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Diversity of in vitro cytokine responses by human macrophages to infection by Mycobacterium tuberculosis strains
Abstract
Macrophages are an important component in the first line of defence of the innate immune system. They are capable of producing cytokines in response to bacterial challenge, as well as in response to cytokine stimuli from other cells in the immune system. The microbicidal response of human monocyte-derived macrophages in vitro, induced by exogenously added cytokines, is highly variable. We found that tumour necrosis factor-alpha (TNF-alpha) and granulocyte-macrophage colony-stimulating factor (GM-CSF) could have either stimulatory or inhibitory effects on intracellular BCG killing, depending on the macrophage donor. Macrophages infected in vitro by various clinical isolates of Mycobacterium tuberculosis or the laboratory strain H37Rv, produced varying levels of both TNF-alpha and IFN-gamma. Certain M. tuberculosis strains tended to be associated with high cytokine production in each of three independent experiments, indicating that strains may differ in the host response elicited to infection.
... Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis (TB), emerged as a pathogen in Africa and has co-evolved with humans following migration to Europe and Asia some 70,000 years ago [1]. Distinct phylogenetic lineages of MTB consistently associate with human populations of different genetic ancestry in a variety of settings2345 and elicit differing immune responses from antigen-presenting cells of healthy donors in vitro67891011 . Antimycobacterial immune responses might therefore be expected to vary between TB patients of different ethnic origin; however, studies investigating this question have not been conducted. ...
... variation in inflammatory profile is not explained by variation in MTB strain lineage MTB has co-evolved with humans, and different bacillary strains associate with different ethnic groups [2]; moreover, MTB strains of different lineage elicit differing immune responses in vitro67891011 _ENREF_8. Ethnic variation in inflammatory profile in PTB might therefore be explained by differential representation of MTB strain lineages between ethnic groups. ...
Distinct phylogenetic lineages of Mycobacterium tuberculosis (MTB) cause disease in patients of particular genetic ancestry, and elicit different patterns of cytokine and chemokine secretion when cultured with human macrophages in vitro. Circulating and antigen-stimulated concentrations of these inflammatory mediators might therefore be expected to vary significantly between tuberculosis patients of different ethnic origin. Studies to characterise such variation, and to determine whether it relates to host or bacillary factors, have not been conducted. We therefore compared circulating and antigen-stimulated concentrations of 43 inflammatory mediators and 14 haematological parameters (inflammatory profile) in 45 pulmonary tuberculosis patients of African ancestry vs. 83 patients of Eurasian ancestry in London, UK, and investigated the influence of bacillary and host genotype on these profiles. Despite having similar demographic and clinical characteristics, patients of differing ancestry exhibited distinct inflammatory profiles at presentation: those of African ancestry had lower neutrophil counts, lower serum concentrations of CCL2, CCL11 and vitamin D binding protein (DBP) but higher serum CCL5 concentrations and higher antigen-stimulated IL-1 receptor antagonist and IL-12 secretion. These differences associated with ethnic variation in host DBP genotype, but not with ethnic variation in MTB strain. Ethnic differences in inflammatory profile became more marked following initiation of antimicrobial therapy, and immunological correlates of speed of elimination of MTB from the sputum differed between patients of African vs. Eurasian ancestry. Our study demonstrates a hitherto unappreciated degree of ethnic heterogeneity in inflammatory profile in tuberculosis patients that associates primarily with ethnic variation in host, rather than bacillary, genotype. Candidate immunodiagnostics and immunological biomarkers of response to antimicrobial therapy should be derived and validated in tuberculosis patients of different ethnic origin.
... In contrast, the low inflammatory phenotype of M. tuberculosis CAS, responsible for an outbreak in Leicester, was linked to a chromosomal deletion and could be reversed by restoration of the functional gene [15]. Several other studies have described differences in the inflammatory response induced by different isolates of M. tuberculosis [12,13,17,18,19]. In the present study, we used a collection of strains covering the global genetic diversity of MTBC to test the hypothesis that inflammatory phenotype would be linked to genotype. ...
... Overall there was a significantly lower response to evolutionarily modern lineages as compared to ancient lineages. Previous reports have described differences amongst M. tuberculosis isolates in their inflammatory phenotype [10,12,13,15,18,19] but the present study is the first to link this to MTBC phylogeny. Consistent with previous reports, we observed a higher inflammatory phenotype for the laboratory strain H37Rv in comparison to the Beijing strain, HN878 [11]. ...
The aim of the present study was to determine whether there is a correlation between phylogenetic relationship and inflammatory response amongst a panel of clinical isolates representative of the global diversity of the human Mycobacterium tuberculosis Complex (MTBC). Measurement of cytokines from infected human peripheral blood monocyte-derived macrophages revealed a wide variation in the response to different strains. The same pattern of high or low response to individual strains was observed for different pro-inflammatory cytokines and chemokines, and was conserved across multiple human donors. Although each major phylogenetic lineage of MTBC included strains inducing a range of cytokine responses, we found that overall inflammatory phenotypes differed significantly across lineages. In particular, comparison of evolutionarily modern lineages demonstrated a significant skewing towards lower early inflammatory response. The differential response to ancient and modern lineages observed using GM-CSF derived macrophages was also observed in autologous monocyte-derived dendritic cells and murine bone marrow-derived macrophages, but not in human unfractionated peripheral blood mononuclear cells. We hypothesize that the reduced immune responses to modern lineages contribute to more rapid disease progression and transmission, which might be a selective advantage in the context of expanding human populations. In addition to the lineage effects, the large strain-to-strain variation in innate immune responses elicited by MTBC will need to be considered in tuberculosis vaccine development.
... We wonder whether use primary macrophages or a macrophage cell line because of the functional differences and most intense response against Mycobacterium challenge exerted by primary macrophages compared to macrophage cell lines, so finally we choose the primary source [42]. Then because primary macrophages could present a high level of variability under the culture conditions [43] and strain of intracellular pathogens [44] compared to cell lines of macrophages, we decide to use a single donor of cells for avoiding variability due to donor source. ...
Study background: The study was designed to evaluate if the addition of Dexamethasone, IFN-g, or LPS into culture media of primary bovine Monocyte-Derived Macrophages (MDMs), could support in vitro infection with Mycobacterium avium subspecies paratuberculosis.
Methods: Primary bovine Monocyte-Derived Macrophages (MDMs) were infected in vitro with a reference strain of Map at 5:1 MOI for 2h. Map-infected MDMs were stimulated with IFN-g, LPS, Dexamethasone or medium alone for 24h. At 0, 6, 72 and 120h of culture, it was evaluated the presence of Map by bacterial culture and amplification of the IS900 fragment by real-time PCR. The function of Map-infected MDM was evaluated by measurement of TNF-a, IL-6, IL-8, IL-10, IL-12, IP-10, and CCL3 in culture supernatants by Luminex. Data were analyzed by Kruskal-Wallis test.
Results: The IS900 segment was amplified in samples of Map-infected MDM from all stimuli. The growth of Map in bacterial culture was observed at each time-point evaluated without statistically significant differences between groups. Map-infected-MDMs stimulated with Dexamethasone significantly reduced cytokine production compared with control, excepting for IP-10 production from 6 to 120h (P<0.01). Overall cytokine production at 72h was significantly higher in Map-infected MDM treated with LPS (P<0.01) excepting for IP-10 and CCL3 production at 120h. IL-8 and IL-12 production at 72h and IP-10 production at 120h were significantly higher in Map-infected MDM treated with IFN-g (P<0.01).
Conclusion: Primary bovine MDM obtained from peripheral blood mononuclear cells could be used for growth of Map in vitro. The addition of LPS or IFN-gamma reduced the capability of MDM for sustaining the growth of Map until 120h post-infection, although Dexamethasone
... Cell-mediated immunity, characterized by activation of Th1 subsets of CD4 + T-cells with an increase in TNF-a and IFN-c is also important in host defense against M avium (Jouanguy et al. 1999;Ehrt et al. 2001;Manca et al. 2001;Nau et al. 2002). IFN-c-activated macrophages have enhanced ability to kill mycobacteria (Bonay et al. 1999;Jouanguy et al. 1999;Beltan et al. 2000;Ehrt et al. 2001;Giacomini et al. 2001;Hoal-van Helden et al. 2001;Manca et al. 2001;Qiao et al. 2002;Karcher et al. 2008;Herbst et al. 2011). Introducing an IFN-c transgene into the lungs of SCID mice reduced their susceptibility to BCG infection (Xing et al. 2001). ...
Mycobacterium avium (M. avium) causes significant pulmonary infection, especially in immunocompromised hosts. Alveolar macrophages (AMs) represent the first line of host defense against infection in the lung. Interferon gamma (IFN-γ) activation of AMs enhances in vitro killing of pathogens such as M. avium. We hypothesized that airway delivery of AMs into the lungs of immunodeficient mice infected with M. avium will inhibit M. avium growth in the lung and that this macrophage function is in part IFN-γ dependent. In this study, normal BALB/c and BALB/c SCID mice received M. avium intratracheally while on mechanical ventilation. After 30 days, M. avium numbers increased in a concentration-dependent manner in SCID mice compared with normal BALB/c mice. Airway delivery of IFN-γ-activated BALB/c AMs or J774A.1 macrophages overexpressing IFN-γ into the lungs of SCID mice resulted in a significant decrease in M. avium growth (P < 0.01, both comparisons) and limited dissemination to other organs. In addition, airway delivery of IFN-γ activated AMs and macrophages overexpressing IFN-γ increased the levels of IFN-γ and TNF-α in SCID mice. A similar protective effect against M. avium infection using J774A.1 macrophages overexpressing IFN-γ was observed in IFN-γ knockout mice. These data suggest that administration of IFN-γ activated AMs or macrophages overexpressing IFN-γ may partially restore local alveolar host defense against infections like M. avium, even in the presence of ongoing systemic immunosuppression.
... We wonder whether use primary macrophages or a macrophage cell line because of the functional differences and most intense response against Mycobacterium challenge exerted by primary macrophages compared to macrophage cell lines, so finally we choose the primary source [42]. Then because primary macrophages could present a high level of variability under the culture conditions [43] and strain of intracellular pathogens [44] compared to cell lines of macrophages, we decide to use a single donor of cells for avoiding variability due to donor source. ...
... It was also reported that lineages 1, 5 and 6 were ancient and; 2, 3 and 4 were modern on the basis of TbD1 analysis. The genetically diverse MTB strains from different lineages have been shown to induce variable host responses in macrophages , cell lines and mouse models [12,13,14,15,16,17,18]. These strains are also known to vary with respect to their growth, virulence and immunopathology [19]. ...
Mycobacterium tuberculosis (MTB) persistently infects and survives within the host macrophages. Substantial genotypic variation exists among MTB strains which correlate with their interactions with the host. The present study was designed to establish a correlation, if any, between infection and induction of innate immune response by genetically diverse drug resistant MTB isolates from India. For this purpose, three clinical isolates from ancient and modern lineages, along with H37Ra and H37Rv were evaluated for intracellular growth, phagocytic index, induction of proinflammatory cytokines and apoptosis following infection in THP-1 cell line. A wide variation in the induction of cytokines was revealed subsequent to infection with different strains. EAI-5 strain from ancient lineage 1, induced higher proinflammatory responses, higher apoptosis and moderate intracellular growth compared to other strains, in contrast, for Beijing strain of modern lineage 2, all three parameters were lowest among the clinical isolates. Further, the responses induced by LAM-6 from modern lineage 4 were at a moderate level, similar to the laboratory strain H37Rv which also belongs to lineage 4. Thus, these profiles were specific to their respective lineages and/or genotypes and independent of their drug resistance status. Further, a positive correlation, among TNF-α, IL-1β, IL-6 and IL-12 induced in infected THP-1 cells was demonstrated. In addition, induction of all pro-inflammatory cytokines correlated well with the host cell apoptosis. A positive correlation was observed between phagocytic index in the category of '>10 bacilli/cell' and induction of apoptosis, only for virulent strains, indicating that initial accumulation of MTB strains inside the host cell may be an important determining factor for different innate responses.
... We also chose to study macrophages rather than whole blood in order to concentrate on a single cell population that is most relevant for Tb pathogenesis. A number of studies have shown that the strain of Mtb induces different immune responses [25,26]. Although the choice of Mtb strain could stimulate different gene expression profiles, we chose to study the commonly used laboratory strain (Mtb H37Rv). ...
Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB.
... Often, these strains are responsible for a considerable proportion of disease episodes in those regions (1,13,18). This and other observations (19,20) has led to the notion that some strains may be more virulent than others (21), although it must be cautioned that simple abundance (1,18) is not necessarily an indicator of virulence (22). Although it is possible to assess virulence in animal models, most animal models do not reflect the human disease situation precisely. ...
Traditional epidemiological methods provide insight into the dynamics of diseases such as tuberculosis. These traditional techniques have limitations and rely on a number of assumptions. The application of molecular techniques to the study of epidemiology has allowed us to gain new insights into the biology of the organism Mycobacterium tuberculosis and the dynamics of the disease. We have been enabled to push the limits of understanding of the epidemiology of this disease, allowing us to challenge the old clinical dogmas, ask new questions, design new strategies, and measure the efficacy of such new interventions to combat this age-old scourge. Among the dogmas challenged are that infection outside the home is commonplace, so-called relapse cases may in fact be largely reinfection, and active transmission may be more common than previously thought and reactivation disease relatively uncommon. These findings alone demand urgent attention and the design of optimal intervention strategies to reduce the burden of disease.
... Through evolutionary selection, pathogenic mycobacteria have acquired means to evade specific host immune effector mechanisms, presumably including those of the innate response. The propensity of certain M. tuberculosis isolates to cause outbreaks, for example, has been linked to increased virulence in macrophages or mice in association with altered host cytokine expression profiles (2,3,6,8,12). However, even these virulent outbreak-associated isolates cause disease in only a small proportion of infected individuals. ...
The initial host response to Mycobacterium tuberculosis is driven by innate immunity. For this study, we examined the ability of 18 recent clinical isolates and 5 reference strains
to survive and replicate in the context of host innate immunity by using whole blood culture. Six healthy tuberculin-negative
volunteers served as subjects. H37Ra showed the least capacity to replicate of any of the strains tested, decreasing in viability 1.3 log CFU during 72 h of
whole blood culture, whereas H37Rv increased 0.32 log. Clinical isolates varied greatly in their ability to replicate in blood cells, ranging from −0.4 to
+0.8 log (P < 0.001). Four showed significantly more growth than H37Rv, and one showed significantly reduced growth. Host mechanisms for restricting intracellular mycobacterial growth were more
effective during the first 24 h of whole blood culture than during the 24- to 72-h period. Certain mycobacterial isolates
appeared preferentially able to withstand host defenses during each of these intervals. Although there was relatively more
homogeneity among subjects than among strains, one of the six subjects showed a reduced capacity to restrict intracellular
mycobacterial growth due to a defect expressed during the first 24 h of culture. Our findings indicate substantial variability
in the capacity of clinical tuberculosis isolates to replicate in host cells in the face of innate host immunity.
... Despite the fact that hosts have only a limited number of pathways in which they respond to pathogens, macrophages show both pathogen-specific gene expression profiles and a shared gene expression pattern when infected with diverse bacterial pathogens [5]. An in vitro study of human macrophage responses to a repertoire of genotypically and epidemiologically well defined clinical isolates of Mycobacterium tuberculosis (MTB) showed a strain dependent host response [6]. A more recent study has shown a shared and a unique gene expression signature by human macrophages stimulated with four isolates of M. avium that varied in growth characteristics [7]. ...
Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease (JD) persistently infects and survives within the host macrophages. While it is established that substantial genotypic variation exists among MAP, evidence for the correlates that associate specific MAP genotypes with clinical or sub-clinical disease phenotypes is presently unknown. Thus we studied strain differences in intracellular MAP survival and host responses in a bovine monocyte derived macrophage (MDM) system.
Intracellular survival studies showed that a bovine MAP isolate (B1018) and a human MAP isolate (Hu6) persisted in relatively higher numbers when compared with a sheep MAP isolate (S7565) at 24-hr, 48-hr and 96-hr post infection (PI). MDMs stimulated with B1018 up-regulated IL-10 at the transcript level and down-regulated TNFalpha at the protein and transcript levels compared with stimulations by the S7565 and Hu6. MDMs infected with Hu6 showed a down regulatory pattern of IL-10 and TNFalpha compared to stimulations by S7565. Cells stimulated with B1018 and Hu6 had low levels of matrix metalloprotease-3 (MMP3) and high levels of tissue inhibitor of metalloprotease-1 (TIMP1) at 96-hr PI relative to MDMs stimulated by S7565.
Taken together, results suggest that the bovine (B1018) and the human (Hu6) MAP isolates lead to anti-inflammatory and anti-invasive pathways in the macrophage environment whereas the sheep (S7565) MAP isolate induces a pro-inflammatory pathway. Thus the infecting strain genotype may play a role in polarizing the host immune responses and dictate the clinicopathological outcomes in this economically important disease.
The Innate Immune Response to Mycobacterium Tuberculosis is Dependent on Strain Lineage and on Host Population
publication description.
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publication description : The genome structure of Mycobacterium tuberculosis is strongly clonal, in the absence
of horizontal gene transfer. Thus it is feasible that clonal lineages may exhibit
particular phenotypic characteristics, which may, in turn, result in differences in virulence or influence their association with particular host populations.
Indeed, the global distribution of M. tuberculosis strains is not uniform and certain strain lineages predominate in particular geographical areas. Further, there is evidence that some strain lineages are emerging, suggesting differences in virulence. Firstly, we investigated the association between strain genotype of M. tuberculosis and
in vitro correlates of virulence such as growth phenotype and cytokine induction in the monocyte-derived macrophage (MDM) model.
Secondly, in order to study the interaction between host genetic background and the innate immune response to different strains of M. tuberculosis we conducted a cross sectional study comparing cytokine responses to in vitro infection of healthy donor monocyte derived macrophages (MDM) from individuals from different population groups with strains from different M. tuberculosis lineages. The inflammatory cytokines TNF, IL-12p40, IL-6, IL-1b and GMCSF were secreted at higher levels in response to infection with lineage 4 and lineage 3 strains as compared to lineage 2 and lineage 1 strains. Principal component analysis and linear modeling identified three inflammatory cytokines (IL-6, IL-12p40, and GM-CSF) to be differentially secreted in all four-population groups.
All research datas' are analyzed by Rajesh Sarkar, myself as per guidance of UCT statistic department and expert acknowledged in the thesis and dissertation sections. One postgraduate student from statistic department got postgraduate degree from these data analysis. In this regard, any misleading information will not be acceptable.
The genome structure of Mycobacterium tuberculosis is strongly clonal, in the absence of horizontal gene transfer. Thus it is feasible that clonal lineages may exhibit particular phenotypic characteristics, which may, in turn, result in differences in virulence or influence their association with particular host populations.
Indeed, the global distribution of M. tuberculosis strains is not uniform and certain strain lineages predominate in particular geographical areas. Further, there is evidence that some strain lineages are emerging, suggesting differences in virulence.
Firstly, we investigated the association between strain genotype of M. tuberculosis and in vitro correlates of virulence such as growth phenotype and cytokine induction in the monocyte-derived macrophage (MDM) model. We report that ‘modern’ clinical M.
tuberculosis isolates from Cape Town (Lineage 2 and Lineage 4 strains) exhibit both lineage-specific patterns of growth in vitro (in broth and MDM) as well as cytokine responses in MDM.
Secondly, in order to study the interaction between host genetic background and the innate immune response to different strains of M. tuberculosis we conducted a cross sectional study comparing cytokine responses to in vitro infection of healthy donor MDM from individuals from different population groups with strains from different M. tuberculosis lineages. The inflammatory cytokines TNF, IL-12p40, IL-6, IL-1b and GMCSF were secreted at higher levels in response to infection with lineage 4 and lineage 3 strains as compared to lineage 2 and lineage 1 strains.
Principal component analysis and linear modeling identified three inflammatory cytokines (IL-6, IL-12p40, and GM-CSF) to be differentially secreted in all four-population groups. In addition, we noted evidence of possible strain lineage-host ethnicity interactions for several cytokines. Together, these studies suggests that genetic diversity of M. tuberculosis might influence the early innate immune response during tuberculosis infection, and that lineage-specific responses may be modulated by host genetic background.
This study characterized the immune responses in early Mycobacterium tuberculosis (Mtb) H37Ra infection of human peripheral blood mononuclear cell (PBMC)-collagen matrix culture and the impact of Bacille Calmette-Guérin (BCG) vaccination history of donor PBMCs on the immune responses to Mtb infection. Aggregates of PBMCs were initially observed on day 3 and the size of aggregates continued to increase on day 8 post-infection, where macrophages and T cell subsets were identified to be present. Similarly, mycobacterial load progressively increased in infected PBMCs during the 8 days of culture but were significantly lower in infected PBMCs from BCG vaccinated (BCG+) donors compared to unvaccinated (BCG-) donors. The levels of INF-γ, TNF-α, IL-4, IL-6, IL-10 and IL-17 in the supernatants of Mtb-infected PBMCs peaked at day 3 and decreased on days 5 and 8. The levels of these cytokines except IL-10 were significantly lower in Mtb-infected PBMCs from BCG+ donors compared to infected PBMCs from BCG- donors. The percentages of activated naïve Th cells, activated effector memory Th cells and activated central memory Tc cells were significantly higher in Mtb-infected PBMCs compared to uninfected PBMCs at day 8 post-infection. Further, the proportion of activated central memory Tc cells was significantly higher in infected PBMCs from BCG+ donors compared to the BCG- donors. This study highlights the possibility that BCG vaccination may confound results that utilize human PBMCs to study Mtb infection.
For those involved in infectious disease medicine, it seems that, to paraphrase an old adage, there are three certain things in life: taxes, death, and drug resistance. The variation in drug resistance detected in isolates of well-known bacteria (e.g., Pneumococcus) between different nations with different prescribing policies is well known. This is perhaps not surprising because the treatment of most infectious bacterial diseases is by monotherapy. Thus, logically, one might not expect much drug resistance in Mycobacterium tuberculosis (tuberculosis, TB) because the therapy for TB involves 2–4 different drugs administered simultaneously, and the risk of developing resistance for multiple drugs is additively small, as outlined in Section 3.2.
Genome variation is the main underlying reason for phenotypic differences observed between organisms from the same species. This includes minor variations like single nucleotide polymorphisms, but also large sequence polymorphisms due to deletions and insertions. Some of these variations are of insignificant functional importance, but others may lead to an increase in viability and, in the case of microbes, possibly an increase in pathogenicity. However, the influence of variations on phenotype is not always dependent on the size of the mutated domain, and even single nucleotide polymorphisms can cause significant changes in an organism. This is of course an important area of research into studying pathogenic organisms and their epidemiological features. Mycobacterium tuberculosis strains also display a degree of strain variation, which has been previously suggested to be important for the outcome of disease. Certain strains are postulated to be more or less virulent, persistent, transmissible, or immunopathological. These variations do not only have important implications for the spread of the disease, but provide us with tools to characterize transmission, which have been used extensively for a number of years in tuberculosis molecular epidemiology. In this article, we discuss the different methods which are available for detecting genome variation; we look at the different mechanisms which cause this variation (including insertions, deletions, duplications and single nucleotide polymorphisms), and review the consequences and implications of this genome variation with regard to Mycobacterium tuberculosis pathogenicity.
Mycobacterium tuberculosis is a potent inducer of cytokine production by mononuclear phagocytes, which are an important cellular component in the first line immune defence. In this study, the cell wall-associated Triton X-100 soluble protein (TSP) antigens, TSP-H37Rv, TSP-H37Ra, TSP-K, and TSP-BCG, were isolated from M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. tuberculosis K-strain, and M. bovis BCG, respectively. The monocytes were isolated from the peripheral blood mononuclear cells of healthy individuals and were co-cultured with each TSP antigens and the secretory proteins of M. tuberculosis (PPD and 30-kDa antigen) to measure the production of cytokines; tumor necrosis factor (TNF)-α, interleukin (IL)-12, IL-8 and monocyte chemotactic protein-1 (MCP-1). The TSP-H37Rv antigen-stimulated monocytes showed higher level of TNF-α and IL-12 production compared to those of other TSP antigens and PPD. Especially, IL-12 production in response to the TSP-H37Rv antigen was significantly elevated in comparison with that of PPD-stimulated monocytes (TSP-H37Rv, 255.5±256.9 pg/ml; PPD, 55.7±55.4 pg/ml). However, the 30-kDa antigen did not induce TNF-α expression and also showed the lowest level of cytokine and chemokine production by monocytes. MCP-1 and IL-8 production were similarly increased in response to all TSP antigens and the PPD antigen. The production of IL-12 by the TSP-H37Rv antigen stimulation was significantly increased in PPD reactors than that in the non-reactor group, while the levels of other cytokines stimulated with each TSP antigens, 30-kDa and PPD antigen were not significantly different between the tuberculin reactor and the non-reactor groups. These results suggest that the cell wall-associated TSP antigen isolated from M. tuberculosis H37Rv acts as a more potent IL-12 inducer than the PPD antigen in innate immune response and thus it could further activate the Th1-mediated immune responses effectively against M. tuberculosis infection.
The outcome of tuberculosis infection and disease is highly variable. This variation has been attributed primarily to host and environmental factors, but better understanding of the global genomic diversity in the M. tuberculosis complex (MTBC) suggests that bacterial factors could also be involved. Review of nearly 100 published reports shows that MTBC strains differ in their virulence and immunogenicity in experimental models, but whether this phenotypic variation plays a role in human disease remains unclear. Given the complex interactions between the host, the pathogen and the environment, linking MTBC genotypic diversity to experimental and clinical phenotypes requires an integrated systems epidemiology approach embedded in a robust evolutionary framework.
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The existence of a genetic component in mycobacterial disease susceptibility is no longer in doubt and the investigations now being conducted aim to determine which genes are involved, to what extent, and in which disease phenotype they are relevant. In certain rare instances of susceptibility to poorly pathogenic mycobacteria, the genetic component is clear. The approaches employed to elucidate common disease susceptibility include linkage studies, particularly genome-wide linkage analysis of both tuberculosis and leprosy, and association studies. A number of candidate genes have shown association with tuberculosis, and in many cases, on replication of the study, association has been confirmed in a disparate population, indicating the wider importance of the gene in the disease process. In other instances, associations appear to be particular to a population or a subtype of disease.
Different phenotypes are displayed by Mycobacterium tuberculosis (M. tuberculosis) strains, fuelling speculation that certain strains are "hypervirulent" and able to evade host defenses better than others. Furthermore, differential antigen expression by M. tuberculosis strains may explain why certain patients are susceptible to a repeat episode of tuberculosis. The objective of this study was to compare protein expression by M. tuberculosis H37Rv and clinical isolates in order to determine whether differential protein expression contributes to the different phenotypes expressed by these strains. Expression of alpha-crystallin, the antigen 85 complex, PstS-1, L-alanine dehydrogenase and the 65 kDa antigen was analysed by Western blotting and enzyme-linked immunosorbent assays, using mouse monoclonal antibodies. We found no significant difference in the growth rate of the M. tuberculosis strains in vitro, and although M. tuberculosis protein expression showed phase variation during growth, expression seemed to be qualitatively, but not quantitatively, conserved in the strains investigated. These results have potentially important implications for vaccine development and serodiagnosis.
The success of Mycobacterium tuberculosis as a human pathogen depends on its ability to tolerate and perhaps manipulate host defense mechanisms.
To determine the induction of tumour necrosis factor-alpha (TNF alpha), a central mediator of immunity, by human monocytes infected with virulent M. tuberculosis, M. leprae and attenuated M. bovis BCG.
Mycobacteria-induced cellular activation pathways of TNF alpha production was investigated using an inhibitor of protein tyrosine kinase (PTKs) and an inhibitor of mitogen-activated protein (MAP) kinases.
TNF alpha production was significantly lower during infection with virulent M. tuberculosis than with BCG and this differential response was independent of mycobacterial viability. TNF alpha production involved the PTK and MAP kinase pathways. Reduced TNF alpha induction by M. tuberculosis was associated with a reduction in the extent and duration of phosphorylation of extracellular-signal regulated kinases (ERK 1/2). Infection with M. leprae triggered low and transient ERK 1/2 activation as well as low TNF alpha production.
Maintenance of the differential response in both live and heat-killed preparations suggests that the reduced TNF alpha response associated with virulent mycobacteria is due to differences in the presence of components capable of triggering host pattern recognition receptors, rather than events associated with phagosome trafficking or the active release of intracellular modulators.
The impact of tuberculosis (TB) is considerably lower than one may expect, since in the absence of immunosuppression, fewer than 10% of infected individuals will develop active disease. The relatively low proportion of individuals who progress to active disease after infection can probably be ascribed to innate resistance in most infected individuals, since vaccination using BCG or a previous episode of TB does not work reliably or effectively to confer protection in high burden parts of the world. Innate factors affecting resistance or susceptibility can be modulated by the environment and such external influences cannot be ignored. Specifically, we will address bacterial variability as well as environmental factors such as diet, smoking, helminths and hormones. We will also discuss host genes that may be involved in susceptibility or resistance at various stages of infection or disease. The discovery of as yet unknown genes impacting on TB susceptibility or disease course may lead to new insights into mechanisms of disease and novel therapies. With adaptive immunity being of little value and good TB control programmes being rare, innate resistance is still our best defence against this
Four agents, thalidomide, oxpentifylline, dexamethasone and a polyclonal anti-TNF-alpha antibody, were all shown by specific Elisa to block endogenous TNF-alpha production by Bacillus Calmette Guerin (BCG)-infected human monocyte-derived macrophages in in vitro culture. There was however no significant enhancement of intracellular BCG growth, over a 7-day incubation, in human monocyte-derived macrophages in the presence of any of the TNF-alpha-blocking agents, as determined by both radiometric and CFU counting methods of assessing bacterial viability and growth. The result suggests that the action of TNF-alpha alone is unlikely to be an important effector mechanism in antimycobacterial immunity within human cells.
We investigated the effects of certain macrophage-active cytokines on the phagocytosis and growth inhibition of Mycobacterium avium complex (MAC) by human alveolar macrophages (AM). We also evaluated the effects of pretreatment with each cytokine on the superoxide anion release (O2-) from AM. The cytokines that we used were recombinant GM-CSF, natural type TNF-alpha, recombinant interferon-gamma (IFN-gamma), and recombinant IL-2. We found that phagocytosis by the various cytokine-stimulated AM was similar to that of unstimulated AM. On the other hand, significant growth inhibition of MAC was observed in the macrophages treated with GM-CSF or TNF-alpha, while no growth inhibition of MAC was observed in the macrophages treated with IFN-gamma or IL-2. Pretreatment with all cytokines tested enhanced the O2- release from AM, but there was no correlation between the enhancement of O2- release and the growth inhibition of MAC. Thus, we concluded that GM-CSF or TNF-alpha could activate AM to inhibit growth of MAC, probably not through the enhanced production of reactive oxygen intermediates.
We have examined the effect of killing of host monocytes infected with bacillus Calmette-Guérin (BCG) on the viability of the intracellular mycobacteria. Peripheral blood monocytes were infected in vitro with a single bacillus per cell and maintained in culture for 6-8 d to allow the bacilli to replicate. Replicating viable BCG were found singly in perinuclear vacuoles bounded by tightly apposed lipid bilayers. Monocytes were then exposed to toxic mediators that induced killing of cells as evaluated by 51Cr release into the culture medium. Both hydrogen peroxide (H2O2) (an inducer of cell necrosis) and adenosine triphosphate (ATP4-) (an inducer of cell apoptosis) treatment killed infected monocytes. H2O2-induced killing had no effect on BCG viability. ATP-induced cell death was accompanied by DNA fragmentation and nuclear condensation. Apoptosis was associated with a swelling of the phagocytic vacuoles which became multibacillary and with a reduction of BCG viability as enumerated by colony-forming units.
The ability of Mycobacterium tuberculosis H37Rv and H37Ra, M. bovis BCG and M. smegmatis to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by cultured murine peritoneal macrophages is inversely related to their virulence. The avirulent species of mycobacteria which were unable to persist in macrophages were capable of inducing significant levels of TNF-alpha compared to that formed in cultures infected with the virulent M. tuberculosis H37Rv. This difference was also associated with an inherent toxicity by live H37Rv for macrophage cultures. Heat-killed H37Rv was non-toxic and induced significant levels of TNF-alpha; in contrast, live and heat-killed suspensions of avirulent mycobacteria had an equivalent ability to trigger TNF-alpha secretion. The TNF-alpha response was dose-dependent, related directly to the percentage of infected cells, and peaked 6-12 h post-infection. An early and vigorous TNF-alpha response appears to be a marker of macrophage resistance, while the downregulation of this response seems associated with macrophage toxicity and unrestricted mycobacterial growth.
Interferon gamma (IFN-gamma) exerts a variety of immunoregulatory effects on several cell targets. It is generally assumed that IFN-gamma is specifically produced by T and large granular lymphocytes. In this study, we show that IFN-gamma is constitutively expressed in resting mouse peritoneal macrophages (PM). Treatment of PM with cycloheximide results in a significant accumulation of IFN-gamma mRNA, suggesting that a short-lived IFN-gamma mRNA accumulates when protein synthesis is inhibited. Moreover, treatment of PM with IFN-gamma also results in a clear-cut accumulation of this mRNA. This effect is not observed in murine lymphocytes from mesenteric lymph nodes (which instead produce IFN-gamma after phytohemagglutinin treatment) and in mouse cell lines. The treatment of PM with IFN-gamma also results in secretion of IFN-gamma after 24-48 h. The upregulation of IFN-gamma expression is also found in PM from anti-asialo GM1-treated nude mice. We suggest that the ability of PM to produce this IFN-gamma is indicative of an autocrine mechanism. The macrophage IFN-gamma may play a role in the regulation of cell differentiation and immune response.
To determine the geographical distribution of tuberculosis in the two Western Cape suburbs with the highest reported incidence of tuberculosis.
Descriptive illustrative study.
Two adjacent Western Cape suburbs covering 2.42 km2 with a population of 34,294 and a reported tuberculosis incidence of > 1,000/100,000.
All patients notified as having tuberculosis over a 10-year period (1985-1994).
None
The geographical distribution of the cases was determined using a geographical information system (GIS) and the National Population Census (1991).
One thousand eight hundred and thirty-five of the 5,345 dwelling units (34.3%) housed at least 1 case of tuberculosis during the past decade and in 483 houses 3 or more cases occurred. These cases were distributed unevenly through the community, with the tuberculosis incidence per enumerator subdistrict (ESD) varying from 78 to 3,150/100,000 population.
In a small area with a high incidence of tuberculosis, the cases are spread unevenly through the community and there are certain houses where tuberculosis occurs repeatedly. This information should be used to direct health services to concentrate on certain high-risk areas.
Gamma interferon (IFN-gamma) is a cytokine which plays a critical role in resistance to Mycobacterium tuberculosis infection. While T lymphocytes and natural killer cells are a major source of IFN-gamma, previous demonstrations that it can be produced by murine macrophages prompted us to examine the capacity of human alveolar macrophages to express IFN-gamma. Here we report that in vitro infection of alveolar macrophages with M. tuberculosis induces both the release of IFN-gamma protein and a transient increase in IFN-gamma mRNA levels. The IFN-producing cells were shown to be macrophages by reverse transcription-in situ PCR. We also observed that M. tuberculosis stimulation resulted in IFN-gamma-dependent expression of the chemokines IFN-gamma-inducible protein 10 and monokine induced by IFN-gamma, suggesting that macrophage-derived IFN-gamma can function in an autocrine and/or paracrine manner. The existence of a positive regulatory loop was suggested by the observation that exogenous IFN-gamma protein could induce IFN-gamma mRNA expression in uninfected alveolar macrophages. Interleukin-12 was also found to be a potent inducer of IFN-gamma production, and M. tuberculosis-induced IFN-gamma production appears to be mediated, at least in part, by IL-12. In contrast, M. tuberculosis-induced IFN-gamma production by alveolar macrophages could be blocked by exogenous interleukin-10. These studies are the first to demonstrate an autoregulatory role for IFN-gamma produced by alveolar macrophages infected in vitro with M. tuberculosis.
Regulation of interleukin (IL)-12 production by coexpression of tumor necrosis factor (TNF)-α, IL-10, and transforming growth
factor (TGF)-β in human monocytes infected with Mycobacterium tuberculosis H37Ra was analyzed. Also, since IL-12 induces interferon (IFN)-γ, the effect of IFN-γ on IL-12 expression was examined. IL-12
mRNA was measured by reverse transcriptase-polymerase chain reaction and IL-12 protein by ELISA. IL-12 p35 mRNA was constitutive
and inducible. IL-12 p70 protein paralleled IL-12 p40 protein expression. TNF-α protein expression occurred earlier than IL-12
p40 protein but was not required for IL-12 induction. Addition or neutralization of TGF-β did not significantly alter IL-12
induction. In contrast, recombinant IL-10 reduced IL-12 and neutralization of IL-10 minimally enhanced IL-12. A pronounced
increase in IL-12 followed IFN-γ pretreatment, which selectively up-regulated IL-12 p35 mRNA. Further understanding of operative
cytokine networks during M. tuberculosis infection may improve strategies for vaccine development and immunotherapy.
Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.
Interferon gamma (IFN-gamma) exerts a variety of immunoregulatory effects on several cell targets. It is generally assumed that IFN-gamma is specifically produced by T and large granular lymphocytes. In this study, we show that IFN-gamma is constitutively expressed in resting mouse peritoneal macrophages (PM). Treatment of PM with cycloheximide results in a significant accumulation of IFN-gamma mRNA, suggesting that a short-lived IFN-gamma mRNA accumulates when protein synthesis is inhibited. Moreover, treatment of PM with IFN-gamma also results in a clear-cut accumulation of this mRNA. This effect is not observed in murine lymphocytes from mesenteric lymph nodes (which instead produce IFN-gamma after phytohemagglutinin treatment) and in mouse cell lines. The treatment of PM with IFN-gamma also results in secretion of IFN-gamma after 24-48 h. The upregulation of IFN-gamma expression is also found in PM from anti-asialo GM1-treated nude mice. We suggest that the ability of PM to produce this IFN-gamma is indicative of an autocrine mechanism. The macrophage IFN-gamma may play a role in the regulation of cell differentiation and immune response.
We used Mycobacterium avium infection in severe combined immunodeficiency (SCID) mice to examine T-cell-independent mechanisms of inflammatory cell recruitment. SCID mice infected with a virulent strain of M. avium (TMC724) were able to recruit macrophages to sites of mycobacterial replication and formed organized and coherent granulomas in the absence of functional T cells. Phagocyte recruitment was almost totally ablated by neutralization of either tumour necrosis factor-alpha (TNF-alpha) or interferon-gamma (IFN-gamma) in vivo demonstrating that granuloma formation was dependent on the presence of these cytokines. This was concomitant with a reduction in the in situ cytokine mRNA levels otherwise induced in infected mice, for chemokines, pro-inflammatory and regulatory cytokines, including TNF-alpha, IFN-gamma, macrophage inflammatory protein-1 alpha, interleukin-1 beta (IL-1 beta) and IL-10. Furthermore, in vivo treatment of infected mice with anti-asialo GM-1 antisera, which depletes natural killer (NK) cells, prevented recruitment of inflammatory cells. In vitro studies confirmed that M. avium was able to elicit IFN-gamma from SCID spleen in a dose-dependent manner. These data show for the first time that secretion of IFN-gamma from NK cells can mediate a T-cell-independent pathway of granuloma formation and cellular infiltration in response to mycobacteria.
Mycobacterium tuberculosis H37Rv causes progressive disease in animals, whereas the H37Ra strain does not. The relevance of this difference in virulence to human infection is uncertain because these strains have been shown to have similar growth rates in human macrophages. To evaluate the intracellular growth of M. tuberculosis strains in macrophages under conditions similar to those encountered in vivo, we infected human monocyte-derived macrophages with H37Ra, H37Rv, or one of four isolates from tuberculosis patients at a low bacillus-to-macrophage ratio. H37Rv and the patient isolates grew significantly faster than H37Ra, based on the numbers of CFU and acid-fast bacilli. These findings did not result from extracellular mycobacterial growth, differential macrophage viability, or bacillary clumping. In contrast to other published results, these findings indicate that the virulence characteristics of M. tuberculosis strains in animal models are relevant to human tuberculosis infection.
Organisms belonging to the Mycobacterium avium complex (MAC) are associated with life-threatening bacteremia in patients with the acquired immunodeficiency syndrome (AIDS). As these organisms survive within macrophages, we examined the ability of recombinant human granulocyte-monocyte colony-stimulating factor (GM-CSF) to activate human monocyte-derived macrophages to inhibit the intracellular growth or kill the most mouse-virulent MAC strain in our collection that belongs to serotype 1. While unstimulated cells did not inhibit intracellular growth of MAC, macrophages activated by GM-CSF (10-10(4) U/ml) inhibited or killed up to 58 +/- 5% of the initial inoculum. This activation was dose-dependent, with maximal change occurring with a dose of 100 U/ml after 72 hr exposure. Inhibition or killing was demonstrated if GM-CSF was given both before or after establishment of infection. The combination of GM-CSF (10(2) U/ml) plus TNF (10(2) U/ml) augmented macrophage killing (range, 31 +/- 4%) compared with GM-CSF (10(2) U/ml) alone, but the combination of recombinant human interferon-gamma (IFN gamma) plus GM-CSF resulted in a significant decrease in intracellular inhibition of growth or killing (13.3 +/- 2%) compared with 57.7 +/- 5% obtained with GM-CSF alone. These results indicate that: 1) GM-CSF can activate macrophages to inhibit intracellular growth or kill MAC; 2) killing may be augmented by TNF; and 3) IFN gamma may impair GM-CSF-dependent macrophage activation.
The bactericidal function of macrophages was investigated in congenic mice expressing the phenotype of susceptibility (B10.A, Bcgs) or resistance (B10.ABcgr) to mycobacterial infection. When splenic and peritoneal macrophages from these two mouse strains were infected in vitro with Mycobacterium smegmatis, the Bcgr macrophages were shown to inactivate M. smegmatis more efficiently than their Bcgs congenic counterparts. The mechanisms of this superior antimycobacterial activity was studied further. Addition of catalase did not abolish killing to a significant degree in either allelic type of macrophage, suggesting that hydrogen peroxide production was not involved in the killing activity controlled by the Bcg gene. Activation of Bcgs macrophages by exposure to crude lymphokines rendered them equally as efficient as their Bcgr counterparts in their capacity to destroy M. smegmatis. This finding suggests that both the genetically resistant and susceptible macrophages have the potential to kill M. smegmatis in vitro. This potential is expressed constitutively by the Bcgr but not Bcgs macrophages and can be induced, by lymphokine treatment, in the Bcgs macrophages. In a final set of experiments, the macrophage killing of M. smegmatis was evaluated as a test system to type for the Bcg gene allelic type in vitro, using a set of AXB and BXA recombinant inbred strains of mice. Results obtained show that typing of AXB/BXA recombinant inbred strains for the trait of bactericidal activity vs. M. smegmatis in vitro revealed a perfect match with the strain distribution pattern of resistance/susceptibility to Mycobacterium bovis BCG in vivo.
Organisms belonging to the Mycobacterium avium complex (MAC) are the most common bacterial pathogens in patients with AIDS but factors associated with the activation of cellular defense mechanisms against this atypical mycobacterium have not been defined. Peritoneal macrophages harvested from a chronic MAC infection in C57 black mice are able to kill approximately 86% of intracellular MAC in contrast to 0 to 20% killing by unstimulated human and mouse macrophages in vitro. The availability of human rTNF-alpha, rIFN-gamma, and rIL-2 permitted evaluation of the role of each of these lymphokines/monokines, alone or in combination, in activating macrophages in vitro to kill MAC. Human monocyte-derived macrophages were cultured in vitro, stimulated with rIL-2, rIFN-gamma, or rTNF, and then infected with MAC (serovars 1 and 8). Mouse peritoneal macrophages were harvested, cultured in vitro, and stimulated with rIFN-gamma. rTNF (10(4) U/ml) was associated with a modest increase of intracellular killing of MAC (58 +/- 5%) even when utilized 24 or 48 h after macrophage infection or when administered for 5 consecutive days after infection (78.1 +/- 4%). Both human and murine IFN-gamma were associated with increased intracellular growth of MAC (32 +/- 4% for murine and 38 +/- 3% for human macrophages). However, intracellular killing (53 +/- 6% compared with control) was observed after 6 days of treatment with IFN-gamma. This latter effect was fully blocked by anti-TNF antibody, whereas rIL-2 alone did not augment the intracellular killing of MAC by human macrophages. rTNF plus either rIFN-gamma or rIL-2 triggered significant increases in superoxide anion production, but subsequent MAC killing was no greater than with rTNF alone. Treatment of macrophages with 10 U/ml of rTNF followed by rIL-2 (200 U/ml) was associated with 68% of intracellular killing. TNF seems to be an important monokine, promoting activation of mycobactericidal mechanisms in human macrophages.
Given the critical antimicrobial properties of mononuclear phagocytes, an important concern in cell biology and immunology has been to understand how intracellular microbes are able to establish states of chronic infection within these cells. Recent studies indicate that mononuclear phagocytes become functionally deactivated during intracellular infection. Here, Neil Reiner considers the experimental evidence to indicate that this is a frequent event that may be accounted for by induced defects in the signaling pathways required to bring cells to an activated state.
A protocol using combined exposure to interferon-gamma (IFN-gamma), calcitriol and tumour necrosis factor-alpha (TNF-alpha) has been reported to activate human monocytes in vitro to kill Mycobacterium tuberculosis. We have attempted to repeat the findings in two laboratories, with negative results; treated cells were no different from untreated cells in this respect. However, the treated cells were more sensitive to a toxic effect of the bacteria. We suggest that the reported dramatic mycobacterial killing may have been an illusory consequence of the toxicity leading to cell lysis and loss of the liberated bacteria from the assay.
In this paper we report that macrophages can be stimulated to express detectable levels of IFN-γ-specific mRNA. Macrophages
from lipopolysaccharide (LPS)-responsive, C3H/OuJ mice are induced by LPS to increase steady-state levels of IFN-γ-specific
mRNA, while those from LPS-hyporesponsive C3H/HeJ mice are not. This interstrain variation is apparently the result of LPS-specific
signal differences since macrophages derived from both Lpsn and Lpsd mouse strains are able to produce comparable levels of IFN-γ-specific mRNA following stimulation with polyinosinic-polycytidylic
acid. The identity of the cell type responsible for this IFN-γ message appears to be the macrophage as IFN-γ-specific mRNA
was also detectable following T and natural killer cell depletion, in the LPS-stimulated RAW 264.7 cell line, and in a homogeneous
population of mature macrophages propagated in vitro by stimulation of bone marrow progenitors with recombinant colony stimulating factor-1. Immunofluorescent staining of fixed
and permeabilized LPS-stimulated macrophages confirmed the presence of immunoreactive IFN-γ protein. The possible significance
of IFN-γ production by macrophages is discussed in the context of normal macrophage differentiation as well as the inflammatory
immune response.
The relative phagocytosis and intracellular fate of Mycobacterium tuberculosis (MTB) (H37Ra) in human alveolar macrophages (AM) and their precursors blood monocytes (MN) was investigated. Uptake of MTB by MN and AM was confirmed by electron microscopy. At an infection ratio of 100:1 (MTB:target cell), the percentage of infected AM and the number of MTB per AM was > MN (p < 0.001, p < 0.0001, respectively). Uptake of MTB was increased by increasing concentrations of serum and decreased in the presence of heat-inactivated serum. Among complement receptors (CR) CR1, CR3, and CR4, the major CR mediating uptake of MTB by MN were CR1 and CR3, whereas for AM, CR4 was the major CR. When MN and AM were infected with MTB and cultured for up to 7 days, AM limited intracellular growth of MTB more effectively than MN as determined by a CFU assay. MTB stimulated production of TNF-alpha by mononuclear phagocytes and by AM > MN (p < 0.007). Pentoxifylline inhibited TNF-alpha production by mononuclear phagocytes and concurrently increased MTB growth (AM > MN). A polyclonal neutralizing antibody to TNF-alpha also increased MTB growth in AM. Thus, AM are more efficient in phagocytosis of MTB than MN, and uptake is mediated through CR4 to a greater extent than CR1 or CR3. The slowed replication of MTB in AM is associated with an increase in TNF-alpha production, and intracellular growth is promoted by pentoxifylline and neutralizing antibody to TNF-alpha. These data suggest that AM may play a prominent and efficient role in the primary defense of the lung in tuberculosis through CR-mediated uptake, predominantly CR4, and TNF-alpha-mediated killing of MTB.
Mycobacterial infection leads to the development of specific cell-mediated immune responses that have been measured clinically by assessing delayed-type hypersensitivity with Mantoux skin testing. Several characteristics of Mantoux skin testing for tuberculosis infection can make the procedure inaccurate, inconvenient, and sometimes misleading. It is also a poor predictor of immunity to tuberculosis in bacille Calmette-Gúerin vaccinees, yet decisions to revaccinate often are based on skin test responses after initial immunization. Skin testing with other mycobacterial antigens has similar limitations. In vitro assessment of cellular immunity to mycobacteria offers multiple, potential advantages over skin testing and has become technically feasible in recent years. Measurement of the effector functions that comprise cell-mediated immunity (eg, cytokine secretion and cytotoxicity) rather than cutaneous delayed-type hypersensitivity responses is more likely to reflect meaningfully specific mycobacterial immunity and, therefore, provide a means for determining mycobacterial immunity after immunization. Eliminating the variability in placement and interpretation inherent in skin testing could provide a more stable foundation for comparative studies in populations and improve decision making for individuals. Finally, in vitro testing permits the use of discrete mycobacterial antigens instead of crude protein preparations, allowing greater specificity in the detection of infection as well as assessment of responses to defined candidate vaccine antigens. Several studies have compared skin testing with in vitro proliferation of lymphocytes stimulated by mycobacterial antigens for the detection of Mycobacterium tuberculosis infection. Preliminary veterinary and human studies suggest that in vitro assessment of gamma-interferon production in response to mycobacterial antigens can be used to detect prior infection with organisms of the M tuberculosis complex. Streamlined techniques for in vitro testing of cellular immunity may allow its practical adoption in the clinical setting and lead to its use as a replacement for Mantoux skin testing.
Studies of patients with severe infections arising from poorly pathogenic mycobacterium or Salmonella species have revealed genetic mutations in type-1 cytokine (IL-12p40) or type-1 cytokine receptor (IFN-γR1, IFN-γR2, IL-12Rβ1) genes. This article summarizes the findings and discusses their implications.
The macrophage plays an important role in both the innate and acquired (humoral and cellular) immune responses. Their specialized derivatives, the dendritic cells (DCs), are uniquely potent in induction of naive T and B lymphocytes, whereas macrophages influence a range of immune responses by antigen recognition, capture, clearance and transport. They recruit haemopoietic cells to local sites of inflammation and immunity and regulate their activities. We have used various myeloid-restricted membrane antigens and receptors as markers and functional contributors to these activities, and briefly review their role in immune regulation in vivo and in vitro.
The microbicidal capacity of the macrophage is frequently evaded by mycobacteria, leading to tuberculosis (TB). We investigated a number of parameters affecting the rate of growth of mycobacteria in human monocyte-derived macrophages (MDM). The results show a great deal of variation in the growth of both Mycobacterium bovis BCG and M. tuberculosis H37Rv, using a large number of human macrophage donors, (132 and 40, respectively), but no correlation was seen with the TB status of the MDM donor. Clumping of the mycobacteria resulted in more vigorous growth in MDM, suggesting that inoculum size could affect disease progression. The growth rates of 17 clinical isolates of M. tuberculosis were measured in macrophages derived from three donors and no consistent or marked differences between isolates were observed over the 5-day period of growth measurement. However, all 17 clinical strains grew consistently faster than H37Rv in the same experiments.
Differential mechanisms of intra-cellular killing of Mycobacterium avium and Listeria monocytogenes by activated human and murine macro-phages. The role of nitric oxide
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