Mycobacterial sulfolipid shows a virulence by inhibiting cord factor induced granuloma formation and TNF-alpha release

Virulence mechanism of infection with Mycobacterium tuberculosis is currently focused to be clarified in the context of cell surface lipid molecule. Comparing two mycobacterial glycolipids, we observed toxicity and prominent granulomatogenic activity of trehalose 6,6'-dimycolate (TDM) injection in mice, evident by delayed body weight gain and histological observations, whereas 2,3,6,6'-tetraacyl trehalose 2'-sulfate (SL) was non-toxic and non-granulomatogenic. Likewise, TDM but not SL caused temporarily, but marked increase of lung indices, indicative of massive granuloma formation. Interestingly, co-administration of TDM and SL prevented these symptoms distinctively and SL inhibited TDM-induced release of tumor necrosis factor alpha (TNF-alpha) in a dose-dependent manner. Histological findings and organ index changes also showed marked inhibition of TDM induced granuloma formation by co-administration of SL. Simultaneous injection of SL together with TDM was highly effective for this protection, as neither injection 1h before nor after TDM injection showed highly inhibitory. In parallel studies on a cellular level, TDM elicited strong TNF-alpha release from alveolar but not from peritoneal macrophages in vitro. This effect was blocked when alveolar macrophages were incubated in wells simultaneously coated with TDM and SL, indicating that SL suppresses TDM-induced TNF-alpha release from macrophages. Our results suggest a novel mechanism by which SL could contribute to virulence at early stage of mycobacterial infection or stimulation with the glycolipids by counteracting the immunopotentiating effect of TDM.     

Cytokine message and protein expression during lung granuloma formation and resolution induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Trehalose-6,6'-dimycolate (TDM), or cord factor, is a mycobacterial cell wall component that induces granuloma formation and proinflammatory cytokine production in vivo and in vitro. The purpose of this work was to better understand the mechanisms by which TDM promotes lung granuloma formation. This was accomplished by characterizing cytokine mRNA expression during TDM-induced alveolitis culminating in cohesive granuloma development. A single intravenous injection of TDM given to C57BL/6 mice produced lung granulomas that peaked in number 5 days after challenge and were nearly resolved by 14 days. mRNA in whole lung preparations was quantitated by bioluminescent RT-PCR. Tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 were significantly elevated during granuloma development and decreased during granuloma resolution. There were no detectable changes in mRNA for interferon-y (IFN-y), IL-2, IL-4, IL-5, IL-10, and IL-12(p40). The level of TNF-alpha protein extracted from lung minces highly correlated with morphologic indices of granulomatous inflammation, indicating that it may be an important modulator of the inflammatory intensity induced by TDM. TDM may interact specifically with macrophages in vivo, as evidenced by induction of TNF-alpha, IL-1beta, and IL-6, but not IFN-gamma, protein in bone marrow-derived macrophages from C57BL/6 mice. TDM may therefore play an important role early in macrophage activation during the host granulomatous response to mycobacteria.     

Pulmonary TCR γδ T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6′-dimycolate (TDM) isolated from Mycobacterium tuberculosis

We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6′-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.     

Pulmonary TCR γδ T cells induce the early inflammation of granuloma formation by a glycolipid trehalose 6,6'-dimycolate (TDM) isolated from Mycobacterium tuberculosis

We previously showed that formation of pulmonary granulomas in mice in response to a mycobacterial glycolipid, trehalose 6,6'-dimycolate (TDM) is due to the action of TNF-α and not of IFN-γ. However, the mechanisms of formation and maintenance of pulmonary granulomas are not yet clear. The purpose of the present study is to evaluate the mechanisms of granuloma formation by TDM at the early phase. Histological analysis showed that inflammatory cells infiltrated the murine pulmonary interstitium on day 2 after an intravenous injection with TDM as a w/o/w emulsion. Clear granuloma formation was observed on day 7 after the injection. The mRNA expression of IL-17, IFN-γ and macrophage inflammatory protein 2 was found in lung mononuclear cells at the day after TDM injection. The major IL-17-producing cells were T-cell receptor (TCR) γδ T cells expressing Vγ6. In mice depleted of γδ T cells by treatment with anti-TCR γδ monoclonal antibody, the number of TDM-induced granuloma was decreased, but the size of granuloma was not affected. Our results suggest that the mycobacterial glycolipid TDM causes activation of IL-17-producing TCR γδ T cells and stimulates chemotaxis of inflammatory cells including neutrophils in to lung.     

Augmentation of pulmonary foreign body granulomatous inflammation in mice by lipopolysaccharide: involvement of macrophage activation and tumor necrosis factor-alpha.

We examined the effect of lipopolysaccharide (LPS) on dextran bead-induced lung granulomas using LPS responder (C3H/HeN) and nonresponder (C3H/HeJ) mice. LPS augmented granuloma sizes, TNF-alpha and N-acetyl-beta-D-glucosaminidase levels of lung extracts in C3H/HeN, but not in C3H/HeJ mice. Granuloma macrophages of C3H/HeN mice produced higher levels of superoxide anion and TNF-alpha than those of C3H/HeJ mice. Taken together with our previous report that macrophages and macrophage-derived cytokines are essential for the development of lesions, the present results suggest that activation of macrophages plays an important role in the development and augmentation of pulmonary granulomas.     

Requisite role for complement C5 and the C5a receptor in granulomatous response to mycobacterial glycolipid trehalose 6,6'-dimycolate

The development of pulmonary granulomatous lesions during mycobacterial infection is a complex phenomenon, in part caused by responses elicited towards the surface glycolipid trehalose 6,6'-dimycolate (TDM; cord factor). The molecular mechanisms underlying granuloma formation following challenge with TDM are not yet completely understood. The present study defines pathologic differences in acute response to Mycobacterium tuberculosis TDM in C57BL/6 mice and mice lacking the C5a receptor (C5aR-/-). Mice were intravenously injected with TDM prepared in water-in-oil-in-water emulsion and examined for histologic response and changes in proinflammatory cytokines and chemokines in lung tissue. Control C5a receptor-sufficient mice demonstrated a granulomatous response that peaked between days 4 and 7. Increased production of macrophage inflammatory protein-1 alpha (MIP-1alpha), interleukin-1beta (IL-1beta) and CXC chemokine KC (CXCL1) correlated with development of granulomas, along with modest change in tumor necrosis factor-alpha (TNF-alpha). In contrast, the C5aR-/- mice revealed markedly exacerbated inflammatory response. The receptor-deficient mice also demonstrated a lack of coherent granulomatous response, with severe oedema present and instances of lymphocytic cuffing around pulmonary vessels. Lung weight index was increased in the C5aR-/- mice, correlating with increased MIP-1alpha, KC, IL-1beta and TNF-alpha over that identified in the congenic C5aR-sufficient controls. Correlate experiments performed in C5-deficient (B10.D2-H2d H2-T18c Hco/oSnJ) mice revealed similar results, leading to the conclusion that C5 plays a significant role in mediation of chemotactic and activation events that are the basis for maturation of granulomatous responses to TDM.     

Recombinant guinea pig tumor necrosis factor alpha stimulates the expression of interleukin-12 and the inhibition of Mycobacterium tuberculosis growth in macrophages

Tumor necrosis factor alpha (TNF-alpha) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis. It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-alpha (rgpTNF-alpha) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-alpha antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis. The mRNA levels for TNF-alpha and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-alpha alone. In infected macrophages, a lower dose of rgpTNF-alpha intensified the mRNA levels of TNF-alpha and IL-12 p40. However, higher doses of rgpTNF-alpha suppressed TNF-alpha and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [3H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-alpha antibody to block endogenous TNF-alpha exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-alpha is crucial to the stimulation of optimal expression of protective cytokines and that TNF-alpha contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis.     

Different Toll-like receptor agonists induce distinct macrophage responses

We previously reported that gram-negative bacterial lipopolysaccharide (LPS) activates cells via Toll-like receptor (TLR) 4, whereas the mycobacterial cell wall glycolipid lipoarabinomannan (LAM) activates cells via TLR2. We also identified a secreted TLR2 agonist activity in short-term culture filtrates of Mycobacterium tuberculosis bacilli, termed soluble tuberculosis factor (STF). Here we show that STF contains mannosylated phosphatidylinositol (PIM) and that purified PIM possesses TLR2 agonist activity. Stimulation of RAW 264.7 macrophages by LPS, LAM, STF, and PIM rapidly activated nuclear factor (NF)-kappaB, activator protein-1 (AP-1), and mitogen-activated protein (MAP) kinases. These TLR agonists induced similar levels of NF-kappaB and AP-1 DNA-binding activity, as well as trans-activation function. Unexpectedly, these TLR agonists induced tumor necrosis factor alpha secretion, whereas only LPS was capable of inducing interleukin-1beta and nitric oxide secretion. Thus, different TLR proteins are still capable of activating distinct cellular responses, in spite of their shared capacities to activate NF-kappaB, AP-1, and MAP kinases.     

Effect of lipoarabinomannan and mycobacteria on tumour necrosis factor production by different populations of murine macrophages.

Tumour necrosis factor (TNF) production is an important pathological mediator in mycobacterial infections, and yet little is known of the factors which influence its production. We have studied the influence of murine macrophage heterogeneity and activation state on TNF production following mycobacterial stimulation in vitro. Lipoarabinomannan (LAM) from strains of Mycobacterium tuberculosis and Myco. avium differentially stimulated TNF production in thioglycollate-elicited macrophages in a dose-dependent manner. In comparison, resident peritoneal macrophages produced much less TNF when stimulated with LAM, dead mycobacteria or lipopolysaccharide (LPS). In contrast, zymosan stimulated resident macrophages to a higher degree than thioglycollate-elicited cells. Another comparison between bone marrow and thioglycollate-elicited macrophages showed that both responded to LPS, but only the latter was stimulated significantly by H37Rv LAM. This may indicate that LAM stimulation of macrophages takes place through a different pathway than both zymosan- and LPS-stimulated TNF production. Also, in vitro activation of peritoneal macrophages with interferon-gamma (IFN-gamma), increased TNF response to several stimuli. Our studies indicate that the pathology of mycobacterial infections through TNF production may be influenced by the type and activation state of the macrophage which responds to that infection.     

Macrophage scavenger receptor down-regulates mycobacterial cord factor-induced proinflammatory cytokine production by alveolar and hepatic macrophages

We aimed to reveal the regulatory function of macrophage scavenger receptor-A (MSR-A) in proinflammatory cytokine production by macrophages stimulated with mycobacterial cord factor (CF). By the culture with CF, MSR-A (+/+) alveolar macrophages and Kupffer cells produced TNF-alpha/MIP-1alpha in a time- and dose-dependent manner. However, the amounts of cytokines produced by them were much less compared to those produced by MSR-A (-/-) macrophages. Consistent with this, treatment of MSR-A (+/+) macrophages with anti-MSR-A antibody increased TNF-alpha production. Binding of CF to MSR-A was demonstrated by measuring the binding affinity. These results indicate that CF binds MSR-A, and MSR-A down-regulates TNF-alpha/MIP-1alpha production by activated macrophages, suggesting the role of this receptor in suppression of excessive inflammatory responses during mycobacterial infection.     

Low response of BALB/c macrophages to priming and activating signals.

Trehalose dimycolate (TDM), a mycobacterial glycolipid, is a powerful macrophage-priming agent. However, its efficiency seems limited in the case of BALB/c mice. Peritoneal macrophages harvested from TDM-treated BALB/c mice did not control BCG growth in vitro as efficiently as similar macrophages from two other mouse strains, (B6 x D2)F1 and C57BL/6, which are respectively Bcgr and Bcgs. BALB/c macrophages elicited by TDM also exhibited a low capacity to produce hydrogen peroxide and, after activation by lipopolysaccharide (LPS), weak cytostatic activity against P815 mastocytoma cells. Finally, alkaline phosphodiesterase, a marker of resident and inflammatory macrophages, was still expressed at a high level in macrophages of BALB/c mice treated with TDM. Low responsiveness of BALB/c macrophages to stimuli was not observed with TDM only; activation for tumor cytotoxicity of thioglycolate-elicited macrophages from BALB/c mice required also higher doses of interferon-gamma, and LPS. L-Arginine-dependent production of nitric oxide was inducible in macrophages from BALB/c mice, but the conditions required for its induction were more stringent. Thus, the reduced antiproliferative effects of BALB/c macrophages may be due to uncomplete induction of NO synthase after suboptimal stimulation.     

Dysregulated response to mycobacterial cord factor trehalose-6,6'-dimycolate in CD1D-/- mice.

The biologic effects of the mycobacterial glycolipid trehalose-6,6'-dimycolate (TDM) include granuloma formation and macrophage activation and are dependent on physical conformation. In mice, the group II CD1 surface molecule CD1d has been implicated in glycolipid presentation. The importance of CD1d interactions in pathology has yet to be established. We hypothesized that mice lacking CD1d (CD1D(-/-)) would demonstrate dysregulated granulomatous response to TDM, compared with CD1D(+/-) heterozygous controls. Mice were intravenously injected with TDM-coated polystyrene-divinylbenzene beads and examined for histologic response and for changes in inflammatory cytokine and chemokine mRNA. Control CD1D heterozygous mice demonstrated a granulomatous response, which peaked at day 5. Increased mRNA for tumor necrosis factor-alpha (TNF-alpha) and macrophage inflammatory protein-1alpha (MIP-1alpha) correlated with development of granulomas, with very little change in interleukin-1beta (IL-1beta) and monocyte chemoattractant protein-1 (MCP-1). In contrast, the CD1D(-/-) mice revealed markedly different responses. Five days after administration, severe pulmonary hemorrhage was induced. The relative size of inflammation surrounding coated bead in the CD1D(-/-) mice was nearly double that induced in the CD1D(+/-) mice. CD1D(-/-) mice also demonstrated elevated mRNA for both inflammatory cytokines and chemokines by day 1 after administration, significantly earlier than responses seen in the heterozygous controls.     

Role of tumor necrosis factor-alpha in Mycobacterium-induced granuloma formation in tumor necrosis factor-alpha-deficient mice

To study the role of TNF-alpha in mycobacterial infection, we generated TNF-alpha-knockout (KO) mice, in which the third and fourth exons of the TNF-alpha gene were disrupted. The C57BL/6 KO mice were injected with virulent Mycobacterium tuberculosis strain Kurono or avirulent bacillus Calmette-Guérin (BCG) Pasteur (10(6) colony-forming units), through the tail veins. The major organs were removed at weekly intervals, and morphologic observation, assays of IL-1, IL-12, IFN-gamma, and inducible nitric oxide synthase mRNA expression, and colony counts in the lungs and spleen were performed. Peritoneal macrophages from BCG- and H37Rv strain-treated mice produced significant levels of nitric oxide after stimulation in vitro. Formation of abscesses was seen only in the Kurono-treated groups, and these abscesses contained large numbers of mycobacteria. The administration of recombinant TNF-alpha significantly ameliorated the mycobacterial lesions. IFN-gamma mRNA was expressed significantly in virulent H37Rv-treated groups with time, and the number of mycobacterial colonies per unit weight increased remarkably with time. Nitric oxide production was not observed in H37Rv-treated groups but was seen in BCG-treated groups. We concluded that TNF-alpha played an important role in protective immunity against virulent mycobacteria. Because avirulent mycobacteria did not induce granulomas in TNF-alpha-KO mice, TNF-alpha played an indirect role in granuloma formation.     

Role of tumor necrosis factor-alpha in the spontaneous development of pulmonary fibrosis in viable motheaten mutant mice.

The viable motheaten mutant mouse is severely immunodeficient and dies from a naturally occurring progressive pulmonary inflammation at approximately 10 weeks of age. The pulmonary disease is characterized by excessive macrophage accumulation in the lung and fibrosis. We correlated the development of lung injury in viable motheaten mice with tumor necrosis factor-alpha (TNF-alpha) levels in serum and lung. Significantly increased serum TNF-alpha levels were observed by enzyme-linked immunosorbent assay in viable motheaten mice at 4, 6, and 10 weeks of age as compared with normal control littermate mice. These ages correlated well with observed changes in lung wet weights, lung collagen content, and histological evidence of pulmonary inflammation and fibrosis. Qualitative assessment of lung tissue TNF-alpha levels was performed by immunohistochemical staining using immunoperoxidase techniques. These studies revealed increased levels of TNF-alpha in macrophage-like cells in viable motheaten mice from 5 to 10 weeks of age as compared with littermate control animals. Alveolar macrophages isolated from viable motheaten mice produced significantly greater amounts of TNF-alpha when stimulated with lipopolysaccharide compared with alveolar macrophages from control animals. In addition, administration of anti-TNF-alpha antibody to motheaten bone marrow recipient mice decreased the severity of acute lung injury. The results demonstrate a close correlation between the development of pulmonary injury and TNF-alpha levels in this model of spontaneously developing fibrotic lung disease.     

T-cell-independent granuloma formation in response to Mycobacterium avium: role of tumour necrosis factor-alpha and interferon-gamma.

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.     

Different types of pulmonary granuloma necrosis in immunocompetent vs. TNFRp55-gene-deficient mice aerogenically infected with highly virulent Mycobacterium avium

The immunopathogenesis of mycobacterial infections frequently involves the formation of caseating granulomas which cause tissue destruction and, in the case of tuberculosis (TB), may lead to cavity formation. Both intravenous and aerosol models of Mycobacterium tuberculosis infection in mice do not reflect the pulmonary lesions characteristic of TB patients. Using both low-dose (10² colony-forming units, cfu) and high-dose (10⁵ cfu) aerosol infection with a highly virulent strain of Mycobacterium avium (TMC724) in C57BL/6 mice, it is now shown that these mice are capable of developing centrally caseating necrosis in lung granulomas after approximately 4 months of infection. In contrast, mice infected intravenously with the high dose never developed this type of lesion, although bacterial counts in their lungs reached levels comparable to those attained by aerosol-infected mice (10¹⁰ cfu). To study the relevance of events signalled by tumour necrosis factor (TNF) in this model, TNFRp55 gene-deficient and syngeneic C57BL/6 immunocompetent mice were infected with 10⁵ cfu M. avium via aerosol. In gene-deficient mice, newly formed pulmonary granulomas acutely disintegrated, showing signs of apoptotic cell death and neutrophil influx, and TNFRp55 knock-out mice all succumbed to infection just beyond the stage of granuloma initiation. Aerogenic infection with M. avium in mice is a suitable model to study the immunopathogenesis of granuloma necrosis because it closely mimicks the histopathology of mycobacterial infections in humans, including TB. Furthermore, the use of TNFRp55 gene-deficient mice in this model establishes a role for TNF in maintaining the integrity of a developing pulmonary granuloma. Copyright © 1999 John Wiley & Sons, Ltd.     

In vivo induction of apoptosis in the thymus by administration of mycobacterial cord factor (trehalose 6,6'-dimycolate).

It is reported that some bacteria or bacterial components cause thymic atrophy via the apoptotic process. The present study demonstrated for the first time in vivo induction of apoptosis in the mouse thymus by mycobacterial cord factor (CF) (trehalose 6,6'-dimycolate). When 300 microg of purified CF from Mycobacterium tuberculosis was intravenously administered to BALB/c mice in the form of water-in-oil-in-water (w/o/w) emulsion, thymic atrophy and pulmonary granulomas were induced with a peak on day 7, whereas, in the form of liposomes, CF induced thymic atrophy on days 14 to 21 in parallel with the development of hepatic granulomas. Thymic atrophy resulted from the depletion of cortical lymphocytes via apoptosis as revealed by DNA fragmentation and karyorrhectic changes. In contrast, mycobacterial sulfatide (2,3,6,6'-tetraacyl trehalose 2'-sulfate) caused neither thymic atrophy nor granuloma formation. Compared to lipopolysaccharide-induced thymocyte apoptosis, CF (w/o/w)-induced thymocyte apoptosis developed more slowly, reached a maximum later, and lasted longer but was less intense. Although serum tumor necrosis factor alpha (TNF-alpha) levels in CF-treated mice were not significantly elevated, administration of anti-TNF-alpha antibody almost completely inhibited thymic atrophy and granuloma formation. Serum corticosterone levels were only slightly elevated by CF administration. The present results indicate that mycobacterial CF induces thymic atrophy via apoptosis, which is closely linked with granuloma formation.     

Tumor necrosis factor signaling mediates resistance to mycobacteria by inhibiting bacterial growth and macrophage death

Tumor necrosis factor (TNF), a key effector in controlling tuberculosis, is thought to exert protection by directing formation of granulomas, organized aggregates of macrophages and other immune cells. Loss of TNF signaling causes progression of tuberculosis in humans, and the increased mortality of Mycobacterium tuberculosis-infected mice is associated with disorganized necrotic granulomas, although the precise roles of TNF signaling preceding this endpoint remain undefined. We monitored transparent Mycobacterium marinum-infected zebrafish live to conduct a stepwise dissection of how TNF signaling operates in mycobacterial pathogenesis. We found that loss of TNF signaling caused increased mortality even when only innate immunity was operant. In the absence of TNF, intracellular bacterial growth and granuloma formation were accelerated and was followed by necrotic death of overladen macrophages and granuloma breakdown. Thus, TNF is not required for tuberculous granuloma formation, but maintains granuloma integrity indirectly by restricting mycobacterial growth within macrophages and preventing their necrosis.     

Monocyte chemoattractant protein 1 in a rat model of pulmonary granulomatosis.

Monocyte chemoattractant protein 1 (MCP1), also known as monocyte chemotactic and activating factor, possesses potent chemotactic activity for monocytes and can augment monocyte tumoristatic activity against some tumor cell lines. While these activities suggest a role in inflammatory and immunologic processes, the biologic role of MCP1 has not been studied in vivo. Glucan-induced pulmonary granulomatosis in the rat is an ideal model in which to study the role of MCP1 because the granulomas are monocyte/macrophage rich. Intravenous infusion of particulate yeast cell wall glucan resulted in the synchronous development of angiocentric pulmonary granulomas. Early lesions (6 hours) were characterized by intravascular glucan aggregates surrounded by neutrophils and foci of alveolar hemorrhage while later appearing granulomatous lesions (48 to 96 hours) were dominated by monocytes and macrophages. Granuloma formation was paralleled by a peripheral blood monocytosis. Analysis of bronchoalveolar lavage (BAL) fluid revealed an early, transient rise in tumor necrosis factor activity followed by a marked rise in monocyte-specific chemotactic activity. The rise in BAL fluid monocyte chemotactic activity, which coincided with the development of the monocyte/macrophage-rich granulomas, was preceded by a marked increase in whole lung MCP1 mRNA expression. BAL fluid monocyte chemotactic activity could be nearly completely neutralized with antibody directed against rat MCP1. These studies demonstrate that MCP1 mRNA expression is upregulated in glucan-induced pulmonary granulomatosis and that MCP1 is present in BAL fluid. Intrapulmonary granulomatosis may be important in the pathogenesis of granuloma formation.