Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation.

Young, John Ding‐E; Leong, Lauren Gee; Liu, Chau‐Ching; Damiano, Angela; Cohn, Zanvil A.

doi:10.1073/pnas.83.15.5668

Cited by 57 publications

(29 citation statements)

References 29 publications

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“…NK cells function to produce cytokines that can modulate innate and adaptive immune responses (40 -42). YT cells are known to secrete high levels of granzyme B into the culture supernatant (43), and NK cells release small amounts of perforin into the culture medium (44). Therefore, experiments were performed to determine whether soluble factors or direct cell contact are responsible for the anticryptococcal activity.…”

Section: Discussionmentioning

confidence: 99%

NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity

Ling

Wang²,

Neely

et al. 2004

The Journal of Immunology

103

111

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Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown. Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation. When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes. However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated. Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone. These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.

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Section: Discussionmentioning

confidence: 99%

NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity

Ling

Wang²,

Neely

et al. 2004

The Journal of Immunology

103

111

View full text Add to dashboard Cite

show abstract

“…Recent results from our laboratory indicate that PFP/P1 is a secretory protein (31). Ca 2+ appears to be required for the cellular release of the pore-former (31). Ca 2+ is therefore thought to be required for both the fusion of granules with plasma membranes (i.e., degranulation) and the assembly of PFP/P1 into tubular lesions.…”

Section: Single-channel Fluctuations and Different Pore Sizes Slow Smentioning

confidence: 99%

“…It is possible that the release of PFP/P1 into restricted intercellular spaces between the effector and target cells may have a concentration effect, raising its local concentration many-fold. Recent results from our laboratory indicate that PFP/P1 is a secretory protein (31). Ca 2+ appears to be required for the cellular release of the pore-former (31).…”

Section: Single-channel Fluctuations and Different Pore Sizes Slow Smentioning

confidence: 99%

Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

Young¹,

Podack²,

Cohn³

1986

The Journal of Experimental Medicine

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Histocompatibility-restricted cytotoxic T lymphocytes produce circular lesions on target cell membranes. The pore-forming protein (PFP or perforin 1) that forms these membrane lesions has been purified from lymphocytes. At 37 degrees C, in the presence of Ca2+, this protein polymerizes into a supramolecular tubular complex of Mr greater than 10(6) that partially resists dissociation by SDS and reducing agents. It incorporates spontaneously into planar lipid bilayers during polymerization to form nonselective ion channels, showing heterogeneous size distribution, the smallest conductance per unit being identified as 400 pS in 0.1 M NaCl. PFP/P1 that had been assembled in lipid vesicles before incorporation into planar bilayer show much larger single channel conductance, ranging from 1 to 6 nS in 0.1 M NaCl, suggesting that PFP/P1 may assume multiple functional sizes in proportion to its state of polymerization. The reconstituted channels are relatively voltage-insensitive, with most channels persisting in the open state for seconds to minutes. Nucleated cells are rapidly depolarized by this protein. The purified protein lyses a variety of tumor cells. Polymerization and functional channel activity are absolutely Ca2+-dependent. The activity of this protein may play a direct role in T lymphocyte-mediated cytolysis.

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“…Ca 21 may be required for this form of membrane fusion . Previous studies (30) have already shown that stimulation of lymphocytes with A23187, a calcium ionophore, triggers cells to secrete PFP. It would be interesting to determine whether the interaction of lymphocytes with targets also leads to an abrupt rise of the cytoplasmic levels of free calcium that initiates granule fusion and degranulation .…”

Section: Discussionmentioning

confidence: 99%

Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

Young¹,

Damiano²,

Dinome³

et al. 1987

The Journal of Experimental Medicine

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Granules isolated from CTL and NK cells contain a cytolytic pore-forming protein (PFP/perforin). At low temperatures (on ice), PFP binds to erythrocyte membranes without producing hemolysis. Hemolysis occurs when the PFP-bound erythrocytes are warmed up to 37 degrees C, which defines a temperature-dependent, lytic (pore-formation) step distinct from the membrane-binding event. Ca2+ and neutral pH are required for both membrane binding and pore formation by PFP. Serum, LDL, HDL, and heparin inhibit the hemolytic activity of PFP by blocking its binding to lipid membranes. Lysis by PFP that has bound to erythrocyte membranes is no longer susceptible to the effect of these inhibitors. The hemolytic activities associated with intact granules and solubilized PFP show different requirements for Ca2+ and pH, indicating that cytolysis produced by isolated granules may involve an additional step, possibly fusion of granules with membranes. It is suggested that three distinct Ca2+- and pH-dependent events may be involved during cell killing by CTL and NK cells: fusion of cytoplasmic granules of effector cells with their plasma membrane, releasing PFP from cells; binding of the released PFP to target membranes; and insertion of monomers and the subsequent formation of lytic pores in the target membrane. The serum-mediated inhibition of membrane binding by PFP could prevent the accidental injury of bystander cells by cell-released PFP, but would allow cytolysis to proceed to completion once PFP has bound to the target membrane.

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Extracellular release of lymphocyte cytolytic pore-forming protein (perforin) after ionophore stimulation.

Cited by 57 publications

References 29 publications

NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity

NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity

Properties of a purified pore-forming protein (perforin 1) isolated from H-2-restricted cytotoxic T cell granules.

Dissociation of membrane binding and lytic activities of the lymphocyte pore-forming protein (perforin).

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