Ring The Alarm! Granulysin’s Role in Recruiting Dendritic Cells

Our body puts up a tough fight against a constant barrage of invaders. Our first line of defense against invading pathogens is referred to as our “innate defense” and consists of a rapid response by NK, NKT and ϒδ T cells (1). These cells respond much quicker than T and B cells which are activated later if the infection persists. However, this secondary “adaptive response” requires the recruitment of dendritic cells (DCs). DCs are able to activate T and B cells highly specific for that antigen to order to mount a more focused response (1).

A recent study by Tewary et al. 2011 looks specifically at the role NK cells plays in the secondary adaptive immune response, specifically via the release of granulysin.

The GNLY gene produces the antimicrobial protein granulysin, which is initially 15 kDa in size. NK cells secrete this initial form that can be further cleaved into a smaller form, 9-kDa. The 9-kDa form is stored in the granular compartment of NK cells. The contents of the granular compartment contain toxins that cause cell death when released (degranulation) onto their pathogen targets (1) as part of the innate response.

However, a previous study also showed that GNLY participates in the adaptive response by recruiting dendritic cells (DCs), NK cells, and T cells (2), though the mechanism by which it was able to do so was unclear. Molecules that exhibit this recruitment capacity are referred to as alarmins.

In Tewary et al. 2011, the mechanism by which GNLY is able to recruit DCs and T cells and promote an immune response is elucidated.

Researchers were first interested in whether GNLY was an attractant (chemotactic) for human monocyte-derived immature DCs (Mo-iDCs). This was done using a chemotaxis assay which involves use of a microchamber with a lower and upper compartment separated by a filter. The Mo-iDCs were placed in the upper compartment, with concentrations of GNLY in the lower. Afterward, cells are stained and counted in order to quantify migration. Both the 9-kDa and 15-kDa forms were able to induce MO-iDC migration in a dose-dependent manner.

A subsequent experiment was performed to examine whether this migration was mediated through a G-protein coupled receptor (GPCR). GPCRs can mediate their signaling cascade via their alpha sub-unit. Therefore, Mo-iDCs were pretreated with PTx (alters the alpha sub-unit) prior to the assay. Treatment with PTx effectively inhibited migration suggesting that granulysin mediates its recruitment of immature DCs through one of their GPCRs.

Researchers then wanted to examine whether GNLY could not only recruit immature DCs but also activate them. Activated DCs exhibit a “mature phenotype” which essentially means it begins to express specific cell surface markers and produce certain cytokines (molecules that promote inflammation). Human Mo-DCs were treated with 9 and 15-kDa granulysin. Both forms were able to cause the upregulation of mature cell surface markers CD80, CD83, CD86, and MHC Class II. Furthermore, GNLY-treated DCs produced elevated amounts of cytokines IL-6, IL-8, IL-10, and IL-12, and TNFα. CD1c+ peripheral blood DCs also were treated with granulysin and produced similar proinflammatory cytokines: Il-6, Il-8, and TNFα. Interestingly, the 15-kDa form of granulysin was more potent at inducing proinflammatory cytokine IL-12. To ensure the specificity of GNLY, anti-GNLY antibodies were employed in the supernatant which diminished the aforementioned effects.

To determine whether GNLY-mediated DC recruitment and activation was functional, GNLY-treated Mo-DCs were tested for their ability to induce T cell proliferation. A mixed lymphocyte reaction (MLR) assay was used in which T cells and GNLY-treated-DCs were both placed in a well plate. T cells then were then marked and counted. Results showed that GNLY-treated-DCs exhibited an enhanced capacity to present antigen to T cells and cause significant proliferation in comparison to controls. Taken together, these results suggest that GNLY induces a phenotypic and functional maturation in Mo-DCs which would aid in initiating the adaptive immune response (Tewary et al. 2010).

The proinflammatory behavior of granulysin seen in the above experiments led researchers to try and identify the specific GPCR mechanism. As discussed before, GPCRs mediate their effects through an intracellular signaling cascade. Adaptor proteins often aid in this process. Specifically, MyD88 is an adaptor protein that was of interest to researchers. Macrophage cell lines from mice who expressed this protein (MyD88^+/+) and those that didn’t (MyD88^-/-) were taken and treated with the 15-kDa form of GNLY. The supernatant was then analyzed for Il-6. Production of IL-6 was shown to be dependent on MyD88, seeing as the macrophages lacking this protein did not produce high amounts of this cytokine. Macrophages were also treated with Pam 3-cys, an agonist for the TLR 1/2 receptor. This agonist was similarly able to induce IL-6 production. To ensure that the reason macrophages from MyD88^-/- mice didn’t produce IL-6 was from an inability to produce it at all, these macrophages were stimulated with poly 1:C. Poly 1:C binds and activates TLR-3 receptor but doesn’t utilize the MyD88 adaptor protein. Upon stimulation with poly 1:C, these cells produced sufficient amounts of IL-6.

It’s important to mention that TLR receptors are expressed on innate immune cells as part of the innate immune response (1).

To then further specify whether GNLY was exerting its effects through a TLR receptor, different bone-marrow derived DCs (BMDCs) were isolated from TLR4 mutant mice: C3H/HeJ (mutation that renders TLR4 non-functional) and wild type C3H/HeN mice (functional TLR4). Only the DCs from the wild-type C3H/HeN exhibited a mature phenotype (upregulation of CD80) and produced proinflammatory cytokines (IL-6, TNFα) when treated with GNLY. The C3H/HeJ did not exhibit mature cell surface markers or cytokine production. These results suggest that GNLY mediates its effects through the TLR4 receptor.

Finally, researchers wondered whether the recruitment and activation of DCs via GNLY was physiologically relevant. As discussed earlier, GNLY is released normally through degranulation of NK cells. Researchers therefore wanted to examine whether the observed behavior would occur when degranulation was induced. The supernatant was taken from cells of the NK92 cell line as well as from primary NK cells after stimulation with SrCl₂. SrCl₂ is a potent inducer of degranulation. When the supernatant was applied to Mo-DCs it resulted in phenotypic and cytokine activation. This effect was partially diminished when anti-GNLY antibodies were produced confirming the notion that GNLY contributed to these effects.

In conclusion, researchers were able to illuminate the capacity for GNLY to exhibit alarmin properties. GNLY was capable of activating and recruiting human Mo-DCs. This interaction with DCs demonstrates a direct proinflammatory role for GNLY. However, further research needs to be done to further specify the exact GPCR activated in this inflammatory response. Ultimately, knowing more about the behavior exhibited by NK cells, as well as the immune system in general, allows researchers to better take advantage of this knowledge in order to better combat pathogen invaders.

Primary Reference:

Tewary, P., Yang, D., Rosa, G., Li, Y., Finn, MW., Krensky, AM., Clayberger, C., & Oppenheim, JJ. (2011) Granulysin activates antigen-presenting cells through TLR4 and acts as an immune alarmin. Journal of Immunobiology. 116(18). 3465-73.

Other Citations:

(1) Mak, Tak & Saunders, Mary. Primer to the Immune Response. California. Elsevier, 2011. Print.

(2) Deng, A., Chen, S., Li, Q., Lyu, SC., Clayberger, C., & Krensky, AM. (2005) Granulysin, a cytolytic molecule, is also a chemoattractant and proinflammatory activator. Journal of Immunology. 174(9), 5243-8.