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Published ahead of print on December 30, 2002, doi:10.1165/rcmb.2002-0163OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 28-38, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0163OC

Associations between Toll-Like Receptors and Interleukin-4 in the Lungs of Patients with Tuberculosis

Gael Fenhalls, Ginette R. Squires, Liesel Stevens-Muller, Juanita Bezuidenhout, Gillian Amphlett, Ken Duncan and Pauline T. Lukey

MRC Center for Molecular and Cellular Biology, and Departments of Medical Biochemistry and Anatomical Pathology, University of Stellenbosch, Cape Town, South Africa; and Asthma Cell Biology, Statistical Sciences, and Diseases of the Developing World, GlaxoSmithKline R&D, Stevenage, United Kingdom

Address correspondence to: Pauline Lukey, Ph.D., Diseases of the Developing World, GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, Hertfordshire SG1 2NY, UK. E-mail: pauline.t.lukey{at}gsk.com


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Toll-like receptors (TLRs) are implicated in the intracellular killing of Mycobacterium tuberculosis, and their expression is modulated by interleukin-4 (IL-4) in vitro. Our aim was to examine the expression of TLRs at the site of pathology in tuberculous lung granulomas and to explore the effect of the immune response on TLR expression. Immunohistochemistry was performed on lung granulomas from nine patients with tuberculosis undergoing lobectomy for haemoptysis. All nine patients expressed all of the TLRs studied (TLRs 1–5 and 9), whereas only five out of the nine patients had any granulomas positive for IL-4. Statistical analysis of TLR and cytokine staining patterns in 183 individual granulomas from the nine patients revealed significant associations between pairs of receptors and IL-4. A positive association between TLR2 and TLR4 (P < 0.0001) and a negative association between TLR2 and IL-4 (P < 0.0001) was observed. The associations between TLRs 1, 5, and 9 were significantly different in IL-4–negative compared with IL-4–positive patients. In conclusion, TLRs are expressed by various cell types in the human tuberculous lung, and their expression patterns are reflected by differences in the immune response.

Abbreviations: acid-fast bacilli, AFB • dendritic cells, DCs • interferon, IFN • interleukin, IL • lipopolysaccharide, LPS • purified protein derivative, PPD • T helper, Th • Toll-like receptors, TLRs • tumor necrosis factor, TNF


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ten mammalian homologs of the Drosophila Toll protein have been identified so far (1) and are characterized by an intracellular signaling domain related to the interleukin (IL)-1 Receptor (Toll/IL-1R "TIR" domain) and an extracellular domain consisting of leucine-rich repeats. Mammalian Toll-like receptors (TLRs) mediate responses to a variety of putative ligands generated by invading pathogens. TLRs form a vital part of the innate immune system (2), and their activation induces a signal transduction cascade that ultimately influences the type of specific immune response mounted against the pathogen (3).

TLRs respond to both specific and diverse microbial stimuli (4). TLR2 appears to recognize components of gram-positive bacteria such as peptidoglycan, as well as mycobacterial lipoproteins and yeast zymosan (4, 5). TLR2 may form heterodimers with TLR1 or TLR6, broadening both the range of bacterial components recognized and the signaling pathways induced (6, 7). TLR3 is stimulated by dsRNA and may be important in viral infections (8). TLR4 appears to be important in the recognition of gram-negative bacteria, as its putative ligands include lipopolysaccharide (LPS) (9). TLR4 forms a homodimer and can associate with CD14 to enhance recognition of LPS (10). TLR5 is activated by bacterial flagellin (11). Recently, TLR7 and TLR8 have been identified as the probable receptors for antiviral immune modifying compounds (imiquimod and R-848) (12). TLR9 is activated by unmethylated CpG oligodeoxynucleotides specific for bacterial DNA (13). Lastly, TLR10 is preferentially expressed on cells of the immune system and is most homologous to TLRs 1 and 6 (14). TLRs are expressed on the surface of immune cells (phagocytes, lymphocytes, and dendritic cells) (3, 15–18) and have also been detected on epithelial and endothelial cells (19, 20).

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is a facultative intracellular pathogen that can survive and replicate within the host macrophage. Transmission is via the aerosol route and droplets containing live bacilli are ingested by alveolar macrophages, which are unable to eliminate the pathogen. Thereafter, a chronic cell-mediated immune response results in the development of granulomas consisting of aggregates of macrophages and lymphocytes (21). A cell-mediated immune response is characterized by macrophage activation due to T helper type 1 (Th1) cells producing interferon (IFN)-{gamma}, and is considered beneficial in tuberculosis (22). On the other hand, controversy exists as to the role of the Th2 cytokine IL-4 (23, 24). This cytokine may be expressed in patients with tuberculosis due to an inappropriate antimycobacterial immune response (25) or to other underlying pathologies such as parasite infestation or atopy (26–28). In patients with an effective immune response the granulomas ultimately resolve, and the pathogen remains quiescent. However, in susceptible patients, the granulomas may become necrotic at the center, liquefy and rupture into the bronchus, releasing live infectious bacilli into the airways (29). It is possible that both types of granuloma (resolving and disseminating) may be present in the same patient at the same time.

There is evidence that TLRs may play a role in the host immune response to M. tuberculosis. Exposure of murine macrophages to heat-killed M. tuberculosis or components of the bacterial cell wall (such as lipoarrabinomannan and lipids) results in expression of the inflammatory cytokine tumor necrosis factor (TNF)-{alpha} via TLR2 signaling (30). When live M. tuberculosis was used to stimulate murine macrophages, however, both TLR2- and TLR4-dependent responses were observed (31). An opportunistic pathogen, M. avium, was only able to stimulate TLR2 and not TLR4 (31). Therefore, live virulent mycobacteria can stimulate through TLR2, as expected, but also through TLR4 in a CD14-independent manner (31). Exposure of immature monocyte-derived dendritic cells (DCs) to peptidoglycan, arabinogalactan, and mycolic acids resulted in DC maturation and production of TNF-{alpha}, IL-6, and IL-12p40 (32). Activation of TLR2 by the 19-kD lipoprotein of M. tuberculosis results not only in production of TNF-{alpha} and IL-12, but also in killing of the intracellular pathogen (33). Thus TLR2 may play a role in protective immunity to tuberculosis by upregulating the Th1-inducing cytokine IL-12 (32) as well as the killing mechanisms of infected macrophages (33). It is, however, also possible that M. tuberculosis can exploit TLRs to its own advantage. The 19-kD lipoprotein also causes downregulation of MHC class II molecules on the surface of macrophages by a TLR2-dependent mechanism, and this may mask the presence of M. tuberculosis from CD4+ Th1 cells (34). This may contribute to the ability of M. tuberculosis to evade the host immune response and establish a chronic infection.

Our previous studies show that TNF-{alpha}, IFN-{gamma}, and IL-12 p40 are readily detectable in lung tuberculous granulomas from patients with severe cavitating disease (35). Different granulomas within the same individual express different combinations of these cytokines, and therefore each granuloma is an immune microenvironment with a specific cytokine profile (29, 35). Furthermore, these patients can be polarized into two groups according to the presence or absence of IL-4 in the lungs, with seven out of the 12 patients studied positive for IL-4 (29, 35). In at least two of these IL-4–positive patients, atopy or parasite infestation was found in addition to tuberculosis (35). IL-4 did not appear to have prognostic significance in these severely ill patients; however, the presence or absence of IL-4 may indicate subtle underlying differences in the immune response of these two groups in response to M. tuberculosis infection (35).

To investigate expression of TLRs at the site of pathology in patients with pulmonary tuberculosis, we performed immunohistochemistry for TLRs 1, 2, 3, 4, 5, and 9 on paraffin-embedded tissue from nine patients with pulmonary tuberculosis undergoing therapeutic lobectomy for potentially fatal haemoptysis. All of the TLRs studied were readily detectable in the patients, except TLR3, where expression was relatively sparse. The tissue sections were also stained for IL-4, and as expected from previous studies (29, 35), the patients could be grouped into IL-4–positive and IL-4–negative patients. Differences in the expression patterns of TLRs 1, 5, and 9 were seen between these two groups. Furthermore, at the level of the individual granuloma, the presence of IL-4 significantly decreased the probability that the granuloma would be TLR2- or TLR4-positive. Therefore, heterogeneity between different granulomas, even within the same patient, was observed and TLR expression patterns varied in patients with different immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients
Resected lung tissue was collected from nine patients with tuberculosis who presented with life-threatening hemoptysis at Tygerberg Hospital, Cape Town, South Africa (Table 1). It is likely that these patients had had tuberculosis previously, but this was not clear from the clinical records available. Seven out of the nine patients were positive for acid-fast bacilli (AFB) as measured by Ziehl Neelsen staining of paraffin-embedded sections, though the number of AFBs was low. All nine patients, including the four who were negative for AFB (TB14, TB16, TB18, and TB22), were positive for the presence of M. tuberculosis DNA as demonstrated by DNA:DNA in situ hybridization (36). All patients were culture-positive for drug-sensitive M. tuberculosis. Patients were HIV–negative, and they all received a blood transfusion before the surgery. Directly after surgery, tissue was selectively dissected for formaldehyde fixation. All the patients successfully completed their antituberculosis therapy.


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  		TABLE 1 Patient details and the number of granulomas positive for the TLRs and IL-4

 
Nontuberculous lung tissue was obtained from two patients with malignancies. One underwent pneumonectomy for pulmonary blastoma (con1, 27-yr-old female) and the other had a lobectomy for synovial sarcoma (con2, 38–yr-old female). Both of these patients were negative for M. tuberculosis, and lung tissue distal to the tumors was used for the analysis of TLR expression

Ethical approval was obtained for the study from the Ethics Committee of the University of Stellenbosch.

TLR Antibodies
To generate anti-TLR antibodies for use in immunohistochemistry, unique peptide sequences with no potential glycosylation sites were identified within the leucine-rich repeat loops of the extracellular domain of TLR 1–5 and 9. The peptide sequences were vlgetygekedpeglqdfnteslhi for TLR1, frasdndrvidpgkvetltiorrlhipr for TLR2, kqknlitldlshnglsstklgtqvq for TLR3, fkeirlhkltlrnnfdslnvmkt for TLR4, gfhnikdpdqntfaglarssvrhld for TLR5, and glryvdlsdnrisgaseltatmge for TLR9. The peptides were prepared using tuberculin purified protein derivative (PPD) as a carrier protein, the anti-peptide antibodies raised in rabbits and the resulting antibodies affinity-purified against their respective peptides. Antibody specificity was confirmed by ELISA (against the original peptide and recombinant TLR2 and TLR4). Chinese hamster ovary (CHO) cells were transiently transfected with expression plasmids encoding human TLRs 1–6, 8, and 9. These were used for immunocytochemistry and Western blot analyses using the rabbit polyclonals produced.

Immunohistochemistry of TLRs 1, 2, 3, 4, 5, and 9
Consecutive 5-µm formalin-fixed tissue sections were used to detect the presence of TLRs 1, 2, 3, 4, 5, and 9 proteins. Prior to the addition of the TLR antibodies the sections were dewaxed in xylene, rehydrated in graded alcohols, and microwaved on high power for 20 min in 0.01 M citrate buffer pH 6.0. Nonspecific proteins were blocked with 5% goat serum (Dako, Carpinteria, CA) for 10 min. The TLR antibodies were incubated with the sections overnight at 4°C after being diluted in 5% goat serum. Antibody concentrations were as follows: 330 ng/ml for TLR1, 560 ng/ml for TLR2, 325 ng/ml for TLR3, 945 ng/ml for TLR4, 300 ng/ml for TLR5, and 270 ng/ml for TLR9. Biotinylated goat anti rabbit (Dako) was diluted 1:250 and incubated with the sections for 1 h at 4°C. Incubation of the sections with streptavidin–HRP (horseradish peroxidase) as well as the DAB (3'3 diaminobenzedine) was performed using the DAB detection system essentially as described by the manufacturer's instructions (Dako). The slides were then immersed in hematoxylin (Dako) for 2 min, rinsed in distilled water for 5 min, and then mounted with Faramount (Dako). As a control for nonspecific binding, appropriate dilutions of the TLR peptides were mixed together with the appropriate TLR antibody and then incubated with the slides. Immunohistochemistry was then performed as described above. As an additional control, immunohistochemistry was performed using Protein G–purified rabbit IgG at 1 µg/ml. To rule out the possibility that the affinity-purified antibodies were recognizing the carrier protein, 1% PPD was added to the diluted TLR antibodies 30 min before performing immunohistochemistry.

Immunohistochemistry of IL-4
Immunohistochemistry for the detection of IL-4 protein was essentially performed as described (29).

Microscopy and Scoring
The images were captured using a Zeiss microscope (Axioskop 2) fitted with a Sony 3CCV video camera. The images were saved using Axiovision from Zeiss (Baden-Württemburg, Germany). All the slides were analyzed blind and in duplicate by a consultant pathologist at Tygerberg Medical School, Cape Town (Dr. J. Bezuidenhout). Immunohistochemistry is an empirical staining technique and cannot be accurately quantitated. The results were therefore analyzed according to the presence or absence of the brown color reaction. The slides for each TLR and IL-4 were assessed for the presence or absence of signal and necrosis by three independent observers and analyzed for each patient in duplicate. A total of 183 granulomas from all of the patients were scored positive (1) or negative (0) for each TLR and IL-4.

Statistical Analysis
A two-tailed Fisher's exact test was used to test for association between all possible pairwise comparisons of TLRs and IL-4. To check for any differences in expression patterns between individual patients within IL-4–positive and IL-4–negative groups, the Mantel Haenszel chi-square test was used. Expression patterns between pairs of TLRs were compared between IL-4–positive and –negative groups using a generalized linear model. A chi-squared test was used to compare double positive and double negative staining patterns in concordant granulomas. McNemar's test was employed to test for discordant patterns of TLR expression.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Antibody Characterization
By ELISA, anti-TLRs 1–5 and 9 recognized the peptide from which they were generated and anti-TLRs 2 and 4 recognized their respective recombinant proteins (no cross-reactivity was observed; results not shown). Fluorescent immunocytochemistry showed that the antibodies recognized CHO cells transiently transfected with TLRs 1, 2, 4, 5, or 9, but not mock-transfected cells (see ONLINE SUPPLEMENT). Moreover, no cross-reactivity was seen with any of the antibodies using immunocytochemistry (results not shown). Slight background staining of mock-transfected cells with anti-TLRs 2 and 9 was observed (see ONLINE SUPPLEMENT). This may be due to cross-reactivity with endogenous TLRs; however, staining was significantly enhanced in transfected CHOs (see ONLINE SUPPLEMENT). The specificity of the anti-TLRs 1–4 and 9 antibodies was further confirmed by Western blot of lysates from transiently transfected cells (see ONLINE SUPPLEMENT). Each anti-TLR antibody gave a band at the correct molecular weight which could be competed out by peptide pre-absorption. No cross-reactivity was seen (see ONLINE SUPPLEMENT). TLRs 2 and 4 displayed a doublet band, probably due to heterogeneity in glycosylation (37). Successful transfection of TLR6 and TLR8 was confirmed using anti-flag antibodies (not shown). Only antibodies shown to recognize the TLRs by either Western blotting or immunocytochemistry were used in further studies.

Analysis of TLRs 1–5 and 9 at Low-Power Magnification
Consecutive sections through the lung tissue from Patient TB17 is shown at low magnification (x100; Figure 1). A non-necrotic granuloma is depicted which is surrounded by a well-defined lymphocyte cuff (Figure 1). Alveolar macrophages were observed in the alveolar spaces, and a giant cell was present at the periphery. This granuloma stained positive for TLRs 1, 2, 4, 5, and 9 proteins (Figures 1a, 1b, 1d, 1e, and 1f, respectively) and negative for TLR3 protein (Figure 1c).



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  		Figure 1. Immunohistochemical staining of TLRs in serial sections through a non-necrotic granuloma. A granuloma (Gr) with epithelioid macrophages at the center surrounded by the lymphocytic infiltrate (Lc) is shown. Nuclei stain blue and positive immunostaining is brown. Alveolar macrophages (AM) are visible in the alveolar spaces, and a giant cell (Gc) is at the periphery of the granuloma. Edema is visible in the alveolar spaces. This granuloma stains positive for TLR1 (a), TLR2 (b), TLR4 (d), TLR5 (e), and TLR9 (f), but negative for TLR3 (c). Staining with the rabbit IgG control is negative (g). PPD did not inhibit the positive staining for TLR5 (h). Original pictures were taken at x100 magnification.

 
Rabbit IgG controls, which were used to detect nonspecific binding, were negative (Figure 1g). The specificity of the purified antibodies was confirmed, as pre-incubation with relevant TLR peptides eliminated the positive signal (not shown). The anti-TLR antibodies did not bind to mycobacterial proteins as pre-incubation with PPD did not affect the positive signal (result for TLR5 is shown Figure 1h).

No staining for any TLR was observed in the lungs of the control patients with malignancies (supplementary data, [see ONLINE SUPPLEMENT] shows representative staining of TLR2 and TLR4 in con1). Specifically, no TLR staining was observed in alveolar macrophages, type II pneumocytes, or pericytes. Unfortunately, no respiratory epithelium was present in these particular tissue sections, and therefore the presence of TLRs on this cell type is not known.

Each individual granuloma from each patient was scored for the presence or absence of each TLR (Table 1). A total of 183 granulomas were scored, and the most abundant TLRs were 1, 2, 4, and 9 (Table 1). TLR 3 was found to be associated with only 14 of the 183 (8%) granulomas.

Analysis of TLRs 1–5 and 9 at High-Power Magnification
Higher magnification (x400) of the resected lung tissue allowed us to identify cells with different morphologies. Myeloid cells, viz. alveolar macrophages in the alveolar spaces (Figure 2a), epitheliod macrophages within granulomas (Figure 2b), as well as giant cells within and at the periphery of granulomas (Figure 2c), all expressed TLR4. It was also expressed on cells with lymphocyte morphology (Figure 2d). The lymphocytes were concentrated in the lymphocyte cuff, which surrounds the granuloma, and were interspersed with macrophages. Type II pneumocytes, which line the alveolar spaces, were also positive for TLR4 (Figure 2e), as were pericytes, which surround capillaries (Figure 2g). The respiratory epithelium also expressed TLR4, and the expression was concentrated at the apex of the cells (Figure 2f). TLR2 had a similar expression pattern, except that it was not expressed on pericytes. TLR1 was not expressed on the type II pneumocytes or the pericytes, but was expressed on the other cell types mentioned. TLR9 was expressed only on the immune cells, viz. the myeloid cells and lymphocytes, but was absent from other cell types. TLR3 was only found on myeloid cells and pericytes, but not on lymphocytes, pneumocytes, or epithelium. TLR5 was exclusively expressed on myeloid cells.



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  		Figure 2. TLR4 expression at high power magnification. TLR4 is expressed on alveolar macrophages in the alveolar spaces (a), epitheliod macrophages within granulomas (b), giant cells in and adjacent to granulomas (c), cells with lymphocyte morphology in the lymphocyte-rich region of granulomas (d), type II pneumocytes which line the alveoli (e), the apical region of respiratory epithelium (f), and the pericytes surrounding capillaries. Inserts focusing in on the area of interest are included for epitheliod macrophages (b), lymphocytes (d), and pericytes (g). Original pictures taken at x400 magnification, and the arrows indicate the cells stained for TLR4, which exhibit the brown color. Nuclei stain blue.

 
Expression of IL-4
The expression of IL-4 in the human lung tuberculous granuloma has been well-described (29, 35) and will be briefly discussed here. Four out of the nine patients exhibited no staining for IL-4 in any of their 54 granulomas, and were classified as IL-4–negative patients. The remaining five patients had 30 IL-4–positive granulomas out of a total of 129 (23%) and were classified as IL-4–positive patients (Table 1).

Central Necrosis, TLRs, and IL-4
Only 38 out of the total of 183 (~20%) granulomas had caseous necrosis (Table 1). None of the 14 granulomas staining positive for TLR3 was necrotic (Table 1). In necrotic granulomas, TLR1 was always co-expressed with TLR5 and TLR9. However, the frequency of TLR1, TLR5, and TLR9 co-expression in necrotic granulomas varied between IL-4–positive and –negative patients. In the IL-4–positive patients, 22 out of 24 (92%) necrotic granulomas were positive for TLR1, TLR5, and TLR9. However, in the IL-4–negative group, only four out of 14 (28%) necrotic granulomas were positive for TLR1, TLR5, and TLR9. This difference between the two patient groups was significant (P < 0.001). This means that in the IL-4–positive patients, necrotic granulomas were more likely to be positive for TLRs 1, 5, and 9, whereas in IL-4–negative patients necrotic granulomas were more likely to be negative for these TLRs.

Expression of TLRs in IL-4–Positive and IL-4–Negative Patients
IL-4–positive patients had significantly more granulomas positive for TLR1 (P < 0.0001) or TLR9 (P < 0.005) relative to IL-4–negative patients. IL-4–positive patients had 110 out of 129 (85%) granulomas positive for TLR1, whereas IL-4–negative patients had only 31 out of 54 (57%) positive for TLR1 (Table 1). In the case of TLR9, IL-4–positive patients had 78% positive granulomas, whereas IL-4–negative patients had only 56% (Table 1). The relative number of granulomas positive for the other TLRs was not significantly different between the two patient groups.

Relationships between TLRs
All pairwise comparisons between the TLRs and IL-4 were performed and statistically analyzed. Because so many comparisons were being performed, and to prevent spurious differences being declared, only those comparisons significant at P < 0.001 are mentioned. Furthermore, the Mantel Haenszel chi-square test revealed no significant difference among patients, and so the fact that some patients contributed more granulomas than others should not invalidate conclusions from this granuloma-based analysis. Eighty-four out of the 183 granulomas (46%) from the nine patients were positive for five of the TLRs studied, the missing receptor being TLR3 in most cases (no granulomas were positive for all six of the TLRs studied). There was a positive relationship between TLR2 and TLR4 (P < 0.0001), with 87% of all granulomas being concordant for these receptors (Figure 3). In fact, 131 out of 183 (72%) of the granulomas are positive for both TLR2 and TLR4. Those granulomas with discordant expression patterns tend to be TLR4-positive and TLR2-negative (P < 0.005; Figure 3). This relationship is valid for both IL-4–positive and –negative patient groups.



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  		Figure 3. Contingency table of TLR2 and TLR4 for all patients. All 183 granulomas from nine patients were scored for the presence (pos) or absence (neg) of TLR2 and TLR4. Results are shown as the number of granulomas with each phenotype. In the left hand corner the data is presented graphically as percentage of total granulomas.

 
Differences were observed between the IL-4–positive and –negative patients in the relationships between TLR1 and TLR9 (P < 0.0001; Figures 4a and 4b), between TLR5 and TLR9 (P < 0.005; Figures 4c and 4d), and between TLR1 and TLR5 (P < 0.01; Figures 4e and 4f). In the IL-4–negative patients, 99% of the granulomas were concordant for TLR1 and TLR9 (Figure 4a), 96% concordant for TLR5 and TLR9 (Figure 4c), and 95% concordant for TLR1 and TLR5 (Figure 4e). Furthermore, in the IL-4–negative patients their granulomas were equally likely to be negative for both TLRs as positive for both (Figures 4a, 4c, and 4e). For example, 43% of granulomas were negative for both TLR1 and TLR9, and 56% were positive for both TLRs (Figure 4a). A similar trend was observed for TLR5 and TLR9 (Figure 4c) as well as for TLR1 and TLR5 (Figure 4e). In the IL-4–positive patients, > 75% of granulomas were concordant (Figures 4b, 4d, and 4f). However, in this patient group, only 8% of granulomas were negative for TLR1 and TLR9, whereas 70% were positive for both TLRs, and this difference was significant (P < 0.0001; Figure 4b). A similar trend was observed for TLR5 and TLR9 (Figure 4d) and TLR1 and TLR5 (Figure 4f). The discordant granulomas of the IL-4–positive patients were more likely to be TLR1-positive than TLR5-positive (P < 0.0001).



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  		Figure 4. Associations between TLRs 1, 5, and 9 in the IL-4–positive and –negative patient groups. Each of the 183 granulomas from all the patients were scored as positive or negative for TLR1, TLR5, and TLR9. Associations between TLR1 and TLR9 (a and b), TLR5 and TLR9 (c and d) and TLR1 and TLR5 (e and f) were plotted for the IL-4–negative patients (a, c, and e) and the IL-4–positive patients (b, d, and f). Results are presented as a percentage of granulomas. Significant differences between the two patient groups were observed with P < 0.0001 for TLR1 and TLR9, P < 0.005 for TLR5 and TLR9, and P < 0.01 for TLR1 and TLR5.

 
Relationships between TLRs and IL-4
IL-4 was used as a marker to discriminate between two groups of patients with different immune responses to M. tuberculosis as described above. However, we also wished to determine if there was any relationship between IL-4 itself and the TLRs in the 129 individual granulomas of the five IL-4–positive patients. Negative relationships between IL-4 and TLR2 (P < 0.0001; Figure 5), TLR4 (P < 0.0001; Figure 5) and TLR9 (P < 0.01; Figure 5) were observed. In fact, 125 out of 129 (97%) granulomas were discordant for IL-4 and TLR2 expression, with 97 out of 129 (75%) being TLR2-positive and IL-4–negative (Figure 5). Only 2 granulomas were positive for both IL-4 and TLR2 (Figure 5). Similar trends were observed for IL-4 and TLR4 and between IL-4 and TLR9 (Figure 5).



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  		Figure 5. Contingency tables of IL-4 and TLR2, IL-4 and TLR4, and IL-4 and TLR9 for IL-4–positive patients. One hundred twenty-nine granulomas from five IL-4–positive patients were scored for the presence (pos) or absence (neg) of TLR2, TLR4, TLR9, and IL-4. Pairwise comparisons between the TLRs and IL-4 are shown as the number of granulomas with each phenotype. In the left hand column the data is presented graphically as percentage of total granulomas.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The patients with pulmonary tuberculosis described in this study had severe disease with lung cavitation and life-threatening hemoptysis requiring therapeutic lobectomy. Immunohistochemical analysis of their tissue sections was performed, and the results analyzed at different levels. First, at the level of the whole patient, we examined TLR expression, distribution, and morphology of the cells expressing the TLRs. Second, we analyzed the relationship between the patient's ongoing immune response and the expression of the TLRs. For this purpose, the patients were divided into two groups according to their immune response to M. tuberculosis using IL-4 expression as a marker. Five patients produced IL-4, and four patients were IL-4–negative. We then investigated the associations between the TLRs and caseous necrosis in the IL-4–positive and –negative groups of patients. Lastly, the tuberculous granuloma is a discrete microenvironment within the lung, and the expression of TLRs within the granuloma and their associations with IL-4 itself were determined.

All nine patients exhibited staining for all of the six TLRs tested (TLRs1, 2, 3, 4, 5, and 9) and all had evidence of caseous necrosis. TLRs1, 2, 4, 5, and 9 were easily detectable, but TLR3 was expressed sparsely. In fact, 183 individual granulomas from the nine patients were scored for the presence or absence of each of the TLRs, and no granuloma expressed all six TLRs, but 46% were positive for five of them. TLR3 was generally the receptor missing from those granulomas expressing five of the TLRs, and it was only expressed in 8% of the granulomas studied; none of these exhibited central necrosis. Double-stranded RNA has been postulated as the ligand for TLR3, suggesting this receptor may have a function in antiviral immunity (8). Therefore, it may not be surprising that it is not expressed at high levels in patients with pulmonary tuberculosis. Furthermore, TLR3 is reported to be expressed on dendritic cells (DCs) (38) and its expression pattern may reflect the distribution of DCs in these patients with pulmonary tuberculosis. The expression of TLR5 within the tuberculous granuloma may imply that bacterial flagellin (the putative ligand for TLR5 [11]) may not be the only ligand but may be substituted for by components of the nonflagellated M. tuberculosis.

All of the TLRs studied were expressed on myeloid cells (alveolar and epitheliod macrophages and giant cells). The ability of a myeloid cell to express a particular TLR appears to be dependent on the microenvironment of the granuloma with which it is associated. Some granulomas were negative for a particular TLR and the myeloid cells associated with these granulomas failed to express that particular TLR. Lymphocytes expressed TLRs 1, 2, 4, and 9, only in those granulomas where myeloid cells were also positive for these TLRs. However, in granulomas where the myeloid cells expressed TLRs 3 or 5, the lymphocytes associated with these granulomas remained TLR3- or TLR5-negative. Previous studies on TLR mRNA levels in peripheral blood leucocytes indicate that TLR1 is expressed by all leucocytes and probably plays a fundamental role in the innate immune response (18, 38), and this is evidently also true in the lungs of patients with tuberculosis. TLRs 2, 4, and 5 are thought to be expressed only in peripheral blood myelomonocytic cells (38); however, in the patients described here TLRs 2 and 4 are also found on lymphocytes and this may be due to upregulation in response to the pathogen itself or to inflammation. TLR9 is expressed by peripheral blood myeloid cells and lymphocytes (18) as is the case in the tuberculous granuloma. TLR3 is specific for dendritic cells (38) and its distribution in the tuberculous lung may reflect the presence of this cell type, although cells with macrophage morphology also expressed TLR3 in some granulomas. There is also evidence of TLR expression in nonimmune cells, with type II pneumocytes staining for TLRs 2 and 4, respiratory epithelium showing apical staining for TLRs 1, 2, and 4, and pericytes expressing TLRs 3 and 4. In the lungs of patients with malignant disease, no TLR expression was detected in the relatively normal tissue adjacent to the tumor. Therefore, the TLRs observed in the lungs of these patients with tuberculosis are likely to have been upregulated due to the inflammatory reponse or perhaps to the pathogen itself. In the normal gut epithelium TLR3 and TLR5 are constitutively expressed and TLR2 and TLR4 are barely detectable (19). However, in patients with inflammatory bowel disease, TLR3 is downregulated, TLR4 is upregulated and TLR2 and TLR5 remain unchanged (19). Therefore, it is likely that the type of inflammatory response taking place at the site of pathology may be reflected by the pattern of expression of the TLRs.

In patients with pulmonary tuberculosis a marked cell-mediated immune response is evident in the lung granulomas with large amounts of IFN-{gamma}, IL-12p40, and TNF-{alpha} being produced (29, 35). However, each granuloma appears to be a microenvironment with its own characteristic cytokine pattern (29, 35). This cytokine pattern may reflect different stages in the development of the granuloma or even in these severely ill patients, the cytokine pattern may relate to the fate of an individual granuloma: containment or dissemination (29, 35). IL-4 expression, on the other hand appears to divide the patients into two groups, those producing IL-4 in some granulomas (IL-4–positive patients) and those without any evidence of IL-4 production (IL-4–negative patients). It is difficult to explain these different types of immune response on the basis of the Th1 versus Th2 dichotomy as IL-4 is only expressed in granulomas positive for IFN-{gamma} and the presence of IL-4 does not appear to influence the clinical outcome of these patients (29, 35). Admittedly, these patients are severely ill and have received antimycobacterial therapy for different periods of time and this may obscure the effects of the ongoing immune response characterized by IL-4 production. IL-4 is therefore considered to be a marker for the different immune responses but does not necessarily imply the presence of a detrimental Th2 response in these patients. Of the nine patients presented here five were IL-4–positive (23% of 129 granulomas positive for IL-4) and four of the patients were IL-4–negative (none of 54 granulomas producing IL-4). This heterogeneity of immune response between patients and between granulomas was reflected in the expression patterns of the TLRs and central necrosis.

There was a significant positive association between the expression of TLR2 and TLR4 in all patients, irrespective of their IL-4 status. In fact 72% of all granulomas were positive for both TLR2 and TLR4. Both of these TLRs have been shown in vitro, to respond to components of M. tuberculosis. Inflammatory cytokines (TNF-{alpha} and IL-12) are produced when TLR2-positive macrophages are exposed to the 19 kD lipoprotein and the ability of macrophages to kill intracellular bacilli is enhanced (33). Although TLR4 has been shown to bind lipopolysaccharide (LPS) of gram-negative bacteria (9), there is evidence that live M. tuberculosis can activate TLR4 on murine macrophages (31). Both of these TLRs were highly expressed in human tuberculous granulomas and the presence of one greatly increased the probability that the other would also be present Therefore, it appears that in all patients, irrespective of their immune response, TLR2 and TLR4 tend to be expressed together in the majority of granulomas (both necrotic and non-necrotic), perhaps supporting a co-ordinated role for them in the immunopathogenesis of tuberculosis.

IL-4–positive patients had significantly more granulomas expressing TLR1 or TLR9 than IL-4–negative patients. Furthermore, there was a significant difference between the patient groups in the relationship between TLR1 and TLR9 as well as with TLR5. TLRs 1, 5, and 9 were co-expressed within a granuloma much more often in IL-4–positive patients than in IL-4–negative patients. This may imply that the immune response (characterized by IL-4 production), promotes the synchronous expression of TLRs 1, 5 and 9 within individual granulomas, while an immune response characterized by a lack of IL-4 allows for more independent expression of these receptors. Alternatively, co-expression of TLRs 1, 5, and 9 may be conducive to the development of an immune response involving the production of IL-4. Admittedly, these observations may not be functionally related but merely provide biomarkers of the ongoing immune response in these patients.

Caseous necrosis occurs at the center of granulomas and is associated with disappearance of acid fast bacilli (21) and probably plays a role in containing the infection (39). The mechanism for this necrotic process is unknown but may involve delayed type hypersensitivity reactions (40), limiting lymphokine availability (41), or oxygen deprivation (42) at the center of large granulomas. However, the solid caseum, under some circumstances, can undergo liquefaction with resultant cavitation and dissemination of mycobacteria (21). Therefore, caseation is a requirement for containment of the bacillary load (39), but it is also a step on the road to liquefaction, cavitation and tissue damage. In the nine severely diseased patients described in the present manuscript, the majority of the granulomas studied (79%) had no evidence of central necrosis although cavitation was seen on the chest radiographs. The association between TLRs 1, 5, and 9 and necrosis differed between IL-4–positive and –negative patients. In IL-4–positive patients necrotic granulomas were more likely to be TLRs 1, 5, and 9 positive, whereas in IL-4–negative patients necrotic granulomas were more likely to be negative for this group of TLRs. Thus necrosis was associated with a distinct set of TLRs depending on the underlying immune response of the patient (as measured by IL-4 production). It is not possible to determine from these data whether the TLRs are playing a role in the induction of necrosis or if necrosis is inducing co-expression of these receptors or indeed if they are causally related at all. However, there may be an interplay between the immune response, TLR expression and the development of necrosis but whether this is detrimental leading to cavitation and dissemination or beneficial resulting in containment is not clear.

IL-4 has been shown to downregulate TLR2 and TLR4 mRNA expression in human monocyte-derived macrophages (huMDM) in vitro (43). This effect was not seen when IL-10, an anti-inflammatory cytokine, was added to the cultures (43). Furthermore, infection of huMDM with Listeria monocytogenes increased TLR2 and decreased TLR4 mRNA expression and this pathogen-mediated receptor modulation did not occur in IL-4–treated huMDM (43). Therefore, IL-4 is capable of directly modulating TLR expression on macrophages and also of abrogating pathogen-driven changes in TLR expression. In the individual granulomas of IL-4–positive patients described here, a marked negative relationship between IL-4 and TLR2 was observed. The majority (97%) of granulomas were discordant for TLR2 and IL-4 and less than 2% were positive for both TLR2 and IL-4. It is therefore possible that IL-4 downregulates TLR2 expression within the human tuberculous granuloma. A similar though slightly less marked negative relationship between TLR4 and IL-4 was observed and may similarly reflect downregulation of this receptor by IL-4.

TLR9 also exhibited a negative relationship with IL-4 within individual granulomas, implying that IL-4 within a granuloma may be downregulating the expression of TLR9, similarly to TLR2 and TLR4. This is despite the observation that IL-4–positive patients tended to have relatively more TLR9-positive granulomas than IL-4–negative patients. This apparent contradiction may be explained by differential effects of the patient's overall immune response and the cytokine microenvironment of granulomas. This serves to illustrate the disparate effects of the immune response prevalent in a patient (as measured by their ability or inability to produce IL-4) and the effect of individual cytokines within the immune microenvironment of the granuloma.

Immunohistochemistry provides a snapshot of the interaction of the host and the pathogen at one point in time without providing any information as to causative relationships. It is possible that the infection by the pathogen has modulated the TLR expression pattern, which in turn influenced the immune response and the development of necrosis. However, in this chronic infection it is also possible that the immune response and the presence of necrosis are modifying the expression of TLRs. Furthermore, it is difficult to determine which of these observations is advantageous to the host and which favors the survival of the pathogen. We have observed a disparity in the expression of TLRs and caseous necrosis between patients exhibiting different immune responses to M. tuberculosis as measured by their ability to produce IL-4. Co-expresion of TLRs appears to divide the TLRs studied into two groups. The first group is TLR2 and TLR4 which are co-expressed in all patients. The second group is TLR1, TLR5, and TLR9 which are co-expressed in IL-4–positive patients and are associated with necrosis in these patients. Furthermore, at the level of the individual granuloma IL-4 may modulate the expression of TLRs 2, 4, and 9. Thus, we conclude that TLRs are expressed by a variety of cell-types in the human lung tuberculous granuloma, the immune response of the host and the pattern of TLR expression and the presence of necrosis are interrelated and that IL-4 may modulate TLR expression within individual granulomas.


   Acknowledgments
 
The authors are grateful to several colleagues at GlaxoSmithKline: Alan Lewis for designing the peptides used to generate the antibodies; Chris Plumpton and Amanda Jowett for producing the antibodies; and Mary Morse, Stephanie Wagner, and Michelle Young for supplying the various TLR expression plasmids. They are also grateful to Mary Morse for critically reading the manuscript. They would also like to thank Daniela Bosisio from the Maria Negri Institute, Milan, Italy for supplying us with extracts of cells transiently transfected with TLR3. This work was supported by the GlaxoSmithKline Action TB Initiative.


   Footnotes
 
This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjurnals.org.

Received in original form August 23, 2002

Received in final form December 20, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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