Cytotoxic T Lymphocyte Granzyme-b mediates neuronal cell death during Plasmodium berghei ANKA induced experimental cerebral malaria.

Cerebral malaria is a complex, acute, neurological disease characterised by a sudden onset of cerebral symptoms. This disease is manifested as initial arousable stage that is followed by an unarousable coma and eventually death. Parasite burden and CD8+ T cell count in the brain determines the disease outcome. Cytotoxic CD8+ T cell-derived Granzyme-b is required for the development of experimental cerebral malaria (ECM), but the mechanism of pathogenesis is not known. Here, we show that CD8+ T cells infiltrate in to the brain during ECM releasing Granzyme-b that is cytotoxic to neuronal cells. Granzyme-b kills neuronal cells through direct cytotoxicity and also by activating neuronal caspase-3 and calpain1 via cytoskeletal breakdown. Our results showed the increased expression of cell adhesion molecules and chemokine receptors in the brain and their associated infiltration of T cells during ECM.

Protease-activated receptor-1 activation by granzyme B causes neurotoxicity that is augmented by interleukin-1ß.

BACKGROUND: The cause of neurodegeneration in progressive forms of multiple sclerosis is unknown. We investigated the impact of specific neuroinflammatory markers on human neurons to identify potential therapeutic targets for neuroprotection against chronic inflammation. METHODS: Surface immunocytochemistry directly visualized protease-activated receptor-1 (PAR1) and interleukin-1 (IL-1) receptors on neurons in human postmortem cortex in patients with and without neuroinflammatory lesions. Viability of cultured neurons was determined after exposure to cerebrospinal fluid from patients with progressive multiple sclerosis or purified granzyme B and IL-1ß. Inhibitors of PAR1 activation and of PAR1-associated second messenger signaling were used to elucidate a mechanism of neurotoxicity. RESULTS: Immunohistochemistry of human post-mortem brain tissue demonstrated cells expressing higher amounts of PAR1 near and within subcortical lesions in patients with multiple sclerosis compared to control tissue. Human cerebrospinal fluid samples containing granzyme B and IL-1ß were toxic to human neuronal cultures. Granzyme B was neurotoxic through activation of PAR1 and subsequently the phospholipase Cß-IP3 second messenger system. Inhibition of PAR1 or IP3 prevented granzyme B toxicity. IL-1ß enhanced granzyme B-mediated neurotoxicity by increasing PAR1 expression. CONCLUSIONS: Neurons within the inflamed central nervous system are imperiled because they express more PAR1 and are exposed to a neurotoxic combination of both granzyme B and IL-1ß. The effects of these inflammatory mediators may be a contributing factor in the progressive brain atrophy associated with neuroinflammatory diseases. Knowledge of how exposure to IL-1ß and granzyme B act synergistically to cause neuronal death yields potential novel neuroprotective treatments for neuroinflammatory diseases.

Granzyme B-induced neurotoxicity is mediated via activation of PAR-1 receptor and Kv1.3 channel.

Increasing evidence supports a critical role of T cells in neurodegeneration associated with acute and subacute brain inflammatory disorders. Granzyme B (GrB), released by activated T cells, is a cytotoxic proteinase which may induce perforin-independent neurotoxicity. Here, we studied the mechanism of perforin-independent GrB toxicity by treating primary cultured human neuronal cells with recombinant GrB. GrBactivated the protease-activated receptor (PAR)-1 receptor on the neuronal cell surface leading to decreased intracellular cyclic AMP levels. This was followed by increased expression and translocation of the voltage gated potassium channel, Kv1.3 to the neuronal cell membrane. Similar expression of Kv1.3 was also seen in neurons of the cerebral cortex adjacent to active inflammatory lesions in patients with multiple sclerosis. Kv1.3 expression was followed by activation of Notch-1 resulting in neurotoxicity. Blocking PAR-1, Kv1.3 or Notch-1 activation using specific pharmacological inhibitors or siRNAs prevented GrB-induced neurotoxicity. Furthermore, clofazimine protected against GrB-induced neurotoxicity in rat hippocampus, in vivo. These observations indicate that GrB released from T cells induced neurotoxicity by interacting with the membrane bound Gi-coupled PAR-1 receptor and subsequently activated Kv1.3 and Notch-1. These pathways provide novel targets to treat T cell-mediated neuroinflammatory disorders. Kv1.3 is of particular interest since it is expressed on the cell surface, only under pathological circumstances, and early in the cascade of events making it an attractive therapeutic target.

Granule-derived granzyme B mediates the vulnerability of human neurons to T cell-induced neurotoxicity.

Multiple sclerosis (MS) is considered an autoimmune disease of the CNS and is characterized by inflammatory cells infiltrating the CNS and inducing demyelination, axonal loss, and neuronal death. Recent evidence strongly suggests that axonal and neuronal degeneration underlie the progression of permanent disability in MS. In this study, we report that human neurons are selectively susceptible to the serine-protease granzyme B (GrB) isolated from cytotoxic T cell granules. In vitro, purified human GrB induced neuronal death to the same extent as the whole activated T cell population. On the contrary, activated T cells isolated from GrB knockout mice failed to induce neuronal injury. We found that following internalization through various parts of neurons, GrB accumulated in the neuronal soma. Within the cell body, GrB diffused out of endosomes possibly through a perforin-independent mechanism and induced subsequent activation of caspases and cleavage of α-tubulin. Inhibition of caspase-3, a well-known substrate for GrB, significantly reduced GrB-mediated neurotoxicity. We demonstrated that treatment of neurons with mannose-6-phosphate prevented GrB entry and inhibited GrB-mediated neuronal death, suggesting mannose-6-phosphate receptor-dependent endocytosis. Together, our data unveil a novel mechanism by which GrB induces selective neuronal injury and suggest potential new targets for the treatment of inflammatory-mediated neurodegeneration in diseases such as MS.

CD40 signaling in CD8+CD40+ T cells turns on contra-T regulatory cell functions.

CD4(+) regulatory T cells (Treg cells) mediate immunosuppression, whereas CD8(+) T cells confer resistance in many diseases. It is unknown whether CD8(+) T cells confer protection by antagonizing the Treg cells. Using a model of stage-specific immune responses against Leishmania donovani infection in susceptible BALB/c mice, we report that CD3(+)CD8(+)CD40(+) T cells executed CD40-dependent cytotoxicity on CD3(+)CD4(+)CD127(dim)GITR(+)CD25(+) Treg cells during the initial phase of the infection but were later apoptosed by IL-10. CD40 signaled through Ras, PI3K, and protein kinase C, resulting in p38MAPK- or ERK-1/2-independent, but NF-kappaB-dependent, induction of the cytotoxic mediators granzyme and perforin. Adoptive transfer of CD3(+)CD8(+)CD40(+) T cells reduced the L. donovani infection in BALB/c mice. These results identify CD3(+)CD8(+)CD40(+) T cells as the contra-Treg cells and imply a novel immunotherapeutic principle.

The biology of cytotoxic cell granule exocytosis pathway: granzymes have evolved to induce cell death and inflammation.

The granule exocytosis pathway of cytotoxic lymphocytes (Tc and NK cells) is critical for control of tumor development and viral infections. Granule-associated perforin and granzymes are key components in Tc cell-mediated function(s). On the basis of studies that showed granzymes A, B, C, K and M, to induce apoptosis in vitro, all granzymes were thought to also induce cell death in vivo. This review summarizes our present understanding of the biological processes elicited by purified granzyme A and granzyme as well as the processes induced by the more physiologically relevant cytotoxic cells secreting these proteases. The combined evidence supports the concept that the granule secretion pathway is not mono-specific but rather poly-functional including induction of pro-inflammatory cytokines, besides their widely appreciated apoptotic properties.

Ex vivo detection of primary leukemia cells resistant to granule cytotoxin-induced cell death: a rapid isolation method to study granzyme-B-mediated cell death.

Cytotoxic T lymphocytes and natural killer cells (CTL/NK) induce cell death in leukemia cells by the granzyme B (grB)-dependent granule cytotoxin (GC) pathway. Resistance to GC may be involved in immune evasion of leukemia cells. The delivery of active grB into the cytoplasma is dependent on the presence of perforin (PFN) and grB complexes. We developed a rapid method for the isolation of GC to investigate GC-mediated cell death in primary leukemia cells. We isolated GC containing grB, grB complexes and PFN by detergent free hypotonic lysis of the human NK cell leukemia line YT. The GC induce grB-mediated, caspase-dependent apoptosis in live cells. The human leukemia cell lines KG-1, U937, K562 (myeloid leukemia), Jurkat, Daudi, and BV173 (lymphoblastic leukemia) treated with GC internalized grB and underwent cell death. In primary leukemia cells analyzed ex vivo, we found GC-resistant leukemia cells in three out of seven patients with acute myeloid leukemia and one out of six patients with acute lymphoblastic leukemia. We conclude that our method is fast (approximately 1 h) and yields active GC that induce grB-dependent cell death. Furthermore, resistance to GC can be observed in acute leukemias and may be an important mechanism contributing to leukemia cell immune evasion.

H is for helper: granzyme H helps granzyme B kill adenovirus-infected cells.

It is commonly held that the various granzymes (lethal proteases produced by cytotoxic lymphocytes) utilize their different substrate preferences to bring about various forms of target cell death. Although a considerable body of evidence supports this view, it has now become clear that human granzyme H could have evolved a proteolytic specificity that both interferes directly with adenovirus replication and prevents the virus from blocking the potent pro-apoptotic activity of granzyme B.