Synthesis and processing of cathepsin L, an elastase, by human alveolar macrophages.
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ABSTRACT:
SUBMITTER: Reilly JJ
PROVIDER: S-EPMC1135606 | biostudies-other | 1989 Jan
REPOSITORIES: biostudies-other
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Project description:Macrophages are aptly positioned to function as the primary line of defence against invading pathogens in many organs, including the lung and peritoneum. Their ability to phagocytose and clear microorganisms has been well documented. Macrophages possess several substances with which they can kill bacteria, including reactive oxygen species, nitric oxide, and antimicrobial proteins. We proposed that macrophage-derived proteinases may contribute to the antimicrobial properties of macrophages. Macrophage elastase (also known as matrix metalloproteinase 12 or MMP12) is an enzyme predominantly expressed in mature tissue macrophages and is implicated in several disease processes, including emphysema. Physiological functions for MMP12 have not been described. Here we show that Mmp12(-/-) mice exhibit impaired bacterial clearance and increased mortality when challenged with both gram-negative and gram-positive bacteria at macrophage-rich portals of entry, such as the peritoneum and lung. Intracellular stores of MMP12 are mobilized to macrophage phagolysosomes after the ingestion of bacterial pathogens. Once inside phagolysosomes, MMP12 adheres to bacterial cell walls where it disrupts cellular membranes resulting in bacterial death. The antimicrobial properties of MMP12 do not reside within its catalytic domain, but rather within the carboxy-terminal domain. This domain contains a unique four amino acid sequence on an exposed beta loop of the protein that is required for the observed antimicrobial activity. The present study represents, to our knowledge, the first report of direct antimicrobial activity by a matrix metallopeptidase, and describes a new antimicrobial peptide that is sequentially and structurally unique in nature.
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Project description:The etiologic agent of inhalational anthrax, Bacillus anthracis, produces virulence toxins that are important in the disease pathogenesis. Current studies suggest that mouse and human macrophages are susceptible to immunosuppressive effects of one of the virulence toxins, lethal toxin (LT). Thus a paradigm has emerged that holds that the alveolar macrophage (AM) does not play a significant role in the innate immune response to B. anthracis or defend against the pathogen as it is disabled by LT. This is inconsistent with animal models and autopsy studies that show minimal disease at the alveolar surface. We examined whether AM are immunosuppressed by LT. We found that human AM were relatively resistant to LT-mediated innate immune cytokine suppression, MEK cleavage, and induction of apoptosis as compared with mouse RAW 264.7 macrophages. Mouse AM and murine bone marrow-derived macrophages were also relatively resistant to LT-mediated apoptosis despite intermediate sensitivity to MEK cleavage. The binding component of LT, protective Ag, does not attach to human AM, although it did bind to mouse AM, murine bone marrow-derived macrophages, and RAW 264.7 macrophages. Human AM do not produce significant amounts of the protective Ag receptor anthrax toxin receptor 1 (TEM8/ANTXR1) and anthrax toxin receptor 2 (CMG2/ANTXR2). Thus, mature and differentiated AM are relatively resistant to the effects of LT as compared with mouse RAW 264.7 macrophages. AM resistance to LT may enhance clearance of the pathogen from the alveolar surface and explain why this surface is relatively free of B. anthracis in animal models and autopsy studies.
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Project description:The intracellular bacterial pathogen Coxiella burnetii is a category B select agent that causes human Q fever. In vivo, C.?burnetii targets alveolar macrophages wherein the pathogen replicates in a lysosome-like parasitophorous vacuole (PV). In vitro, C.?burnetii infects a variety of cultured cell lines that have collectively been used to model the pathogen's infectious cycle. However, differences in the cellular response to infection have been observed, and virulent C.?burnetii isolate infection of host cells has not been well defined. Because alveolar macrophages are routinely implicated in disease, we established primary human alveolar macrophages (hAMs) as an in vitro model of C.?burnetii-host cell interactions. C.?burnetii pathotypes, including acute disease and endocarditis isolates, replicated in hAMs, albeit with unique PV properties. Each isolate replicated in large, typical PV and small, non-fused vacuoles, and lipid droplets were present in avirulent C.?burnetii?PV. Interestingly, a subset of small vacuoles harboured single organisms undergoing degradation. Prototypical PV formation and bacterial growth in hAMs required a functional type IV secretion system, indicating C.?burnetii secretes effector proteins that control macrophage functions. Avirulent C.?burnetii promoted sustained activation of Akt and Erk1/2 pro-survival kinases and short-termphosphorylation of stress-related p38. Avirulent organisms also triggered a robust, early pro-inflammatory response characterized by increased secretion of TNF-? and IL-6, while virulent isolates elicited substantially reduced secretion of these cytokines. A corresponding increase in pro- and mature IL-1? occurred in hAMs infected with avirulent C.?burnetii, while little accumulation was observed following infection with virulent isolates. Finally, treatment of hAMs with IFN-? controlled intracellular replication, supporting a role for this antibacterial insult in the host response to C.?burnetii. Collectively, the current results demonstrate the hAM model is a human disease-relevant platform for defining novel innate immune responses to C.?burnetii.
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Project description:Alveolar macrophages (AM) are one of the key cell types for initiating inflammatory and immune responses to influenza virus in the lung. However, the genome-wide changes in response to influenza infection in AM have not been defined. We performed gene profiling of human AM in response to H1N1 influenza A virus PR/8 using Affymetrix HG-U133 Plus 2.0 chips and verified the changes at both mRNA and protein levels by real-time RT-PCR and ELISA. We confirmed the response with a contemporary H3N2 influenza virus A/New York/238/2005 (NY/238). To understand the local cellular response, we also evaluated the impact of paracrine factors on virus-induced chemokine and cytokine secretion. In addition, we investigated the changes in the expression of macrophage receptors and uptake of pathogens after PR/8 infection. Although macrophages fail to release a large amount of infectious virus, we observed a robust induction of type I and type III interferons and several cytokines and chemokines following influenza infection. CXCL9, 10, and 11 were the most highly induced chemokines by influenza infection. UV-inactivation abolished virus-induced cytokine and chemokine response, with the exception of CXCL10. The contemporary influenza virus NY/238 infection of AM induced a similar response as PR/8. Inhibition of TNF and/or IL-1? activity significantly decreased the secretion of the proinflammatory chemokines CCL5 and CXCL8 by over 50%. PR/8 infection also significantly decreased mRNA levels of macrophage receptors including C-type lectin domain family 7 member A (CLEC7A), macrophage scavenger receptor 1 (MSR1), and CD36, and reduced uptake of zymosan. In conclusion, influenza infection induced an extensive proinflammatory response in human AM. Targeting local components of innate immune response might provide a strategy for controlling influenza A infection-induced proinflammatory response in vivo.
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Project description:BACKGROUND: Phospholipases C (PLCs) are virulence factors found in several bacteria. In Mycobacterium tuberculosis (Mtb) they exhibit cytotoxic effects on macrophages, but the mechanisms involved in PLC-induced cell death are not fully understood. It has been reported that induction of cell necrosis by virulent Mtb is coordinated by subversion of PGE2, an essential factor in cell membrane protection. RESULTS: Using two Mtb clinical isolates carrying genetic variations in PLC genes, we show that the isolate 97-1505, which bears plcA and plcB genes, is more resistant to alveolar macrophage microbicidal activity than the isolate 97-1200, which has all PLC genes deleted. The isolate 97-1505 also induced higher rates of alveolar macrophage necrosis, and likewise inhibited COX-2 expression and PGE2 production. To address the direct effect of mycobacterial PLC on cell necrosis and PGE2 inhibition, both isolates were treated with PLC inhibitors prior to macrophage infection. Interestingly, inhibition of PLCs affected the ability of the isolate 97-1505 to induce necrosis, leading to cell death rates similar to those induced by the isolate 97-1200. Finally, PGE2 production by Mtb 97-1505-infected macrophages was restored to levels similar to those produced by 97-1200-infected cells. CONCLUSIONS: Mycobacterium tuberculosis bearing PLCs genes induces alveolar macrophage necrosis, which is associated to subversion of PGE2 production.
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