Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Aug;17(8):1085-97.
doi: 10.1111/cmi.12428. Epub 2015 Mar 12.

Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

Joshua T Mattila  1 , Pauline Maiello  1 , Tao Sun  2 , Laura E Via  3 , JoAnne L Flynn  1   2
Affiliations

Granzyme B-expressing neutrophils correlate with bacterial load in granulomas from Mycobacterium tuberculosis-infected cynomolgus macaques

Joshua T Mattila et al. Cell Microbiol. 2015 Aug.

Abstract

The role of neutrophils in tuberculosis (TB), and whether neutrophils express granzyme B (grzB), a pro-apoptotic enzyme associated with cytotoxic T cells, is controversial. We examined neutrophils in peripheral blood (PB) and lung granulomas of Mycobacterium tuberculosis-infected cynomolgus macaques and humans to determine whether mycobacterial products or pro-inflammatory factors induce neutrophil grzB expression. We found large numbers of grzB-expressing neutrophils in macaque and human granulomas and these cells contained more grzB+ granules than T cells. Higher neutrophil, but not T cell, grzB expression correlated with increased bacterial load. Although unstimulated PB neutrophils lacked grzB expression, grzB expression increased upon exposure to M.tuberculosis bacilli, M.tuberculosis culture filtrate protein or lipopolysaccharide from Escherichia coli. Perforin is required for granzyme-mediated cytotoxicity by T cells, but was not observed in PB or granuloma neutrophils. Nonetheless, stimulated PB neutrophils secreted grzB as determined by enzyme-linked immunospot assays. Purified grzB was not bactericidal or bacteriostatic, suggesting secreted neutrophil grzB acts on extracellular targets, potentially enhancing neutrophil migration through extracellular matrix and regulating apoptosis or activation in other cell types. These data indicate mycobacterial products and the pro-inflammatory environment of granulomas up-regulates neutrophil grzB expression and suggests a previously unappreciated aspect of neutrophil biology in TB.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Neutrophils in granulomas can express grzB. Immunohistochemistry was performed on formalin-fixed paraffin-embedded granulomas to assess the localization and expression of grzB, CD3+ T cells, CD8+ lymphocytes and calprotectin-expressing neutrophils. A. GrzB (green) localizes with T cells (red) and neutrophils (blue) in a necrotic (caseous) granuloma. The dashed line bisects the epithelioid macrophage region, highlighting a neutrophil-rich, T–cell-poor region adjacent to the caseum with an abundance of grzB expression. Scale bar represents 100 μm. B. CD3-grzB+ cells in granulomas do not express CD8, suggesting they are not natural killer cells or γδ T cells. Scale bar represents 20 μm. C. GrzB+ calprotectin+ cells have multilobate nuclei indicating they are neutrophils. Scale bar represents 10 μm.
Fig. 2
Fig. 2
Frequency and relative quantification of grzB expression by T cells and neutrophils. A. Granuloma fields imaged at 40× contained significantly more T cells than neutrophils. Each point represents one granuloma, n = 12 monkeys, ***P < 0.0001; Mann–Whitney test, bar represents the median. B. Numbers of neutrophils in granulomas correlate with bacterial load per granuloma, while T-cell numbers do not. Each point represents one granuloma, n = 12 monkeys. C. As a percent of the total population, more neutrophils than T cells express grzB in granulomas. Bars represent the mean with SEM of images from two to four granulomas per animal (monkey ID numbers indicated to the right of the graph). D. On a per cell basis, granuloma neutrophils contain more grzB+ granules per cell than T cells. n = 4627 neutrophils and 10 722 T cells, ***P < 0.0001, Mann–Whitney test, bar represents the median.
Fig. 3
Fig. 3
Neutrophils do not express perforin in granulomas. Images of perforin-stained granulomas were examined for perforin expression. A. Perforin co-localizes with grzB in CD3+ T cells but is not observed in calprotectin-stained neutrophils. B. Quantification of perforin-expressing T cells and neutrophils in images of perforin-stained granulomas.
Fig. 4
Fig. 4
Neutrophils in human granulomas express grzB but not perforin. Human granulomas were examined for neutrophil grzB and perforin expression. A. GrzB (red)-expressing neutrophils (green) preferentially localize at the epithelioid macrophage–caseum interface. Nuclei are indicated in blue. B. GrzB expression. C. Perforin expression.
Fig. 5
Fig. 5
Stimulating neutrophils with bacterial ligands and pro-inflammatory activators up-regulates grzB expression. PBMCs from RBC-depleted whole blood were stimulated with a cocktail of M. tuberculosis ESAT6 and 38.1 peptides, CFP, LPS and P + I and grzB expression was assessed by flow cytometry. A. Representative FACS plots showing CD4+ and CD8+ T cell and neutrophil activation by CFP and P + I relative to media-only controls. B. Quantification of grzB expression by neutrophils. Results from PDBu-stimulated cells are represented on the right axis. Each marker represents an individual monkey with n = 23 monkeys from five independent experiments depicted, **P < 0.005, ***P < 0.0005, Wilcoxon matched-pairs signed-rank test for pairwise comparison.
Fig. 6
Fig. 6
Mycobacterial products and pro-inflammatory stimuli elicit grzB secretion by peripheral blood neutrophils. Paired sets of neutrophil-depleted buffy coat PBMCs and purified neutrophils were subject to stimulation in ELISPOT assay and the number of spot-forming units (SFU) were quantified. A. Representative ELISPOT wells showing the differences between cell fractions and appearance of spots. B. Quantification of grzB expression. Each spot represents the mean of two wells per cell fraction from each monkey. Bars represent the median of results from two independent experiments, four monkeys per experiment. *P < 0.05, Wilcoxon matched-pairs signed-rank test for pairwise comparison between unstimulated and stimulated samples.
Fig. 7
Fig. 7
GrzB expression is up-regulated by M. tuberculosis in an antigen-dependent manner. FACS experiments using whole blood co-cultured with M. tuberculosis, ELISPOT assays comparing buffy coat PBMCS with purified neutrophils and immunohistochemistry on tissues with known bacterial burdens demonstrate that M. tuberculosis can cause up-regulated grzB expression in neutrophils. A. FACS experiments using neutrophils from RBC-free PBMCs. Bars represent mean with SEM. *P < 0.05; Wilcoxon matched-pairs signed-rank test. B. ELISPOT assays indicate M. tuberculosis does not significantly up-regulate grzB expression by buffy coat PBMCs but does significantly up-regulate grzB expression by neutrophils. *P < 0.05; Wilcoxon matched-pairs signed-rank test. C. Bacterial burdens correlate with higher numbers of grzB+ granules per cell for neutrophils (Spearman r = 0.3468, P = 0.0827) but not for CD3+ T cells (Spearman r = −0.1115, P = 0.5878). One monkey (indicated by open markers) had normal numbers of neutrophils and T cells per image, but had neutrophils with very few grzB+ granules per cell, but normal T-cell grzB expression.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Abadie V, Badell E, Douillard P, Ensergueix D, Leenen PJ, Tanguy M, et al. Neutrophils rapidly migrate via lymphatics after Mycobacterium bovis BCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. Blood. 2005;106:1843–1850. - PubMed
    1. Afonina IS, Cullen SP, Martin SJ. Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol Rev. 2010;235:105–116. - PubMed
    1. Afonina IS, Tynan GA, Logue SE, Cullen SP, Bots M, Luthi AU, et al. Granzyme B-dependent proteolysis acts as a switch to enhance the proinflammatory activity of IL-1alpha. Mol Cell. 2011;44:265–278. - PMC - PubMed
    1. Aranday-Cortes E, Hogarth PJ, Kaveh DA, Whelan AO, Villarreal-Ramos B, Lalvani A, Vordermeier HM. Transcriptional profiling of disease-induced host responses in bovine tuberculosis and the identification of potential diagnostic biomarkers. PLoS ONE. 2012;7:e30626. - PMC - PubMed
    1. Asehnoune K, Strassheim D, Mitra S, Yeol Kim J, Abraham E. Involvement of PKCalpha/beta in TLR4 and TLR2 dependent activation of NF-kappaB. Cell Signal. 2005;17:385–394. - PubMed

Publication types