Dr C. L. Silva, Centre for Tuberculosis Research, Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Avenida Bandeirantes 3900, 14049–900, Ribeirão Preto, SP, Brazil. E-mail: email@example.com
Development and evaluation of new vaccines and immunotherapy against tuberculosis demand a better understanding of the immune mechanisms in this disease. Costimulatory signals and intercellular contact seem to be pivotal in determining whether recognition of antigen by T cells leads to activation or anergy. In this paper, we show that virulent M. tuberculosis H37Rv downmodulates the ex vivo expression of CD18 and CD86 on peritoneal macrophages and VLA-4 on lymphocytes but does not disturb the in vitro production of interleukin (IL)-12 and interferon (IFN)-γ after intraperitoneal infection. In addition, splenocytes from infected mice produce IL-10, while the expression of cell surface receptors is unchanged. The interplay among IL-12, IFN-γ and IL-10 in vivo and the downmodulation of cell-surface receptors during the infection at the inflammatory site may contribute to the explanation of the maintenance of infection.
Tuberculosis is a chronic intracellular infectious disease that remains a major public health problem worldwide. In developing countries tuberculosis is a leading cause of morbidity and mortality, and spread of the HIV epidemic contributes significantly to aggravate the situation .
Studies with animal models clearly indicate that resistance to Mycobacterium tuberculosis is a cell-mediated process that requires co-operation between T lymphocytes and macrophages . There is a general consensus that CD4+ T lymphocytes play a pivotal role in the protection [3, 4]. More recently, it has been established that CD8+ T lymphocytes are also essential to control the infection [5, 6]. Tumour necrosis factor (TNF)-α, IL-12  and IFN-γ[9, 10] seem to be crucial for the development of protective cellular immune response. Macrophages play an essential role in internalizing the bacilli and presenting mycobacterial antigens to T lymphocytes. Specific receptors on T lymphocytes recognize the antigens and, in conjunction with costimulatory receptors, release activation signals that induce the production of cytokines, like IFN-γ. This cytokine acts in synergism with some macrophage factors and mycobacterial compounds promoting macrophage activation . In the murine model it was demonstrated that the reactive nitrogen intermediates (RNI), such as nitric oxide (NO), is required for the destruction of intracellular M. tuberculosis bacilli [12, 13]. Recently, the effector mechanisms involved in direct M. tuberculosis killing or infected cells lysis by CD4+ T and CD8+ T lymphocytes were described [14–16]. However, M. tuberculosis can still survive in macrophages. Despite the progress in the comprehension of the pathogen-host relationship, the mechanisms of protection and pathogenesis of this disease are not totally clear. This hinders the development of an efficacious vaccine against this pathogen and therapeutic strategies for intervention in the host immune response.
Primary activation and differentiation of T lymphocytes varies depending on the presence of additional costimulatory signals delivered to the T cell by accessory molecules, which are receptors for specific ligants expressed on antigen-presenting cell (APC). Therefore, antigenic stimulation can lead either to a immune response characterized by proliferation, differentiation, clonal expansion and effector function, or in the absence of appropriate costimulation, to a state of long-lasting antigen-specific unresponsiveness termed anergy . The members of the B7 family, B7-1 (CD80), B7-2 (CD86) and their counter receptors CD28 and CTLA-4 (cytotoxic T lymphocyte-associated protein), are critical to induce an immune or anergic state [18–20]. The receptors CD80 and CD86 display a pattern of expression that depends on the type of APC and the presence of a variety of stimuli . CD28 is constitutively expressed on all mouse T cells . Following a T cell receptor (TCR) engagement, the costimulatory signal delivered by the CD28–B7 interaction induces an increase of IL-2 mRNA transcription, IL-2 secretion, upregulation of IL-2 receptor, T-cell proliferation and cytokine secretion [22–24]. CTLA-4 is T-cell restricted and appears after T-cell activation [25, 26]. Recent studies provide compelling support for an inhibitory role for CTLA-4 in the regulation of T-cell responses .
Besides costimulatory receptors, other molecules regulate T-cell activation [28, 29]. Most of them, which belong to the integrins and Igs families, were first described as mediators of the migration process . Besides, recent evidences indicate that both costimulatory and adhesion receptors are essential to provide the intercellular contact [31, 32] that induces an efficient T-cell activation.
In the present study, we investigated the expression of CD28, CTLA-4, very late antigen-4 (VLA-4), CD54 or intercellular adhesion molecule-1 (ICAM-1), CD18 (β2 integrin), CD80 and CD86 in mice infected by intraperitoneal route with M. tuberculosis H37Rv. For this purpose, the expression of CD28, CTLA-4, VLA-4, CD18 and CD54 was evaluated on peritoneal and spleen lymphocytes. Additionally, the CD80, CD86 and CD18 expression was evaluated on peritoneal macrophages after 7, 15 and 42 days of infection and correlated with the IFN-γ, IL-12 and IL-10 production by these cells.
Materials and methods
Infection of mice BALB/c mice, 5–8 weeks old, were bred in the Animal Facility of the School of Medicine of Ribeirão Preto, University of São Paulo, Brazil, and were kept under barrier conditions in a level III biohazard laboratory. Mice were infected by intraperitoneal inoculation (i.p.) with 1 × 106 viable colony-forming units (CFU) of M. tuberculosis H37Rv strain (American Type Culture Collection, Rockville, MD, USA) grown in 7H9 medium. Viability of inoculum was tested previously with fluorescein diacetate (Sigma, St. Louis, MO, USA) and ethidium bromide . Viability was still checked by plating 10-fold serial dilutions in Lowenstein–Jensen solid medium (Difco, Detroit, MI, USA) and counting the colonies (as CFU) after 21 days of culture.
Monoclonal antibodies (MoAbs) To evaluate the expression of costimulatory and adhesion molecules, the following MoAb antimouse cell surface markers were used: (1) labelled with PE: anti-CD28, anti-CTLA-4 and anti-CD80 (hamster IgG), anti-CD86 (rat IgG2a), anti-VLA-4 (rat IgG2b); and (2) labelled with FITC: anti-CD54 (ICAM-1) (hamster IgG) and anti-CD18 (rat IgG2a). Hamster IgG, rat IgG2a and rat IgG2b labelled with PE or FITC were used as control isotypes. All MoAb were acquired from PharMingen (San Diego, CA, USA) and used according to the manufacturer's instructions.
Preparation of peritoneal cells and splenocytes Groups of five infected or noninfected mice were killed by cervical dislocation after slight anaesthesia 7, 15 and 42 days after infection. Cells from the peritoneal cavity were harvested by injection of cold phosphate-buffered saline (PBS). The abdomen was gently massaged and the cell suspension was carefully withdrawn with a syringe. Abdominal washings were pooled in plastic tubes and total cell counts were determined in a Neubauer chamber (Neubauer, Germany). Spleens were removed and homogenized through a fine steel mesh. Red blood cells were lysed with 0.16 m NH4Cl and 0.17 m Tris at 4 °C for 10 min. The splenocytes were then washed and resuspended to 1 × 107 cells/ml in PBS for flow cytometry or to 5 × 106 cells/ml in RPMI 1640 complete medium for cultures.
FACS analysis Peritoneal cells or splenocytes were initially incubated for 40 min at 4 °C with Fc Block (PharMingen, San Diego, CA, USA) to avoid nonspecific binding. Then the cells were incubated with MoAb (0.75 μg/106 cells) for 30 min at 4 °C in the dark. The control of fluorescence was done with unrelated antibodies labelled with fluorochromes. The cells were washed with 2% fetal bovine serum (Gibco, Grand Island, NY, USA) in PBS by centrifugation at 400 × g and fixed with 1% (v/v) formaldehyde in PBS. The cells were acquired by FACSort (Becton & Dickinson, San Jose, CA, USA) and 10 000 cells per sample were collected and analyzed with Lysis II program. Lymphocytes and macrophages were gated by forward (FSC) and side (SSC) scatter (Fig. 1). To assure that lymphocytes and macrophages were gated separately, some experiments were performed by selection with antimouse CD3 or antimouse CD11b, both labelled with fluorescein isothiocyanate (FITC). The selection of the positive population directed at each receptor studied was done with a control isotype. The positive population was analyzed by the percentage of positive cells gated and the level of expression is reported as median of fluorescence intensity (MFI).
Soluble antigens from M. tuberculosis Total soluble antigen from M. tuberculosis H37Rv was prepared from mycobacteria grown in Middlebrook medium 7H9 at 37 °C. After 45 days, the cultures were heat-killed for 2 h, at 80 °C. The mycobacterial suspension was centrifuged for 15 min at 4000 × g. The sediment was washed with PBS solution, centrifuged for an additional 15 min at 4000 × g, resuspensed in PBS and sonicated. After centrifugation the supernatant was considered as a total soluble antigen of M. tuberculosis. The protein content was determined using the Coomassie Protein Assay kit (Pierce, Rockford, IL, USA).
Lymphoproliferation assay and cytokine production Lympho-proliferation assays were done in triplicate by plating 5 × 105 splenocytes in 96 well flat-bottomed plates (Corning, NY, USA) in the presence of Concanavalin A (20 µg/ml) (Con A, Sigma). After 56 h incubation at 37 °C in a humidified incubator with a 5% CO2 atmosphere, the cultures were pulsed for 16 h with [3H] thymidine (0.5 μCi/well, Amersham, UK) and collected onto glass-fibre filters. Radiolabel incorporation was measured by liquid scintillation in a β counter. The levels of IFN-γ, IL-12 and IL-10 were measured by ELISA in supernatants from cultures stimulated (48 h) with Con A or mycobacterial antigen. Briefly, 96-well plates (Corning) were coated with capture antimouse IFN-γ, IL-12 or IL-10 (PharMingen), 1 μg/ml, and incubated overnight at 4 °C. The plates were washed with 0.05% solution of Tween 20 in PBS and blocked with 1% bovine serum albumin (BSA) (Sigma) in PBS for 2 h at room temperature. A hundred µl of culture supernatants and IFN-γ, IL-12 or IL-10 standards were then added and incubated overnight at 4 °C. Cytokine-antibody complexes were detected by addition of biotin antimurine IFN-γ, IL-12 or IL-10 (PharMingen), 1 mg/ml, followed by peroxidase-conjugated streptavidin (Dako, Denmark). After addition of ο-phenylenediamine (Abbott), the colour development was stopped with 16% H2SO4. Absorbance at 492 nm was measured on an ELISA reader (Metertech Σ960) and IFN-γ, IL-12 and IL-10 concentrations were calculated by extrapolating absorbance values from the standard curve. The detection limit for IFN-γ was 40 pg/ml and for IL-12 and IL-10 was 10 pg/ml.
Statistical analysis The statistical significance of data was estimated by the analysis of variance and by the Dunnett's test. P < 0.05 and P < 0.01 were considered statistically significant.
Downmodulation of CD86 expression on peritoneal macrophages
Macrophages and lymphocytes were gated by forward (FSC) and side scatter (SSC) as show in Fig. 1. A parallel analysis was performed with the MoAbs anti-CD3 or anti-CD11b to assure that larger cells gated on R2 were macrophages or monocytes recently migrated and not lymphoblasts.
A significant difference was observed in the percentage of CD86 + peritoneal macrophages obtained from infected and noninfected mice (Fig. 2A), indicating a low influx of CD86 + macrophages at the site of infection. Since the beginning of the infection, these macrophages also exhibited a lower CD86 expression on the cell surface, represented by the MFI score, in comparison to the one observed in cells from control animals (Fig. 2C). The CD80 expression was similar in infected and control animals (Fig. 2D). However, infected mice exhibited a lower percentage of CD80+ macrophages than noninfected mice (Fig. 2B). Variations among the control groups (noninfected mice) analyzed during the experimental kinetics were not expected and may be related to the compensation of fluorescence channels for analyzing macrophage population.
Representative FACS histograms in Fig. 3 show the downmodulation of CD86 (top panel) and CD18 (bottom panel) on macrophages during infection and the similar expression of CD80 between infected and noninfected groups (intermediate panel).
Expression of CD28 and CTLA-4 on peritoneal lymphocytes
As illustrated in Fig. 4(A), there was no significant changes in the CD28 expression by peritoneal lymphocytes during the different phases of infection. Even through the percentage of splenocytes expressing CD28 was low in infected mice (19%, 24% and 16% for 7, 15 and 42 days of infection, respectively), this difference was not significant in comparison with the percentage of lymphocytes from noninfected group (36%, 35% and 38%). Because there was not a significant difference in the CD28 expression at the site of infection and spleen, CTLA-4 expression was evaluated. After infection with M. tuberculosis, there was no ex vivo expression of CTLA-4 in either peritoneal (Fig. 4B) or spleen cells (data not shown).
Downmodulation of VLA-4 expression on peritoneal lymphocytes and CD18 expression on peritoneal macrophages
As we detected no significant difference in the CD28 expression on peritoneal lymphocytes (Fig. 4A), we evaluated the expression of adhesion receptors VLA-4, CD54 and CD18 that contribute to lymphocyte migration  and activation [28, 29]. The results in Fig. 4(C) show that VLA-4 was significantly downregulated on peritoneal lymphocytes from infected mice, since the beginning of the infection. However, the CD54 expression did not presented significant alteration (Fig. 4D). The CD18 expression on peritoneal lymphocytes and macrophages was also evaluated. The CD18 expression on peritoneal lymphocytes was significantly upregulated only in the beginning of the infection (Fig. 4E). On the other hand, CD18 expression was significantly downmodulated on peritoneal macrophages after 15 and 42 days of infection (Fig. 4F). A similar expression of VLA-4, CD54 and CD18 was observed on spleen lymphocytes from infected and noninfected mice (data not shown).
Representative FACS histograms in Fig. 5 show the unchanged levels of CD28 (top panel), the downmodulation of VLA-4 during the infection (intermediate panel) and the upregulation of CD18 on peritoneal lymphocytes only in the beginning of infection (bottom panel).
Splenic cells from infected mice presented a decreased proliferation in response to Con A stimulation, in comparison to the control animals. This reduction was more evident at 7 and 15 days (23% and 38%, respectively) and more discrete (12%) at 42 days of infection (data not shown).
IFN-γ and IL-12 production
In order to evaluate the IFN-γ production during infection, peritoneal and splenic cells were stimulated in vitro with Con A or with the total soluble antigen derived from M. tuberculosis. Infected animals produced higher IFN-γ levels in response to specific antigen stimulation in comparison with the control animals (Fig. 6A). This increase was more pronounced at 42 days of infection (Fig. 6B). Peritoneal cells obtained at this time produced increased levels of IFN-γ in response to Con A stimulation (Fig. 6B). Splenocytes from infected mice also produced IFN-γ in response to Con A stimulation. However, they produced no IFN-γ when stimulated with M. tuberculosis soluble antigen either at the beginning (7 days) or during the chronic phase of infection (42 days) (Figs 6C,D).
The profile of IL-12 production during infection was similar in peritoneal (Figs 7A,B) and splenic cells (Figs 7C,D). Interestingly, cells from infected mice spontaneously produced IL-12. This production increased after the stimulation with soluble antigen. Peritoneal cells and splenocytes from infected mice produced higher IL-12 concentration at the beginning of the infection (7 days), in comparison to 42 days. Cells from normal mice also produced IL-12 in response to mycobacterial antigen stimulation.
The production of IL-10 by peritoneal cells obtained after 7 or 42 days of infection was lower than the control cells when stimulated in vitro with Con A or soluble antigen (Fig. 8A,B). On the other hand, splenocytes obtained 42 days after infection and stimulated with the soluble antigen produced higher levels of IL-10 than cells from noninfected mice (Fig. 8D).
In this paper, we used an experimental model of systemic tuberculosis to evaluate the expression of CD28, CTLA-4, VLA-4, CD54, CD18 on lymphocytes, and CD80, CD86 and CD18 on macrophages at the site of infection and in the spleen. Our results indicate that M. tuberculosis infection downmodulated the CD86 expression on peritoneal macrophages but did not affect CD80 expression on these cells (Figs 2,3). The kinetics of the CD86 expression on macrophages during infection showed that this receptor was downmodulated on peritoneal macrophages since the beginning of infection, as shown in Fig. 2(C).
Many studies support a dominant role for CD86 in primary T-cell responses [34, 35]. This dominance of CD86 in primary T-cell responses may be based on temporal patterns of receptor expression. CD86 is constitutively expressed on resting human peripheral blood monocytes and is upregulated following stimulation . Moreover, the CD86 expression is rapidly upregulated during B-cell and macrophage activation [37, 38]. CD80 is expressed later and not all activation signals upregulate this receptor . In this context, the downmodulation of the CD86 expression on macrophages, at the site of infection in our model, could impair the initial steps of T-lymphocyte activation owing to a deficient contact between T cells and APC. The possible involvement of CD86 in primary immune response is reinforced by recent reports indicating the contribution of CD86 to the signal transduction. It is possible that the cytoplasmic domain of CD86 has a function in signal transduction that would not be expected for CD80 .
Modulation of CD80 and CD86 in tuberculosis and other experimental models has been described. Saha et al. (1994)  showed that macrophages infected in vitro with avirulent M. tuberculosis presented a downmodulation on B7 expression. Leishmania donovani also impairs the upregulation of B7 after in vitro macrophage infection . On the other hand, Subauste et al. (1998)  reported an upregulation of CD80 and CD86 after in vitro infection of human monocytes with Toxoplasma gondii. According to these results, deficient expression of costimulatory or adhesion molecules could impair antigen presentation by infected cells. Our finding shows the existence of a selective mechanism of downregulation of costimulatory molecules ex vivo at the site of infection, since only CD86, but not CD80, was downmodulated on peritoneal macrophages (Figs 2 and 3).
CD18 receptor, characterized by the β chain of Mac-1 molecule, was also downmodulated on peritoneal macrophages. In this way, our data suggests that the uptake and phagocytosis of M. tuberculosis was mediated, at least partly, by Mac-1. This receptor has been related to the uptake of mycobacteria [43, 44]. However, it seems that in the absence of Mac-1, M. tuberculosis is able to gain entry into host cells via alternative phagocytic receptors and establish infection .
We also evaluated the expression of CD28, which is the ligant for CD80 and CD86, on peritoneal lymphocytes. CD28 is expressed at low density on the surface of T lymphocytes and there is evidence of upregulation of this receptor on cell surface after lymphocyte activation . Our results show that there was no significant difference on the level of CD28 expression on cells from either infected or noninfected mice (Fig. 4A).
The expression of CD28 was also evaluated on splenocytes and we did not detect significant differences, despite of lower number of CD28+ lymphocytes in infected mice (data not shown). Because there were no changes in the expression of CD28, the CTLA-4 expression was investigated. The CTLA-4 receptor is a member of the CD28 family and its expression occurs after lymphocyte activation  and is currently associated with lymphocyte deactivation . Besides this, CTLA-4 has a 20-fold higher affinity for CD80 and CD86 receptors than CD28 [25, 26]. There was no expression of CTLA-4 on the surface of peritoneal (Fig. 4B) and spleen lymphocytes (data not shown). The CTLA-4 expression was induced 48 h after in vitro T-lymphocyte activation and then this receptor was quickly internalized in clathrin vesicles . Attempting to detect the CTLA-4 expression within the cells, peritoneal cells were firstly permeabilized and afterwards labelled with antimouse CTLA-4 MoAb. There was no CTLA-4 expression inside peritoneal cells from infected animals (data not shown). To increase the amplification signal and therefore to improve the sensitivity of the assay, CTLA-4 expression was also checked with a monoclonal IgG antibody antimouse CTLA-4 as a first step, a biotin anti-IgG as a second step and a streptoavidin conjugated to PE as a third step. Again, no ex vivo CTLA-4 expression was detected (data not shown).
Cell adhesion molecules can also intensify the contact between T cells and APC, and thereby allow for low avidity TCR-antigen contacts. The integrin interactions are examples of this type of costimulatory mechanism [47, 48]. In addition, the VLA-4  and CD54 [28, 31] can also trigger activation signals to T cells. Our finding that M. tuberculosis infection induces a downmodulation of VLA-4 in all three stages of infection and an upregulation of CD18 on these lymphocytes only in the beginning of the infection could be associated with a more discrete lymphocyte migration to the site of infection, despite antigen specific IFN-γ production by peritoneal cells.
The expression of these adhesion receptors on spleen lymphocytes did not show significant differences. Also, the values of MFI were very similar between lymphocytes from infected and noninfected mice (data not shown). This pattern of similar expression found in spleen lymphocytes for both, adhesion and costimulatory receptors, during the infection, could be related to the migration of specific lymphocytes, activated in secondary lymphoid organs such as the spleen, to the site of infection.
In view of the regulatory role of cytokines on costimulatory and adhesion molecules, we evaluated the production of IFN-γ, IL-12 and IL-10. IFN-γ and IL-12 are closely related with protective immune response in experimental tuberculosis [8–10]. On the other hand, IL-10 knock-out mice infected with M. tuberculosis develop the opposite pattern of immune response in comparison to the wild-type, characterized by increased antimycobacterial immunity .
The increased levels of IFN-γ and IL-12 at the site of infection suggest a preferential differentiation of Th1 response. On the other hand, there was no significant production of IFN-γ by spleen cells stimulated in the same way as peritoneal cells, either at the beginning (7 days) or during the chronic phase of infection (42 days). The IL-12 production by peritoneal and spleen cells was higher in the beginning of infection in relation to the chronic phase. On the other hand, the IL-10 production in infected mice was higher in the chronic phase of infection when compared to the production by spleen cells of noninfected mice stimulated with M. tuberculosis antigen and with the production from infected mice in the beginning of infection. Therefore, it seems that our model favours a compartmentalization of the immune response characterized by an expressive production of IFN-γ and IL-12 at the site of infection. An opposite scenario concerning the IFN-γ production occurs in the spleen.
Our results suggest that in the inflammatory microenvironment at the site of infection, the infected macrophages could be activated and secrete IL-12, which contributes to the differentiation of recruited T lymphocytes to the Th1 pattern, and could be associated with protection in experimental tuberculosis [8–10]. In spite of the IL-12 and IFN-γ secretion in the peritoneal cavity, the infection still persists. It is tempting to hypothesize that this is related to the downmodulation of CD86 and CD18 on macrophages during the infection. In addition, the CD86 is expressed earlier than CD80, which could contribute more to delay the resolution of infection. The downmodulation of VLA-4 and the upregulation of CD18 only in the initial phase of infection may reflect low infiltration of lymphocytes to the site of infection that could also contribute to the impairment of the cellular immune response.
We thank Izaíra Tincani Brandão for technical assistance and Dra. Alexandrina Sartori for helpful discussions.
This study was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP).