Cytolytic T lymphocytes (CTLs) kill intracellular pathogens by a granule-dependent mechanism. Granulysin, a protein found in granules of CTLs, reduced the viability of a broad spectrum of pathogenic bacteria, fungi, and parasites in vitro. Granulysin directly killed extracellular Mycobacterium tuberculosis, altering the membrane integrity of the bacillus, and, in combination with perforin, decreased the viability of intracellular M. tuberculosis. The ability of CTLs to kill intracellular M. tuberculosis was dependent on the presence of granulysin in cytotoxic granules, defining a mechanism by which T cells directly contribute to immunity against intracellular pathogens.

http://science.sciencemag.org/content/sci/282/5386/121.full.pdf

An Antimicrobial Activity of Cytolytic T Cells Mediated by Granulysin

Antimicrobial peptides: premises and promises.

In analyzing mechanisms of protection against intracellular infections, a series of human CD1-restricted T cell lines of two distinct phenotypes were derived. Both CD4 − CD8 − (double-negative) T cells and CD8 + T cells efficiently lysed macrophages infected with Mycobacterium tuberculosis . The cytotoxicity of CD4 − CD8 − T cells was mediated by Fas-FasL interaction and had no effect on the viability of the mycobacteria. The CD8 + T cells lysed infected macrophages by a Fas-independent, granule-dependent mechanism that resulted in killing of bacteria. These data indicate that two phenotypically distinct subsets of human cytolytic T lymphocytes use different mechanisms to kill infected cells and contribute in different ways to host defense against intracellular infection.

/pdf/differential-effects-of-cytolytic-t-cell-subsets-on-2lzy1vsm9w.pdf

Differential effects of cytolytic T cell subsets on intracellular infection

MTB ranks as the first worldwide pathogen latently infecting one third of the population and the second leading cause of death from a single infectious agent, after the human immunodeficiency virus (HIV). The development of vigorous and apparently appropriate immune response upon infection with M.tuberculosis in humans and experimental animals conflict with failure to eradicate the pathogen itself and with its ability to undergo clinical latency from which it may exit. From a clinical standpoint, our views on MTB infection may take advantage from updating the overall perspective, that has quite changed over the last decade, following remarkable advances in our understanding of the manipulation of the immune system by M.tuberculosis and of the role of innate components of the immune response, including macrophages, neutrophils, dendritic cells and NK cells in the initial spread of MTB and in its exit from latency. Scope of this review is to highlight the the major mechanisms of MTB escape from immune control and to provide a supplementary translational perspective for the interpretation of innate immune mechanisms with particular impact on clinical aspects.

/pdf/immunology-of-tuberculosis-55xecqgi4x.pdf

Immunology of Tuberculosis

We extracted a granule-rich sediment from normal human neutrophils and subjected it to chromatographic, electrophoretic, and functional analysis. The extract contained three small (molecular weight less than 3,500) antibiotic peptides that were named human neutrophil peptide (HNP)-1, HNP-2, and HNP-3, and which will be referred to as "defensins." HNP 1-3, a mixture of the three defensins, killed Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli effectively in vitro when tested in 10 mM phosphate buffer containing certain nutrients, but it had little or no bactericidal activity in nutrient-free buffer. In contrast, the nutrient-free buffer supported a high degree of activity by HNP 1-3 against Cryptococcus neoformans. In addition to its antibacterial and antifungal properties, HNP 1-3 directly inactivated herpes simplex virus, Type 1. Two of the individual purified defensins, HNP-1 and HNP-2, were as microbicidal as the mixture HNP 1-3. HNP-3 was less active than the other defensins against most but not all of the microbes tested. Immunoperoxidase stains revealed HNP 1-3 to have a granular localization in the neutrophil's cytoplasm by light microscopy. Frozen thin section immunogold transmission electron microscopy showed HNP 1-3 to be localized in azurophil granules. These studies define a broad-spectrum antimicrobial system in human neutrophils. The defensin system may operate in conjunction with or independently from oxygen-dependent microbicidal processes to enable human neutrophils to inactivate and destroy potential pathogens.

Defensins: natural peptide antibiotics in human neutrophils

Three independent assay methods were used to investigate the activities of antimicrobial peptides (human and rabbit defensins and protegrin from porcine leukocytes) against Mycobacterium tuberculosis in vitro. M. tuberculosis H37Ra was cultured in the presence of human neutrophil peptide 1, synthetic rabbit neutrophil peptide 1, or porcine protegrin 1 at 37 degrees C for 6 to 48 h, and antimycobacterial activity was measured by CFU assay. These peptides at a concentration of 50 microg/ml showed significant antibacterial effects on M. tuberculosis after 24 and 48 h of incubation (85.9 to 97.5% at 24 h and 91.6 to 99.4% at 48 h). A radiometric method and a radial diffusion assay confirmed these observations. Antibacterial activity against M. tuberculosis was independent of calcium (1.0 mM) or magnesium (1.0 mM) and not inhibited by sodium chloride (100 mM). The optimal pH for antibacterial activity against M. tuberculosis was greater than 4.0. Three clinical isolates of M. tuberculosis were also studied, and these peptides showed 86.3 to 99.0% reduction in CFU of these organisms. Morphological studies using scanning electron microscopy showed that defensins caused lesions on the surface of H37Ra. These observations suggest that antimicrobial peptides such as defensins and protegrins may represent an important component of the host defense mechanism against M. tuberculosis and offer a potential new approach to therapy.

In vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against Mycobacterium tuberculosis.

We have examined the activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare. M. avium-M. intracellulare at 2.5 x 10(6)/ml or 2.5 x 10(8)/ml was cultured in the presence of defensins at 37 degrees C from 4 to 48 h. After incubation, CFU were enumerated. Human neutrophil peptide 1 (HNP-1) at 5 micrograms/ml had the ability to kill M. avium-M. intracellulare. Treatment with HNP-1 resulted in significant (96.3 to 97.7%) killing of M. avium-M. intracellulare, even after taking clumping into consideration. This activity was not affected by the presence of calcium (0.5 and 1.0 mM), magnesium (0.5 and 1.0 mM), or sodium chloride (25, 50, and 100 mM). The optimal pH for bactericidal activity was higher than 5. We tested numerous M. avium-M. intracellulare strains, and HNP-1 was successful in killing every strain, although the degree of killing varied among them (34.2 to 87.2%). Additionally, this activity was independent of colonial morphology. We also examined the activity of HNP-2 and HNP-3 against M. avium-M. intracellulare and found that they were as effective in killing M. avium-M. intracellulare as HNP-1 was. These observations suggest that defensins may play an important role in the host defense against M. avium-M. intracellulare.

https://iai.asm.org/content/iai/60/11/4720.full.pdf

R I Lehrer

Papers

In vitro activity of the antimicrobial peptides human and rabbit defensins and porcine leukocyte protegrin against Mycobacterium tuberculosis.

Activity of defensins from human neutrophilic granulocytes against Mycobacterium avium-Mycobacterium intracellulare.