Mycobacteria are the etiologic agents of numerous diseases which account for significant morbidity and mortality in humans and other animal species. Many mycobacteria are intramacrophage pathogens and therefore the macrophage response to infection, which includes synthesis of cytokines such as tumor necrosis factor alpha (TNF-␣) and production of nitric oxide, has important consequences for host immunity. However, very little is known about the macrophage cell signaling pathways initiated upon infection or how pathogenic mycobacteria may modulate the macrophage responses. Using primary murine bone marrow macrophages, we established that p38 and extracellular signal-regulated kinases 1 and 2 of the mitogen-activated protein kinase (MAPK) pathways are activated upon infection with different species of mycobacteria. However, we observed decreased MAPK activity over time in macrophages infected with pathogenic Mycobacterium avium strains relative to infections with nonpathogenic mycobacteria. Furthermore, macrophages infected with M. avium produced lower levels of TNF-␣, interleukin 1␤, and inducible nitric oxide synthase 2 than macrophages infected with nonpathogenic species. Inhibitor studies indicate that the MAPKs are required for the Mycobacterium-mediated induction of these effector proteins. Our data indicate that MAPKs are activated in macrophages upon invasion by mycobacteria and that this activation is diminished in macrophages infected with pathogenic strains of M. avium, resulting in decreased production of important immune effector proteins. The decreased MAPK activation associated with M. avium infections suggests a novel point of immune intervention by this mycobacterial species.Mycobacterium spp. are intramacrophage pathogens and therefore the engagement of macrophage receptors by mycobacteria is one of the initial steps in the infection process. A macrophage may respond to a mycobacterial infection by secreting cytokines such as tumor necrosis factor alpha (TNF-␣) or interleukin 1␤ (IL-1␤) and by producing reactive oxygen and nitrogen intermediates. These effects, among others, are the end result of activating various macrophage-signaling pathways. However, questions remain regarding which pathways are initiated and/or modulated by a mycobacterial infection. Previous studies have shown that mycobacterial components such as lipoarabinomannan (LAM) can stimulate a macrophage response, resulting in the production and secretion of TNF-␣ and IL-1␤ (1). Studies have also indicated that arabinofuranosyl-terminated LAM isolated from nonpathogenic mycobacteria stimulates a stronger cytokine response in treated macrophages than does mannose-capped LAM (ManLAM) from pathogenic mycobacteria (50, 52). Furthermore, recent work by Ting et al. indicates that human macrophages infected with Mycobacterium tuberculosis are less responsive to gamma interferon, due to a disruption in STAT1 binding to the transcription factor CREB (55). These experiments suggest that mycobacteria and mycobacterial components can modulate...

Previous studies have shown the mitogen-activated protein kinases (MAPKs) to be activated in macrophages upon infection with Mycobacterium, and that expression of TNF-α and inducible NO synthase by infected macrophages was dependent on MAPK activation. Additional analysis demonstrated a diminished activation of p38 and extracellular signal-regulated kinase (ERK)1/2 in macrophages infected with pathogenic strains of Mycobacterium avium compared with infections with the fast-growing, nonpathogenic Mycobacterium smegmatis and Mycobacterium phlei. However, the upstream signals required for MAPK activation and the mechanisms behind the differential activation of the MAPKs have not been defined. In this study, using bone marrow-derived macrophages from BALB/c mice, we determined that ERK1/2 activation was dependent on the calcium/calmodulin/calmodulin kinase II pathway in both M. smegmatis- and M. avium-infected macrophages. However, in macrophages infected with M. smegmatis but not M. avium, we observed a marked increase in cAMP production that remained elevated for 8 h postinfection. This M. smegmatis-induced cAMP production was also dependent on the calmodulin/calmodulin kinase pathway. Furthermore, stimulation of the cAMP/protein kinase A pathway in M. smegmatis-infected cells was required for the prolonged ERK1/2 activation and the increased TNF-α production observed in these infected macrophages. Our studies are the first to demonstrate an important role for the calmodulin/calmodulin kinase and cAMP/protein kinase A pathways in macrophage signaling upon mycobacterial infection and to show how cAMP production can facilitate macrophage activation and subsequent cytokine production.

Previous studies in our laboratory have shown a differential activation of the mitogen-activated protein kinases (MAPKs) in primary bone marrow-derived macrophages following infection with pathogenic Mycobacterium avium compared to the activation following infection with nonpathogenic Mycobacterium smegmatis. Additionally, M. smegmatis-infected macrophages produced significantly elevated levels of tumor necrosis factor alpha (TNF-␣) compared to the levels produced by M. avium-infected macrophages. The TNF-␣ production was dependent on both p38 and extracellular signal-regulated kinase 1/2 (ERK 1/2) activation. However, the macrophage transcription factors downstream of the MAPKs, which were required for TNF-␣ production, remained undefined. In this study we determined that the transcription factor cyclic AMP response element binding protein (CREB) is significantly more activated in M. smegmatis-infected macrophages than in M. avium-infected macrophages. We also found that CREB activation was dependent on p38 and protein kinase A but not on ERK 1/2 or calmodulin kinase II. Moreover, mutating the cAMP-responsive element on the TNF-␣ promoter resulted in significantly diminished promoter activity following M. smegmatis infection but not M. avium infection. The inability of macrophages infected with M. avium to sustain MAPK activation and to produce high levels of TNF-␣ was due, in part, to an increase in serine/threonine phosphatase PP2A activity. Our studies are the first to demonstrate an important role for the transcription factor CREB in TNF-␣ production by mycobacterium-infected macrophages, as well as a role for M. avium's induction of PP2A phosphatase activity as a mechanism to limit macrophage activation.Mycobacterium avium is an opportunistic pathogen that affects people with suppressed immune systems, particularly people with late-stage human immunodeficiency virus or chronic lung diseases. In AIDS patients, M. avium is commonly disseminated and can involve almost any internal organ, especially the liver, spleen, and bone marrow. Moreover, M. avium is responsible for increased morbidity and mortality in human immunodeficiency virus-infected individuals (6).M. avium is a facultative intracellular pathogen that resides within the phagosome of the host macrophage. The macrophage is the first line of defense against invading microorganisms, and it functions to phagocytose and subsequently destroy these invaders within a phagolysosome. However, following phagocytosis, M. avium, like other species in the genus Mycobacterium, including Mycobacterium tuberculosis and Mycobacterium leprae, has been shown to halt the maturation of the phagosome through coordinated blocking of lysosome fusion with the phagosome. Thus, the M. avium-containing phagosome fails to acquire lysosomal lytic enzymes, nor does it acidify, due to a scarcity of the proton ATPases (25). However, macrophages have another inherent defense mechanism against mycobacterial diseases. Macrophages are stimulated to secrete a large panel of inflammatory...

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The combination of manual cleaning and ETO sterilization consistently sterilized APC probes. Ninety percent of the probes showed no sign of physical deterioration and 100% maintained their electrical activity after 10 uses. APC probes can potentially be safely and effectively reused up to 10 times, and a significant procedural savings is possible with reuse.