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In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells.

Simon MM, Hausmann M, Tran T, Ebnet K, Tschopp J, ThaHla R, Müllbacher A - J. Exp. Med. (1997)

Bottom Line: Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear.In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays.This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiology, Freiburg, Germany. simon@immunbiol.mpg.de

ABSTRACT
Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

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Analysis of wild-type (C57BL/ 6) and the mutant mice gzmA−/−, gzmB−/−,  gzmA×B−/−, and perforin−/− mice by  PCR. Tail DNA from individual mice was  analyzed by PCR amplification using the  gzmA-, gzmB-, and perforin-specific primer  pairs, as indicated in Materials and Methods.
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Figure 1: Analysis of wild-type (C57BL/ 6) and the mutant mice gzmA−/−, gzmB−/−, gzmA×B−/−, and perforin−/− mice by PCR. Tail DNA from individual mice was analyzed by PCR amplification using the gzmA-, gzmB-, and perforin-specific primer pairs, as indicated in Materials and Methods.

Mentions: For detection of the respective mutations (Fig. 1), DNA was analyzed by PCR, as previously described (22), using the following primers: gzmA−/−: 5′-AGG AGC AAT ATA TAC CAA TGG-3′ and 5′-AGG TAG GTG AAG GAT AGC CAC-3′; neo-primer: 5′-CGG AGA ACC TGC GTG CAA TC-3′. gzmB−/−: 5′-CTG CTA CTG CTG ACC TTG TCT-3′ and 5′-TGA GGA CAG CAA TTC CAT CTA-3′; neo-primer: 5′-TTC CTC GTG CTT TAC GGT ATC-3′. Perforin−/−: 5′-CCA CTC CAC CTT GAC TTC AAA AAG GCG-3′ and 5′-TGG GCA GCA GTC CTG GTT GGT GAC CTT-3′; neo-primer: 5′-CGG AGA ACC TGC GTG CAA TC-3′. The genomic DNA was subjected to amplification by PCR and analyzed as previously described (22).


In vitro- and ex vivo-derived cytolytic leukocytes from granzyme A x B double knockout mice are defective in granule-mediated apoptosis but not lysis of target cells.

Simon MM, Hausmann M, Tran T, Ebnet K, Tschopp J, ThaHla R, Müllbacher A - J. Exp. Med. (1997)

Analysis of wild-type (C57BL/ 6) and the mutant mice gzmA−/−, gzmB−/−,  gzmA×B−/−, and perforin−/− mice by  PCR. Tail DNA from individual mice was  analyzed by PCR amplification using the  gzmA-, gzmB-, and perforin-specific primer  pairs, as indicated in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199142&req=5

Figure 1: Analysis of wild-type (C57BL/ 6) and the mutant mice gzmA−/−, gzmB−/−, gzmA×B−/−, and perforin−/− mice by PCR. Tail DNA from individual mice was analyzed by PCR amplification using the gzmA-, gzmB-, and perforin-specific primer pairs, as indicated in Materials and Methods.
Mentions: For detection of the respective mutations (Fig. 1), DNA was analyzed by PCR, as previously described (22), using the following primers: gzmA−/−: 5′-AGG AGC AAT ATA TAC CAA TGG-3′ and 5′-AGG TAG GTG AAG GAT AGC CAC-3′; neo-primer: 5′-CGG AGA ACC TGC GTG CAA TC-3′. gzmB−/−: 5′-CTG CTA CTG CTG ACC TTG TCT-3′ and 5′-TGA GGA CAG CAA TTC CAT CTA-3′; neo-primer: 5′-TTC CTC GTG CTT TAC GGT ATC-3′. Perforin−/−: 5′-CCA CTC CAC CTT GAC TTC AAA AAG GCG-3′ and 5′-TGG GCA GCA GTC CTG GTT GGT GAC CTT-3′; neo-primer: 5′-CGG AGA ACC TGC GTG CAA TC-3′. The genomic DNA was subjected to amplification by PCR and analyzed as previously described (22).

Bottom Line: Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear.In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays.This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Immunbiology, Freiburg, Germany. simon@immunbiol.mpg.de

ABSTRACT
Granzyme (gzm) A and gzmB have been implicated in Fas-independent nucleolytic and cytolytic processes exerted by cytotoxic T (Tc) cells, but the underlying mechanism(s) remains unclear. In this study, we compare the potential of Tc and natural killer (NK) cells of mice deficient in both gzmA and B (gzmAxB-/-) with those from single knockout mice deficient in gzmA (-/-), gzmB (-/-), or perforin (-/-) to induce nuclear damage and lysis in target cells. With the exception of perforin-/-, all in vitro- and ex vivo-derived Tc and NK cell populations from the mutant strains induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice. This contrasts with their capacity to induce apoptotic nuclear damage in target cells. In gzmAxB-/- mice, Tc/NK-mediated target cell DNA fragmentation was not observed, even after extended incubation periods (10 h), but was normal in gzmA-deficient and only impaired in gzmB-deficient mice in short-term (2-4 h), but not long-term (4-10 h), nucleolytic assays. This suggests that gzmA and B are critical for Tc/NK granule- mediated nucleolysis, with gzmB being the main contributor, while target cell lysis is due solely to perforin and independent of both proteases.

Show MeSH
Related in: MedlinePlus