ, Calaycay J et al. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256: 225–8. 14 Murray PJ, Wang L, Onufryk C, Tepper RI, Young RA. T cell-derived IL-10 antagonizes macrophage function in mycobacterial infection. J Immunol 1997; 158: 315–21. 15 Dalton DK, Pitts-Meek S, Keshav S, Figari IS, Bradley A, Stewart TA. Multiple defects of immune cell function in mice with disrupted interferon- genes. Science 1993; 259: 1739–42. 16 Flynn JL, Chan J, Triebold KJ, Dalton DK, Stewart TA, Bloom BR. An essential role for interferon- in resistance to Mycobacterium tuberculosis infection. J Exp Med 1993; 178: 2249–54. 17 Cooper AM, Dalton DK, Stewart TA, Griffin JP, Russell DG, Orme IM. Disseminated tuberculosis in interferon gene-disrupted mice. J Exp Med 1993; 178: 2243–7. 18 Kamijo R, Le J, Shapiro D et al. Mice that lack the interferon- receptor have profoundly altered responses to infection with Bacillus Calmette-Guerin and subsequent challenge with lipopolysaccharide. J Exp Med 1993; 178: 1435–40. 19 Flynn JL, Goldstein MM, Triebold KJ, Sypek J, Wolf S, Bloom BR. IL-12 increases resistance of BALB/c mice to
185
20
21 22
23 24
25 26
27
28 29 30
31
32
Mycobacterium tuberculosis infection. J Immunol 1995; 155: 2515–24. Manca C, Tsenova L, Barry III CE et al. Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 1999; 162: 6740–6. Moore KW, O’Garra A, de Waal Malefyt R, Vieira P, Mosmann TR. Interleukin-10. Annu Rev Immunol 1993; 11: 165–90. Meraz MA, White JM, Sheehan KC et al. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 1996; 84: 431–42. Kamijo R, Harada H, Matsuyama T et al. Requirement for transcription factor IRF-1 in NO synthase induction in macrophages. Science 1994; 263: 1612–5. Hanano R, Kaufmann SHE. Nitric oxide production and mycobacterial growth inhibition by murine alveolar macrophages: the sequence of rIFN-gamma stimulation and Mycobacterium bovis BCG infection determines macrophage activation. Immunol Letters 1995; 45: 23. Mazzaccaro RJ, Stenger S, Rock KL et al. Cytotoxic T lymphocytes in resistance to tuberculosis. Adv Exp Med Biol 1998; 452: 85–101. Flynn JL, Goldstein MM, Triebold KJ, Koller B, Bloom BR. Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection. Proc Nat Acad Sci USA 1992; 89: 12013–7. Serbina NV, Flynn JL. Early emergence of CD8(+) T cells primed for production of type 1 cytokines in the lungs of Mycobacterium tuberculosis-infected mice. Infect Immun 1999; 67: 3980–8. Stenger S, Mazzaccaro RJ, Uyemura K et al. Differential effects of cytolytic T cell subsets on intracellular infection. Science 1997; 276: 1684–7. Stenger S, Hanson DA, Teitelbaum R et al. An antimicrobial activity of cytolytic T cells mediated by granulysin. Science 1998; 282: 121–5. Murray PJ, Aldovini A, Young RA. Manipulation and potentiation of antimycobacterial immunity using recombinant bacilli Calmette-Guerin strains that secrete cytokines. Proc Natl Acad Sci USA 1996; 93: 934–9. Murray PJ, Young RA. Secretion of mammalian proteins from mycobacteria. In: Parish T, Stoker NG, eds. Methods in Molecular Biology; mycobacteria protocols. Totowa: Humana Press Inc., 1998: 275–84. Moreira AL, Tsenova-Berkova L, Wang J et al. Effect of cytokine modulation by thalidomide on the granulomatous response in murine tuberculosis. Tubercle Lung Dis 1997; 78: 47–55.