Pharmacological Research, Vol . 26, Supplement 2, 1992
26
GLUCOCORTICOID-INDUCED APOPTOSIS OF NATURAL KILLER CELLS AND CYTOTOXIC T LYMPHOCYTES . G. Migliorati, C . Pagliacci*, R. Moraca, F . Crocicchio, I . Nicoletti* and C . Riccardi Institutes of Farmacologia Medica and *Clinica Medica I, Perugia University Medical School, 06100 Perugia, Italy . Key words : Glucocorticoid, apoptosis, interleukins, cytotoxic ymphocytes . The process of apoptosis is operative during embryogenesis, in tumor regression and in the elimination of self-reactive T-lymphocytes in the thymus . The death of cells undergoing apoptosis is preceeded by the chromatin cleavage at the linker nucleosomes by specific endonucleases, which results in extensive DNA fragmentation into oligonucleosomal subunits (1) . Glucocorticoid hormones (GCH) are potent immunosoppressive and antiinflammatory drugs whuch have been shown to influence the development of the lymphohemopoietic system . In particular GCH are able to inhibit the production of a number of cytokines including interleukin-2 (IL-2) and IL-4, and also to induce apoptotic cell death in less mature CD4+CD8+ thymocytes (2) . In the attempt to analyze the possible apoptotic effect of GCH on more mature lymphocytes we used enriched Natural Killer (NK) cell population as well as CD8+ allospecific cytotoxic T-lymphocytes (CTL) . NK cells were obtained by culturing with IL-2 spleen cells of C3H/HeN mice for 7 days in vitro. After culturing cells were enriched (above 98%) by panning with anti-NK1 .1 mAb . Allospecific CTL were obtained by stimulating C3H/HeN spleen cells with allogeneic irradiated C57B1/6 spleen cells for 5 days in vitro . After culturing CD8+ CTL were enriched (above 97%) by panning with anti CD8 mAb . Apoptosis was measured by a recently described flow cytometric assay (3) . Our results indicate that culturing cells at 37'C for 24h, provokes a spontaneous DNA fragmentation. Moreover treatment with dexamethasone (DEX 10 -7 M) induces an extensive DNA fragmentation suggesting that GCH can activate apoptotic cell death both in less mature thymocytes as well as in more differentiated lymphocytes, including NK cell and CTL (Figure 1) . We then tested the possible role of various cytokines in the regulation of DEX-induced NK cell and CTL apoptosis. Results indicate that IL-2 and IL-4 induce a significant inhibition of both NK cell and CTL apoptosis (data not shown) . In the attempt to analyze the possible mechanisms involved in the DEX-induced apoptosis we compared the effect of protein-synthesis and mRNA inhibitors (Cycloheximide, 50 ug/ml) and actinomycin-D, 2 .5 ug/ml), the PKC-inhibitors H7 (50 mM/ml) and staurosporine (10 nM), heat shock (42°C for 30') and the endonuclease inhibitor ZnSO4 (1 mM) on apoptosis of less mature thymocytes and more mature NK cell and CTL .
1043-6618/92/26110026-02/$03 .00/0
© 1992 The Italian Pharmacological Society
27
Pharmacological Research, Vol . 26, Supplement 2, 1992 I. an
32%
57%
a%
NK
I DEX
I CONM
25%
76%
FIGURE 1 : Effects of 10-7M DEX on apoptotic death of NK-cells and CTL
23%
74% CIL V~~
DNA CONTENT
Results show that while all the above treatments strongly inhibit the DEX-induceed apoptosis in thymocytes, only Zn++ inhibits the DEX-induced apoptosis in NK cell and CTL. Moreover both protein synthesis as well as PKC inhibition cause DNA fragmentation and cell death in more mature NK cell and CTL (data not shown) . Taken together these data demonstrate that GCS induce apoptosis in different cell populations with different mechanisms depending on their stage of differentiation . Moreover the above results also suggest that some cytokines modulate NK cell and CTL activity by influencing cell survival . REFERENCES
1) Willie A .H ., Kerr J .F .R., Currie A .R . Cell death : the significance of apoptosis . Int. Rev . Cytol . 1980; 68 :251-265. 2) Willie A .H . Glucorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 1980 ; 284 :555-557 . 3) Nicoletti I ., Migliorati G., Pagliacci M .C ., Grignani F ., Riccardi C . A rapid and simple method for measuring thymocytes apoptosis by propidium iodide staining and flow cytometry . J . Immunol . Methods, 1991 ; 139 :271-279 . Supported by AIRC and by PF ACRO, CNR, Italy
26
GLUCOCORTICOID-INDUCED APOPTOSIS OF NATURAL KILLER CELLS AND CYTOTOXIC T LYMPHOCYTES . G. Migliorati, C . Pagliacci*, R. Moraca, F . Crocicchio, I . Nicoletti* and C . Riccardi Institutes of Farmacologia Medica and *Clinica Medica I, Perugia University Medical School, 06100 Perugia, Italy . Key words : Glucocorticoid, apoptosis, interleukins, cytotoxic ymphocytes . The process of apoptosis is operative during embryogenesis, in tumor regression and in the elimination of self-reactive T-lymphocytes in the thymus . The death of cells undergoing apoptosis is preceeded by the chromatin cleavage at the linker nucleosomes by specific endonucleases, which results in extensive DNA fragmentation into oligonucleosomal subunits (1) . Glucocorticoid hormones (GCH) are potent immunosoppressive and antiinflammatory drugs whuch have been shown to influence the development of the lymphohemopoietic system . In particular GCH are able to inhibit the production of a number of cytokines including interleukin-2 (IL-2) and IL-4, and also to induce apoptotic cell death in less mature CD4+CD8+ thymocytes (2) . In the attempt to analyze the possible apoptotic effect of GCH on more mature lymphocytes we used enriched Natural Killer (NK) cell population as well as CD8+ allospecific cytotoxic T-lymphocytes (CTL) . NK cells were obtained by culturing with IL-2 spleen cells of C3H/HeN mice for 7 days in vitro. After culturing cells were enriched (above 98%) by panning with anti-NK1 .1 mAb . Allospecific CTL were obtained by stimulating C3H/HeN spleen cells with allogeneic irradiated C57B1/6 spleen cells for 5 days in vitro . After culturing CD8+ CTL were enriched (above 97%) by panning with anti CD8 mAb . Apoptosis was measured by a recently described flow cytometric assay (3) . Our results indicate that culturing cells at 37'C for 24h, provokes a spontaneous DNA fragmentation. Moreover treatment with dexamethasone (DEX 10 -7 M) induces an extensive DNA fragmentation suggesting that GCH can activate apoptotic cell death both in less mature thymocytes as well as in more differentiated lymphocytes, including NK cell and CTL (Figure 1) . We then tested the possible role of various cytokines in the regulation of DEX-induced NK cell and CTL apoptosis. Results indicate that IL-2 and IL-4 induce a significant inhibition of both NK cell and CTL apoptosis (data not shown) . In the attempt to analyze the possible mechanisms involved in the DEX-induced apoptosis we compared the effect of protein-synthesis and mRNA inhibitors (Cycloheximide, 50 ug/ml) and actinomycin-D, 2 .5 ug/ml), the PKC-inhibitors H7 (50 mM/ml) and staurosporine (10 nM), heat shock (42°C for 30') and the endonuclease inhibitor ZnSO4 (1 mM) on apoptosis of less mature thymocytes and more mature NK cell and CTL .
1043-6618/92/26110026-02/$03 .00/0
© 1992 The Italian Pharmacological Society
27
Pharmacological Research, Vol . 26, Supplement 2, 1992 I. an
32%
57%
a%
NK
I DEX
I CONM
25%
76%
FIGURE 1 : Effects of 10-7M DEX on apoptotic death of NK-cells and CTL
23%
74% CIL V~~
DNA CONTENT
Results show that while all the above treatments strongly inhibit the DEX-induceed apoptosis in thymocytes, only Zn++ inhibits the DEX-induced apoptosis in NK cell and CTL. Moreover both protein synthesis as well as PKC inhibition cause DNA fragmentation and cell death in more mature NK cell and CTL (data not shown) . Taken together these data demonstrate that GCS induce apoptosis in different cell populations with different mechanisms depending on their stage of differentiation . Moreover the above results also suggest that some cytokines modulate NK cell and CTL activity by influencing cell survival . REFERENCES
1) Willie A .H ., Kerr J .F .R., Currie A .R . Cell death : the significance of apoptosis . Int. Rev . Cytol . 1980; 68 :251-265. 2) Willie A .H . Glucorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature, 1980 ; 284 :555-557 . 3) Nicoletti I ., Migliorati G., Pagliacci M .C ., Grignani F ., Riccardi C . A rapid and simple method for measuring thymocytes apoptosis by propidium iodide staining and flow cytometry . J . Immunol . Methods, 1991 ; 139 :271-279 . Supported by AIRC and by PF ACRO, CNR, Italy
