Granulomas are hallmarks of chronic infectious diseases, such as tuberculosis, brucellosis, and schistosomiasis, and also develop due to the presence of allergens and metals. The granulomatous lesions are generally considered to be the result of chronic antigenic stimulation (10
). A tuberculous granuloma is a focal collection of mononuclear cells surrounded by a halo of lymphocytes and additional monocytes (36
). The reaction occurs when an infected macrophage becomes encircled by other macrophages and the immune system attempts to wall off the microorganisms to prevent the bacteria from spreading locally and throughout the body. This complex cellular structure can be surrounded by connective tissue, including fibroblasts, collagen fibers, and newly formed vessels (36
). A novel morphological characteristic of the granuloma is the presence of epithelioid cells, which occupy the center of each lesion. These epithelioid cells are activated macrophages, which contain increased cytoplasm and disperse chromatin resembling epithelial cells (36
). Langhans multinucleated giant cells are also found in granulomas.
induces a proinflammatory immune response that is characterized by expression of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-12 (IL-12) (12
). Although information has been obtained from bronchoalveolar lavages (BAL) and lung biopsies during advanced disease in humans, only a small number of studies have examined the local cytokine expression patterns associated specifically with granulomas. Examination of BAL cells has indicated that M. tuberculosis
induces a type 1 polarized cytokine response characterized by IFN-γ expression (4
). However, when Fenhalls et al. studied pulmonary granulomas from individuals with active tuberculosis, they found that the expression patterns of IFN-γ relative to those of IL-4 were highly associated with the type of granuloma formed (23
). Granulomas with no evidence of caseation expressed either IFN-γ mRNA or IFN-γ mRNA plus IL-4 mRNA, whereas caseous granulomas expressed little IFN-γ mRNA or IL-4 mRNA (23
). These findings suggest that these cytokines play a role in determining granuloma architecture.
The development of a granuloma likely depends on the movement of cells toward the site of inflammation due to expression of chemotactic molecules, although only one study to date has examined local chemokine expression directly in granulomatous tissue sections (24
). Proinflammatory chemokines are chemotactic cytokines which play a major role in the recruitment of receptor-bearing cells to sites of inflammation (2
). The functions of chemokines include chemotaxis, integrin activation, and degranulation of distinct leukocyte subsets expressing specific chemokine receptors (17
). The expression of proinflammatory chemokines is induced by local environmental signals, such as TNF-α or IFN-γ (2
). Granulomas induced experimentally with mycobacterial agents lead to the development of type 1 cytokine and chemokine expression profiles (12
). IFN-γ induces macrophages and dendritic cells (DC) to produce IFN-γ-inducible CXCR3 ligands, CXCL9/monokine induced by IFN-γ (Mig), CXCL10/IFN-γ-inducible protein with a size of 10 kDa (IP-10), and CXCL11/IFN-γ-inducible T-cellα -chemoattractant (I-TAC), which recruit CXCR3+
cells typically express type 1 cytokines (18
) and therefore can potentially induce further upregulation of CXCR3 ligands, leading to chronic type 1 polarized inflammation, which occurs during simian immunodeficiency virus (SIV) infection of rhesus macaques (Macaca mulatta
In this study we examined local chemokine and cytokine mRNA expression and the local mycobacterial burden in pulmonary granulomatous lesions resulting from intrabronchial infection of cynomolgus macaques with a low dose of virulent M. tuberculosis
. This was one of the first studies of chemokine expression directly in granulomatous tissue sections. We found abundant expression of mRNAs encoding the proinflammatory cytokines IFN-γ and TNF-α, as well as IFN-γ-inducible CXCR3 ligands, within all granulomas regardless of size or structure. The general absence of cells expressing these mRNAs in nongranulomatous regions of lung tissues suggests that they play a local role in granuloma formation and maintenance. Based on these findings we propose a model for M. tuberculosis
-initiated, IFN-γ-driven chronic inflammation in pulmonary granulomas that is similar to a model for the events occurring in lymphoid tissues of SIV-infected rhesus macaques (46
Cynomolgus macaques are susceptible to M. tuberculosis
infection and develop disease that is clinically, immunologically, and pathologically similar to human disease (11
). Interestingly, approximately 40% of cynomolgus macaques infected with a low dose of M. tuberculosis
were able to contain the infection in a subclinical state during the period of study. This subclinical state resembles clinical latency in humans (11
) and indicates that cynomolgus macaques are an appropriate animal model for latency (11
) and reactivation (11
) during M. tuberculosis
infection. Although the proportion of cynomolgus macaques that develop active disease is higher, it is similar to the outcome of infection in humans, in which only 10% of individuals infected with M. tuberculosis
develop overt disease. At the tissue level, M. tuberculosis
-induced granulomas in cynomolgus macaques are structurally similar to granulomas that develop in humans.
We demonstrated by direct analysis of tissue sections using ISH that there are high levels of expression of IFN-γ-inducible chemokine mRNAs and increased numbers of IFN-γ and TNF-α mRNA+
cells in pulmonary granulomas. CXCR3+
cells, which are predominantly activated T and B lymphocytes and NK cells (42
), are likely recruited to the local environment. Our detection of abundant CXCR3+
cells in pulmonary granulomas provides evidence that this recruitment occurs. Although not examined here, local proliferation and apoptosis also likely contribute to accumulation and loss of cells, respectively, and affect overall granuloma size and structure.
Previous studies of chemokine expression during M. tuberculosis
infection included in vivo murine studies and ex vivo and limited tissue-based human studies. Mice infected by the aerosol route exhibited increases in CCL3/MIP-1α, CXCL2/MIP-2, CXCL10/IP-10, and CCL2/MCP-1 expression in lungs (47
). In addition, granulomas elicited in mice by purified protein derivative-coated beads had a polarized type 1 immune response with increased levels of expression of CXCL2/MIP-2, CXCL5/LIX, CXCL10/IP-10, and CXCL9/Mig (14
). In these studies, neutralization of IFN-γ with antibodies greatly reduced the expression of CXCL9/Mig and CXCL10/IP-10. Human studies of patient-derived BAL or alveolar and peripheral macrophages revealed increased expression of CXCL8/IL-8, CCL5/RANTES, CCL3/MIP-1α, CCL2/MCP-1, and CXCL10/IP-10 (24
). The one other ISH and IHC report of chemokine expression in human tuberculous granulomas demonstrated that there was localized expression of CXCL10/IP-10, CXCL8/IL-8, and CCL2/MCP-1 within granulomas (24
The strategy which we have used for detection of cells producing proinflammatory chemokines and cytokines is targeted toward mRNA expressed by the producing cell. This approach reveals the locations and numbers of cells that produce these immunomodulatory proteins, but it does not indicate the locations or amounts of actual proteins. Although this is a limitation of our study, the detection of chemokine and cytokine proteins is complicated by the diffusion of proteins that occurs after release into the extracellular milieu. The detection of different proteins by IHC is also complicated by the unique physiochemical properties of each antigen-antibody pair compared to the properties of RNA-RNA hybrids, which generally have the same physiochemical properties. Our ISH strategy has the additional advantage that it can reveal focal changes in mRNA expression that might not be detected by population analyses of extracted tissue RNAs due to dilution.
Previous in situ studies of human tuberculous granulomas revealed local production of TNF-α and IFN-γ mRNAs in all granulomatous lesions, but only a fraction of these lesions expressed IL-4 mRNA (23
). Our data are consistent with these findings in that IFN-γ mRNA was much more abundant than IL-4 mRNA, but they differ in that we did not detect any IL-4 mRNA by our ISH assay, as also observed by other workers with human granulomas (3
). Limited expression of both IFN-γ and IL-4 mRNAs has been suggested to be associated with progression of granulomas to a necrotic state (23
). However, we observed an association between the numbers of local IFN-γ and TNF-α mRNA+
cells and the sizes (and necrotic states) of pulmonary granulomas. The reasons for the differences between our findings and those of other workers (23
) are not clear but could include differences with respect to (i) the host species studied, (ii) the disease and treatment status of the study subjects, (iii) the tissue-processing protocols, and/or (iv) the ISH probe and detection strategies.
Based on our finding that there was abundant expression of CXCR3 ligands, IFN-γ, and TNF-α in pulmonary granulomatous lesions, we propose a model for the contributions of these molecules to the chronic inflammation that results in granuloma formation and maintenance, which is similar to a model previously proposed for chronic inflammation in SIV-infected lymphoid tissues (46
). First, infection of alveolar macrophages or DC leads to induction of proinflammatory cytokines and chemokines (for example, by interaction of mycobacterial antigen with Toll-like receptors [TLRs]). Additional cells are therefore recruited to the local environment. Immune responses specific to M. tuberculosis
develop following the trafficking of pulmonary DC to draining lymph nodes, which results in the trafficking of type 1 CD4+
T lymphocytes to the site of M. tuberculosis
infection, and these cells produce IFN-γ. IFN-γ function is enhanced in the presence of TNF-α, which is well documented as an important cytokine in the immune response to M. tuberculosis
and in granuloma formation (6
). Both local production of IFN-γ and TLR stimulation induce the expression of CXCR3 ligands and thus recruit CXCR3+
T lymphocytes and NK cells to the site of M. tuberculosis
infection. CXCR3 ligands contribute to further polarization of the local environment because they augment the ability of IFN-γ to induce additional CXCR3 ligand production and lead to a type 1 outcome following stimulation of T lymphocytes with polyclonal activators or specific antigens (28
). In this model, the collective outcome is the establishment and maintenance of a positive feedback loop in which local IFN-γ production leads to the ongoing recruitment of additional IFN-γ-producing cells. Also, this suggests that IFN-γ has a role in granuloma formation and maintenance beyond induction of an antimicrobial state in infected cells. Thus, there are common themes in M. tuberculosis
and SIV infections of macaques that, if recapitulated in humans infected with both organisms, might contribute to a significantly poorer prognosis than the prognosis for individuals infected with either pathogen alone.
In addition to effects of antigen-specific type 1 immune responses, M. tuberculosis
bacilli or mycobacterial components also have direct effects on local cytokine and chemokine expression profiles in and near pulmonary granulomas. In vitro analyses have demonstrated that mycobacterial cell wall components induce expression of proinflammatory cytokines via TLR-2 and TLR-4 (37
). Although alveolar macrophages are initially involved in the uptake of M. tuberculosis
, DC and monocyte-derived interstitial macrophages also phagocytose and process M. tuberculosis
). Interestingly, Lande et al. recently demonstrated that M. tuberculosis
infection of DC leads to induction of CXCL9/Mig and CXCL10/IP-10, the latter in an IFN-αβ-dependent fashion (33
In this model we have not included other complex factors, such as complex multichemokine gradients that lead to simultaneous synergistic or antagonistic signals on cells (26
). In addition, there are likely to be negative regulatory activities that occur at the same time. A candidate for such regulation is the lymphocyte cell surface protein CD26 (also known as dipeptidylpeptidase IV), which catalyzes the removal of N-terminal dipeptides from suitable substrates containing a penultimate proline or alanine (60
). Cleavage by CD26 differentially affects the activity of specific chemokines, rendering some chemokines inactive and others more potent (60
). CD26 proteolytically processes all three CXCR3 ligands and converts them to antagonists of CXCR3-mediated chemotaxis (44
). Determination of the roles played by such complex factors in granuloma formation and maintenance requires further analysis.
To determine whether the immunologic events that occur in granulomatous lesions are due to the abundance of local M. tuberculosis
bacilli or products, we developed an ISH strategy for detection of mycobacterial 16S rRNA directly in tissue sections. One frequently used strategy for in situ detection of M. tuberculosis
is acid-fast staining. However, the sensitivity of this procedure is reduced by the common treatment of tissues with formalin and xylene (27
). Overall, we found a relatively low number of foci hybridizing to the mycobacterial 16S rRNA probe in most animals, although two animals with advanced disease had much higher levels of signal. Most of the mycobacterial 16S rRNA ISH signal was localized to the necrotic portions of the granulomas, although some signal was localized to the cellular regions of caseous and solid granulomas. Our probe for detection of M. tuberculosis
does not discriminate between viable and nonviable organisms or between metabolically active and inactive states of the bacillus, but it detects an RNA target that is stable and abundant (1
). This might explain the different localization of the M. tuberculosis
ISH signal in our study compared to the localization in the studies of Fenhalls et al. (21
), in which the ISH signal was localized to cells in the cellular and surrounding regions of the granulomas. Once they reach a certain size or have a certain fluidic composition, the necrotic centers of caseous granulomas might provide a hospitable environment for the organism. Further studies with additional probes and tuberculosis cases and stages are needed to fully understand the importance of the numbers and states of mycobacterial organisms for granuloma formation and maintenance.
In summary, through examination of over 300 M. tuberculosis-induced pulmonary granulomas in experimentally infected cynomolgus macaques, we demonstrated that there was abundant IFN-γ-inducible chemokine and IFN-γ and TNF-α cytokine mRNA expression within the granulomas. The abundant staining of CXCR3+ cells in the same microenvironments is consistent with recruitment of these cells to the granulomas. These findings, as well as our findings of colocalized M. tuberculosis 16S rRNA in the pulmonary granulomas, suggest that the nature of the type 1 immune response and the direct action of mycobacterial components together lead to the establishment of chronic inflammation. There must be a complex balance among the inflammatory responses, antigen-specific responses, and negative regulatory mechanisms that determines the extent to which a granuloma contains the bacillus or develops into a structure that allows the organism to multiply and spread within and among hosts. Additional definition of the contributions of IFN-γ-inducible and other chemokines to these processes should elucidate mechanisms by which granulomas successfully limit or eliminate M. tuberculosis bacilli. These data should be important for developing additional strategies to combat morbidity and mortality caused by M. tuberculosis.