Iron acquisition by Mycobacterium tuberculosis residing within myeloid dendritic cells

The pathophysiology of Mycobacterium tuberculosis (M.tb) infection is linked to the ability of the organism to grow within macrophages. Lung myeloid dendritic cells are a newly recognized reservoir of M.tb during infection. Iron (Fe) acquisition is critical for M.tb growth. In vivo, extracellular Fe is chelated to transferrin (TF) and lactoferrin (LF). We previously reported that M.tb replicating in human monocyte-dervied macrophages (MDM) can acquire Fe bound to TF, LF, and citrate, as well as from the MDM cytoplasm. Access of M.tb to Fe may influence its growth in macrophages and dendritic cells. In the present work we confirmed the ability of different strains of M.tb to grow in human myeloid dendritic cells in vitro. Fe acquired by M.tb replicating within dendritic cells from externally added Fe chelates varied with the Fe chelate present in the external media: Fe-citrate > Fe-LF > Fe-TF. Fe acquisition rates from each chelate did not vary over 7 days. M.tb within dendritic cells also acquired Fe from the dendritic cell cytoplasm, with the efficiency of Fe acquisition greater from cytoplasmic Fe sources, regardless of the initial Fe chelate from which that cytoplasmic Fe was derived. Growth and Fe acquisition results with human MDM were similar to those with dendritic cells. M.tb grow and replicate within myeloid dendritic cells in vitro. Fe metabolism of M.tb growing in either MDM or dendritic cells in vitro is influenced by the nature of Fe available and the organism appears to preferentially access cytoplasmic rather than extracellular Fe sources. Whether these in vitro data extend to in vivo conditions should be examined in future studies.     

Hereditary hemochromatosis results in decreased iron acquisition and growth by Mycobacterium tuberculosis within human macrophages.

Iron (Fe) acquisition is essential for the growth of intracellular Mycobacterium tuberculosis (M.tb). How this occurs is poorly understood. Hereditary hemochromatosis is an inherited disease in which most cells become overloaded with Fe. However, hereditary hemochromatosis macrophages have lower than normal levels of intracellular Fe. This suggests M.tb growth should be slower in those cells if macrophage intracellular Fe is used by M.tb. Therefore, we compared trafficking and acquisition of transferrin (Tf)- and lactoferrin (Lf)-chelated Fe by M.tb within the phagosome of monocyte-derived macrophages (MDM) from healthy controls and subjects with hereditary hemochromatosis. M.tb in both sets of macrophages acquired more Fe from Lf than Tf. Fe acquisition by M.tb within hereditary hemochromatosis macrophages was decreased by 84% from Tf and 92% from Lf relative to that in healthy control macrophages. There was no difference in Fe acquired from Tf and Lf by the two macrophage phenotypes. Both acquired 3 times more Fe from Lf than Tf. M.tb infection and incubation with interferon gamma (IFN-gamma) reduced macrophage Fe acquisition by 20% and 50%, respectively. Both Tf and Lf colocalized with M.tb phagosomes to a similar extent, independent of macrophage phenotype. M.tb growth was 50% less in hereditary hemochromatosis macrophages. M.tb growing within macrophages from subjects with hereditary hemochromatosis acquire less Fe compared with healthy controls. This is associated with reduced growth of M.tb. These data support a role for macrophage intracellular Fe as a source for M.tb growth.     

Iron uptake from lactoferrin and transferrin by Neisseria gonorrhoeae.

The major iron (Fe) sources available to Neisseria gonorrhoeae in the human host are probably transferrin (TF) and lactoferrin (LF). Although a number of studies have examined Fe uptake by Neisseria meningitidis, no comparable studies have been done on Fe uptake by the gonococcus from TF and LF. We found that, like meningococci, gonococci removed Fe from TF and LF in an energy-dependent manner; uptake was repressed by Fe and did not proceed by a siderophore-mediated uptake system. Unlike published studies examining meningococcal Fe uptake from TF, our study showed that gonococcal Fe uptake from both TF and LF was highly efficient; uptake saturated at 1 microM protein, and growth with 5% saturated TF and LF occurred at maximal rates when the protein was present in appreciable concentrations. We conclude that the availability of protein-bound Fe probably does not limit growth of N. gonorrhoeae in the human body.     

A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein.

We isolated an iron-uptake mutant of Neisseria meningitidis M986-NCV-1 that was severely limited in the ability to use several sources of iron in the form of Fe3+. This mutant, FAM11, grew poorly or not at all with human transferrin (TF) or lactoferrin (LF) as the sole iron source in a defined medium but grew as well as wild-type meningococci with hemin or hemoglobin. Uptake of 55Fe bound to TF, LF, dicitrate complexes, aerobactin, or nitrilotriacetate was reduced to 0 to 4% of the wild-type level. FAM11 did not produce an iron-repressible outer membrane protein (FeRP) of 70 kilodaltons (kDa) found in membranes of iron-stressed M986-NCV-1. Western blot (immunoblot) analysis using rabbit antiserum against this protein revealed that at least 17 of 18 meningococcal and 10 of 14 gonococcal strains produced an FeRP of ca. 70 kDa. The 70-kDa FeRP was shown to be surface exposed by radioimmunoprecipitation with human immune sera. These data suggest that the 70-kDa FeRP is somehow involved in Fe uptake from TF and LF. However, we were unable to transform the iron-uptake phenotype from FAM11 into wild-type meningococci to confirm this. Revertants of FAM11 that grew with TF and LF did not regain the ability to make the 70-kDa FeRP but also did not completely regain the Fe-uptake phenotype of M986-NCV-1.     

Increased Mycobacterium tuberculosis growth in HIV-1-infected human macrophages: role of tumour necrosis factor-alpha

Synergism between Mycobacterium tuberculosis (M. tuberculosis) and HIV-1 infections was demonstrated in several in vitro models and clinical studies. Here, we investigated their reciprocal effects on growth in chronically HIV-1-infected promonocytic U1 cells and in acutely infected monocyte-derived macrophages (MDM). Phagocytosis of M. tuberculosis induced HIV-1 expression in U1 cells, together with increased TNF-alpha production. M. tuberculosis growth, evaluated by competitive PCR, was greater in HIV-1-infected MDM compared to uninfected cells. M. tuberculosis phagocytosis induced greater TNF-alpha and IL-10 production in HIV-1-infected MDM than in uninfected cells. In uninfected MDM, addition of TNF-alpha and IFN-gamma decreased, whereas IL-10 increased M. tuberculosis growth. On the contrary, in HIV-1-infected MDM, addition of TNF-alpha and IFN-gamma increased, whereas IL-10 has no effect on M. tuberculosis growth. TNF-alpha seems to play a pivotal role in the enhanced M. tuberculosis growth observed in HIV-1-infected MDM, being unable to exert its physiological antimycobacterial activity. Here, for the first time we demonstrated an enhanced M. tuberculosis growth in HIV-1-infected MDM, in line with the observed clinical synergism between the two infections.     

Expression of the CopB outer membrane protein by Moraxella catarrhalis is regulated by iron and affects iron acquisition from transferrin and lactoferrin.

The amino acid sequence of the cell-surface-exposed, 81-kDa CopB outer membrane protein of Moraxella catarrhalis was found to be similar to those of TonB-dependent outer membrane proteins of other gram-negative bacteria. Expression of CopB was affected by the availability of iron in the growth medium, and the extent of overexpression of CopB in response to iron limitation varied widely among the M. catarrhalis strains tested. Wild-type M. catarrhalis strains were found to be able to utilize ferric citrate, transferrin, lactoferrin, and heme as sources of iron for growth in vitro. However, an isogenic copB mutant was severely impaired in its ability to utilize transferrin and lactoferrin as sole sources of iron for growth, whereas this same mutant grew similarly to the wild-type parent strain when supplied with ferric citrate as the iron source. The copB mutant was not significantly different from its wild-type parent strain in its ability to bind transferrin and lactoferrin. In addition, the wild-type parent strain and the copB mutant exhibited equivalent rates of uptake of 55Fe from ferric citrate. However, the copB mutant was markedly less able than the wild-type strain to take up 55Fe from transferrin and lactoferrin. These results indicate that lack of expression of the CopB protein exerts a direct or indirect effect on the ability of M. catarrhalis to utilize iron bound to certain carrier proteins.     

Gallium disrupts iron metabolism of mycobacteria residing within human macrophages

Mycobacterium tuberculosis and M. avium complex (MAC) enter and multiply within monocytes and macrophages in phagosomes. In vitro growth studies using standard culture media indicate that siderophore-mediated iron (Fe) acquisition plays a critical role in the growth and metabolism of both M. tuberculosis and MAC. However, the applicability of such studies to conditions within the macrophage phagosome is unclear, due in part to the absence of experimental means to inhibit such a process. Based on the ability of gallium (Ga(3+)) to concentrate within mononuclear phagocytes and on evidence that Ga disrupts cellular Fe-dependent metabolic pathways by substituting for Fe(3+) and failing to undergo redox cycling, we hypothesized that Ga could disrupt Fe acquisition and Fe-dependent metabolic pathways of mycobacteria. We find that Ga(NO(3))(3) and Ga-transferrin produce an Fe-reversible concentration-dependent growth inhibition of M. tuberculosis strains and MAC grown extracellularly and within human macrophages. Ga is bactericidal for M. tuberculosis growing extracellularly and within macrophages. Finally, we provide evidence that exogenously added Fe is acquired by intraphagosomal M. tuberculosis and that Ga inhibits this Fe acquisition. Thus, Ga(NO(3))(3) disruption of mycobacterial Fe metabolism may serve as an experimental means to study the mechanism of Fe acquisition by intracellular mycobacteria and the role of Fe in intracellular survival. Furthermore, given the inability of biological systems to discriminate between Ga and Fe, this approach could have broad applicability to the study of Fe metabolism of other intracellular pathogens.     

Acquisition of iron from transferrin and lactoferrin by the protozoan Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, requires iron for its growth. However, the extent to which different iron sources can be utilized by the parasite is not known. To address this question, we studied acquisition of iron from lactoferrin and transferrin by the extracellular promastigote form of L. chagasi during growth in vitro. A promastigote growth medium based on minimal essential medium supplemented with iron-depleted serum supported promastigote growth only after the addition of exogenous iron. The addition of 8 microM iron chelated to lactoferrin or hemin resulted in normal promastigote growth. Ferritransferrin also supported promastigote growth, but only after a considerable lag. Promastigotes grown in all three iron sources generated similar amounts of hydroxyl radical upon exposure to hydrogen peroxide, indicating that none of these protected parasites against generation of this toxic radical. Promastigotes were able to take up 59Fe chelated to either transferrin or lactoferrin, although uptake from 59Fe-lactoferrin occurred more rapidly. 59Fe uptake from either 59Fe-transferrin or 59Fe-lactoferrin was inhibited by a 10-fold excess of unlabeled ferrilactoferrin, ferritransferrin, apolactoferrin, apotransferrin, or iron nitrilotriacetate but not ferritin or bovine serum albumin. There was no evidence for a role for parasite-derived siderophores or proteolytic cleavage of ferritransferrin or ferrilactoferrin in the acquisition of iron by promastigotes. Thus, L. chagasi promastigotes can acquire iron from hemin, ferrilactoferrin, or ferritransferrin. This capacity to utilize several iron sources may contribute to the organism's ability to survive in the diverse environments it encounters in the insect and mammalian hosts.     

Differences in the uptake of iron from Fe(II) ascorbate and Fe(III) citrate by IEC-6 cells and the involvement of ferroportin/IREG-1/MTP-1/SLC40A1

Dietary iron is present in the intestine as Fe(II) and Fe(III). Since enterocytes take up Fe(II) by the divalent metal transporter (DMT1), Fe(III) must be reduced. Whether other Fe(III) transport processes are present is unknown. Release of iron from the enterocyte into the plasma involves the iron-regulated transporter-1/metal transporter protein-1 (IREG-1/MTP-1, ferroportin) but ferroportin is also found on the apical membrane. We compared the uptake of iron from Fe(II):ascorbate and Fe(III):citrate using the rat intestinal enterocyte cell line-6 (IEC-6), in the presence of ferrous chelators, a blocking antibody to ferroportin, at different pH and during the over-expression of DMT1. Firstly, surface ferrireduction was absent. Secondly, blocking ferroportin partly and totally reduced Fe(II) and Fe(III) uptake, respectively. Thirdly, optimal Fe(II) uptake occurred at pH5.5 but Fe(III) uptake was unaffected by pH and, fourthly, over-expression of DMT1 increased uptake of Fe(II) and Fe(III). This indicates that an increased extracellular H⁺ concentration facilitates DMT1-mediated Fe(II) uptake at the cell membrane. However, since Fe(III) uptake required DMT1, but not cell surface ferrireduction, and was independent of variations in extracellular pH, it appears that Fe(III) is internalised before ferrireduction and transport by DMT1. Ferroportin may function as a modulator of DMT1 activity and play a role in Fe(III) uptake, possibly by affecting the number or affinity of citrate binding sites.     

Transferrins and heme-compounds as iron sources for pathogenic bacteria.

The low concentration of free iron in body fluids creates bacteriostatic conditions for many microorganisms and is therefore an important defense factor of the body against invading bacteria. Pathogenic bacteria have developed several mechanisms for acquiring iron from the host. Siderophore-mediated iron uptake involves the synthesis of low molecular weight iron chelators called siderophores which compete with the host iron-binding glycoproteins lactoferrin (LF) and transferrin (TF) for iron. Other ways to induce iron uptake, without the mediation of siderophores, are the possession of outer membrane protein receptors that actually recognize the complex of TF or LF with iron, resulting in the internalization of this metal, and the use of heme-compounds released into the circulation after lysis of erythrocytes. In this review, the nonsiderophore-mediated iron-uptake systems used by certain pathogenic bacteria are emphasized. The possible contribution of these iron-uptake systems to the virulence of pathogens is also discussed.     

Role of tryptophan degradation in respiratory burst-independent antimicrobial activity of gamma interferon-stimulated human macrophages.

To determine whether extracellular tryptophan degradation represents an oxygen-independent antimicrobial mechanism, we examined the effect of exogenous tryptophan on the intracellular antimicrobial activity of gamma interferon (IFN-gamma)-stimulated human macrophages. IFN-gamma readily induced normal monocyte-derived macrophages (MDM) to express indoleamine 2,3-dioxygenase (IDO) activity and stimulated MDM, alveolar macrophages, and oxidatively deficient chronic granulomatous disease MDM to degrade tryptophan. All IFN-gamma-activated, tryptophan-degrading macrophages killed or inhibited Toxoplasma gondii, Chlamydia psittaci, and Leishmania donovani. Although exogenous tryptophan partially reversed this activity, the increases in intracellular replication were variable for normal MDM (T. gondii [5-fold], C. psittaci [3-fold], L. donovani [2-fold]), chronic granulomatous disease MDM (T. gondii [2.5-fold], C. psittaci [5-fold]), and alveolar macrophages (T. gondii [1.5-fold], C. psittaci [1.5-fold]). In addition, IFN-alpha and IFN-beta also stimulated normal MDM to express IDO and degrade tryptophan but failed to induce antimicrobial activity, and IFN-gamma-treated mouse macrophages showed neither IDO activity nor tryptophan degradation but killed T. gondii and L. donovani. These results suggest that while tryptophan depletion contributes to the oxygen-independent antimicrobial effects of the activated human macrophage, in certain cytokine-stimulated cells, tryptophan degradation may be neither sufficient nor required for antimicrobial activity.     

Reduced in vitro functional activity of human NRAMP1 (SLC11A1) allele that predisposes to increased risk of pediatric tuberculosis disease

Polymorphic variants within the human natural resistance-associated macrophage protein-1 (NRAMP1, also known as SLC11A1) gene have been shown to impact on susceptibility to tuberculosis in different human populations. In the mouse, Nramp1 is expressed at the macrophage phagosomal membrane and its activity can be assayed by the relative acquisition of mannose 6-phosphate receptor (M6PR) in Salmonella-containing vacuoles. Based on this M6PR recruitment assay, we have now developed an assay in primary human macrophages to test the function of human NRAMP1 gene variants. First, we established that M6PR acquisition was significantly higher (P = 0.002) in human U-937 monocytic cell lines transfected with NRAMP1 as compared to untransfected U-937 cells. Second, the M6PR assay was shown to be highly reproducible for NRAMP1 activity in monocyte-derived macrophages (MDM) from healthy volunteers. Finally, the assay was investigated in MDM from pediatric tuberculosis patients and significantly lower NRAMP1 activity was detected in MDM from individuals homozygous for the NRAMP1-274 high-risk allele (CC genotype) in comparison to heterozygous individuals (CT genotype; P=0.013). The present study describes both an assay for human NRAMP1 functional activity and concomitant evidence for reduced NRAMP1 function in the common genetic variant shown to be associated with tuberculosis susceptibility in pediatric patients.     

The distribution of ferritin, lactoferrin and transferrin in granulomatous lymphadenitis of bovine paratuberculosis

Immunohistochemical examination of iron-binding proteins was carried out in the formalin-fixed mesenteric lymph nodes of normal cattle and of cattle with paratuberculosis. Ferritin (FT) and lactoferrin (LF) were found in the granulomas in ileal lymph nodes from six infected cattle. A weak reaction for transferrin (TF) was found in granulomas of a lymph node from one of the infected cattle. FT was found in the macrophages in the medullary sinuses of normal and infected nodes; however, the reaction in infected nodes was generally stronger than that in normal ones. LF in the macrophages was found in only two infected nodes. Neutrophils in both normal and infected cattle always reacted strongly for LF. The TF was always found in the blood vessels and intracellular space. These results suggest that: (1) FT and LF may be important in vivo sources of iron for Mycobacterium paratuberculosis, since their own iron-binding compounds are considered to acquire iron from FT and LF in vitro; (2) the increase in FT and LF in the granulomas may be related to inflammatory hyposideraemia associated with paratuberculosis and (3) epithelioid and giant cells may have a different iron metabolism, from normal macrophages.     

The mechanism for the oxidative polymerization of p-phenylenediamine in aqueous solution of iron chelate

p-Phenylenediamine (PD) was oxidatively polymerized in an aqueous solution of iron chelates, e.g., the Fe(III)-salt of ethylenediaminetetraacetic acid; poly(azophenylene) was formed as a black powder. The ferric chelate is effective for the polymerization, and the polymer yield is proportional to the oxygen consumption. As a rule, the rate of polymerization reaches a maximum at pH 7–8. The electrochemical potential of the system changes rapidly during the initial stage of the polymerization and reaches an equilibrium value within several minutes. This shows that the polymerization proceeds while the equilibrium of ferric and ferrous chelates is maintained. The mechanism of the polymerization is considered to involve an equimolar coordination of the unprotonated species of PD to the ferric chelate, thus forming a mixed complex. PD is activated by electron transfer to the ferric ion which is thereby reduced to the ferrous ion, and the activated PD molecules couple with each other. The ferrous chelate is then oxidized by oxygen to the ferric chelate and may act as a catalyst again. The rate-determining step of the polymerization is the oxidation reaction of ferrous to ferric chelate by oxygen. However, the rate of the polymerization is also affected by the equilibrium between the iron chelate and PD in the polymerization system.     

Utilization of transferrin-bound iron by Haemophilus influenzae requires an intact tonB gene.

Haemophilus influenzae can utilize iron-loaded human transferrin as an iron source for growth in vitro. H. influenzae tonB mutants, containing a chloramphenicol acetyltransferase gene within their tonB genes, could bind iron-charged human transferrin to their cell surfaces, but they were unable to utilize this serum glycoprotein as the sole source of iron for growth in vitro. In contrast, these tonB mutants were able to utilize an iron chelate (ferric ammonium citrate) for growth. Transformation of a tonB mutant with a plasmid encoding a wild-type H. influenzae tonB gene restored the ability of a tonB mutant to utilize iron-charged human transferrin. These results indicate that the uptake of iron from human transferrin by H. influenzae is a TonB-dependent process.     

Interaction of Mycobacterium tuberculosis-Induced Transforming Growth Factor β1 and Interleukin-10

Mycobacterium tuberculosis is associated with the activation of cytokine circuits both at sites of active tuberculosis in vivo and in cultures of mononuclear cells stimulated by M. tuberculosis or its components in vitro. Interactive stimulatory and/or inhibitory pathways are established between cytokines, which may result in potentiation or attenuation of the effects of each molecule on T-cell responses. Here we examined the interaction of transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) in purified protein derivative (PPD)-stimulated human mononuclear cell cultures in vitro. TGF-beta1 induced monocyte IL-10 (but not tumor necrosis factor alpha) production (by 70-fold, P < 0.02) and mRNA expression in the absence but not in the presence of PPD. Both exogenous recombinant (r) IL-10 and rTGF-beta1 independently suppressed the production of PPD-induced gamma interferon (IFN-gamma) in mononuclear cells from PPD skin test-positive individuals. Synergistic suppression of IFN-gamma in cultures containing both rTGF-beta1 and rIL-10 was only seen when the responder cell population were peripheral blood mononuclear cells (PBMC) and not monocyte-depleted mononuclear cells and when PBMC were pretreated with rTGF-beta1 but not with rIL-10. Suppression of PPD-induced IFN-gamma in PBMC containing both rTGF-beta1 (1 ng/ml) and rIL-10 (100 pg/ml) was 1.5-fold higher (P < 0.05) than cultures containing TGF-beta1 alone and 5.7-fold higher (P < 0.004) than cultures containing IL-10 alone. Also, neutralization of endogenous TGF-beta1 and IL-10 together enhanced PPD-induced IFN-gamma in PBMC in a synergistic manner. Thus, TGF-beta1 and IL-10 together potentiate the downmodulatory effect on M. tuberculosis-induced T-cell production of IFN-gamma, and TGF-beta1 alone enhances IL-10 production. At sites of active M. tuberculosis infection, these interactions may be conducive to the suppression of mononuclear cell functions.     

Brucella abortus siderophore 2,3-dihydroxybenzoic acid (DHBA) facilitates intracellular survival of the bacteria

Siderophores are low molecular weight molecules that allow bacteria to acquire iron from host cell proteins. 2,3-dihydroxybenzoic acid (DHBA) is the only known siderophore produced by the intracellular pathogen Brucella abortus. Here its role in virulence was assessed by evaluating the ability of a mutant with a disruption of the entC gene to survive and replicate in vitro in murine and bovine cells and in vivo in resistant and susceptible murine hosts. It was hypothesized that DHBA is vital for bacterial virulence by its ability to chelate intracellular iron thereby preventing generation of anti-bacterial hydroxyl radicals via the Haber-Weiss reaction, to scavenge reactive oxygen intermediates and for acquisition of iron needed for nutritional purposes. The data showed DHBA played a significant role for bacterial survival in host cells after infection including in murine macrophages cultured in the presence and absence of exogenous interferon-gamma (IFN-gamma) and in bovine trophoblasts supplemented with erythritol. In severely iron-depleted conditions, DHBA was also found to be essential for growth in murine macrophages. Despite these deficiencies, the absence of DHBA had no long-term significant effect on the number of CFU recovered in vivo from either the Brucella-resistant C57BL/6 mice or Brucella-susceptible IFN-gamma knock-out C57BL/6 mice.     

Mycobacterium tuberculosis-induced gamma interferon production by natural killer cells requires cross talk with antigen-presenting cells involving Toll-like receptors 2 and 4 and the mannose receptor in tuberculous pleurisy

Tuberculous pleurisy allows the study of human cells at the site of active Mycobacterium tuberculosis infection. In this study, we found that among pleural fluid (PF) lymphocytes, natural killer (NK) cells are a major source of early gamma interferon (IFN-gamma) upon M. tuberculosis stimulation, leading us to investigate the mechanisms and molecules involved in this process. We show that the whole bacterium is the best inducer of IFN-gamma, although a high-molecular-weight fraction of culture filtrate proteins from M. tuberculosis H37Rv and the whole-cell lysate also induce its expression. The mannose receptor seems to mediate the inhibitory effect of mannosylated lipoarabinomannan, and Toll-like receptor 2 and 4 agonists activate NK cells but do not induce IFN-gamma like M. tuberculosis does. Antigen-presenting cells (APC) and NK cells bind M. tuberculosis, and although interleukin-12 is required, it is not sufficient to induce IFN-gamma expression, indicating that NK cell-APC contact takes place. Indeed, major histocompatibility complex class I, adhesion, and costimulatory molecules as well as NK receptors regulate IFN-gamma induction. The signaling pathway is partially inhibited by dexamethasone and sensitive to Ca2+ flux and cyclosporine. Inhibition of p38 and extracellular-regulated kinase mitogen-activated protein kinase pathways reduces the number of IFN-gamma+ NK cells. Phosphorylated p38 (p-p38) is detected in ex vivo PF-NK cells, and M. tuberculosis triggers p-p38 in PF-NK cells at the same time that binding between NK and M. tuberculosis reaches its maximum value. Thus, interplay between M. tuberculosis and NK cells/APC triggering IFN-gamma would be expected to play a beneficial role in tuberculous pleurisy by helping to maintain a type 1 profile.     

活动性结核患者单核来源巨噬细胞C-C基序配体5的表达研究

目的 研究活动性结核患者单核来源巨噬细胞(MDM)趋化因子C-C基序配体5(CCL5)的表达水平.方法 收集309医院的活动性肺结核患者和健康人抗凝血,分离纯化单核细胞并体外培养使其分化为初始型(M0)巨噬细胞.然后分别用细菌脂多糖(LPS)/γ-干扰素(IFN-y)和白细胞介素4刺激24h,使其向促炎症型(M1)巨噬细胞和抗炎症(M2)型巨噬细胞极化,收集细胞并提取总RNA,荧光定量PCR检测CCL5 mRNA的表达.结果 活动性结核患者M0、M1和M2型MDM中CCL5的相对表达量分别为(0.023 ±0.012)、(0.675±0.337)和(0.037 ±0.031),健康人M0、M1和M2型MDM中CCL5的相对表达量分别为(0.051 ±0.026)、(0.727±0.376)和(0.068 ±0.045).与健康人相比,活动性结核患者M0和M2型MDM中CCL5的表达显著降低(U=52.5,P＜0.001;t=2.336,P＜0.05),而M1型MDM中CCL5的表达没有显著变化(t=0.4307,P＞0.05).结论 活动性结核患者MDM细胞中CCL5的表达降低,提示巨噬细胞CCL5参与结核病的感染免疫.