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Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells.

Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L - PLoS ONE (2011)

Bottom Line: Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function.Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response.The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Immunology, Department of Clinical Microbiology, Umeå University, Umeå, Sweden.

ABSTRACT
Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

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Thermal- and oxidative stress increases the release of exosomes by Jurkat and Raji cells.Exosomes were isolated with sequential centrifugations and sucrose gradient from supernatants from the same number of untreated and stressed cells. Measurement of isolated exosomes by A. BCA protein assay, B. fluorescence measurement of Vybrant DiI stainings of exosomal lipid membranes, C. western blot for the exosomal marker CD63. D. Densitometry for the exosomal marker CD63, the density of the bands was normalized to the bands from exosomes released by cells cultured at steady-state conditions ( = 1). * = statistical significance, p<0.05.
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pone-0016899-g003: Thermal- and oxidative stress increases the release of exosomes by Jurkat and Raji cells.Exosomes were isolated with sequential centrifugations and sucrose gradient from supernatants from the same number of untreated and stressed cells. Measurement of isolated exosomes by A. BCA protein assay, B. fluorescence measurement of Vybrant DiI stainings of exosomal lipid membranes, C. western blot for the exosomal marker CD63. D. Densitometry for the exosomal marker CD63, the density of the bands was normalized to the bands from exosomes released by cells cultured at steady-state conditions ( = 1). * = statistical significance, p<0.05.

Mentions: In the next step, we investigated whether thermal and oxidative stress also affected the quantity of exosomes secreted by Jurkat and Raji cells. Using sucrose gradient ultracentrifugation, we isolated exosomes from cell culture supernatants produced by equal amount of Jurkat and Raji cells cultured under steady state and stress conditions, and measured the exosomal yield by three different methods. At present, there is no well-established and recognized method for exosome quantification. The most frequently used methods are based on total exosomal protein measurement by BCA assay and densitometric analysis of Western blot bands [22]. Recently, fluorescence intensity measurement of exosomes labeled with lipophilic fluorescent dyes has also been suggested and used [23]. To enhance the reliability of our measurements we used all three methods - BCA protein assay, fluorescence intensity after exosomal membrane staining with Vybrand DiI and densitometry of Western blots. The results are summarized in Figure 3. Under stress, the exosome secretion from both cell lines was increased as measured by all three methods, reaching a statistical significance in the measurement by BCA assay (Figure 3A, n = 11). A clear tendency of increased exosome quantity was seen by fluorescence intensity (Figure 3B, n = 5). Figure 3C is a Western blot of one representative experiment for the exosomal marker CD63 reflecting the higher protein amount under stressed conditions. Figure 3D shows an increased band density of CD63 after thermal- and oxidative stress, reaching 3-fold increase by thermal- and 15-fold increase by oxidative stress for Jurkat exosomes, and 22-fold increase by thermal- and 32-fold increase by oxidative stress for Raji exosomes. These measurements suggest that oxidative stress seems to enhance exosome secretion to a higher degree than thermal stress, and that Raji cell line seems to be more susceptible to stress-mediated up-regulation of exosome secretion compared to Jurkat. We conclude that cellular stress can up-regulate exosome secretion by both T- and B-cell leukemia/lymphoma cells.


Thermal- and oxidative stress causes enhanced release of NKG2D ligand-bearing immunosuppressive exosomes in leukemia/lymphoma T and B cells.

Hedlund M, Nagaeva O, Kargl D, Baranov V, Mincheva-Nilsson L - PLoS ONE (2011)

Thermal- and oxidative stress increases the release of exosomes by Jurkat and Raji cells.Exosomes were isolated with sequential centrifugations and sucrose gradient from supernatants from the same number of untreated and stressed cells. Measurement of isolated exosomes by A. BCA protein assay, B. fluorescence measurement of Vybrant DiI stainings of exosomal lipid membranes, C. western blot for the exosomal marker CD63. D. Densitometry for the exosomal marker CD63, the density of the bands was normalized to the bands from exosomes released by cells cultured at steady-state conditions ( = 1). * = statistical significance, p<0.05.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045385&req=5

pone-0016899-g003: Thermal- and oxidative stress increases the release of exosomes by Jurkat and Raji cells.Exosomes were isolated with sequential centrifugations and sucrose gradient from supernatants from the same number of untreated and stressed cells. Measurement of isolated exosomes by A. BCA protein assay, B. fluorescence measurement of Vybrant DiI stainings of exosomal lipid membranes, C. western blot for the exosomal marker CD63. D. Densitometry for the exosomal marker CD63, the density of the bands was normalized to the bands from exosomes released by cells cultured at steady-state conditions ( = 1). * = statistical significance, p<0.05.
Mentions: In the next step, we investigated whether thermal and oxidative stress also affected the quantity of exosomes secreted by Jurkat and Raji cells. Using sucrose gradient ultracentrifugation, we isolated exosomes from cell culture supernatants produced by equal amount of Jurkat and Raji cells cultured under steady state and stress conditions, and measured the exosomal yield by three different methods. At present, there is no well-established and recognized method for exosome quantification. The most frequently used methods are based on total exosomal protein measurement by BCA assay and densitometric analysis of Western blot bands [22]. Recently, fluorescence intensity measurement of exosomes labeled with lipophilic fluorescent dyes has also been suggested and used [23]. To enhance the reliability of our measurements we used all three methods - BCA protein assay, fluorescence intensity after exosomal membrane staining with Vybrand DiI and densitometry of Western blots. The results are summarized in Figure 3. Under stress, the exosome secretion from both cell lines was increased as measured by all three methods, reaching a statistical significance in the measurement by BCA assay (Figure 3A, n = 11). A clear tendency of increased exosome quantity was seen by fluorescence intensity (Figure 3B, n = 5). Figure 3C is a Western blot of one representative experiment for the exosomal marker CD63 reflecting the higher protein amount under stressed conditions. Figure 3D shows an increased band density of CD63 after thermal- and oxidative stress, reaching 3-fold increase by thermal- and 15-fold increase by oxidative stress for Jurkat exosomes, and 22-fold increase by thermal- and 32-fold increase by oxidative stress for Raji exosomes. These measurements suggest that oxidative stress seems to enhance exosome secretion to a higher degree than thermal stress, and that Raji cell line seems to be more susceptible to stress-mediated up-regulation of exosome secretion compared to Jurkat. We conclude that cellular stress can up-regulate exosome secretion by both T- and B-cell leukemia/lymphoma cells.

Bottom Line: Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function.Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response.The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Immunology, Department of Clinical Microbiology, Umeå University, Umeå, Sweden.

ABSTRACT
Immune evasion from NK surveillance related to inadequate NK-cell function has been suggested as an explanation of the high incidence of relapse and fatal outcome of many blood malignancies. In this report we have used Jurkat and Raji cell lines as a model for studies of the NKG2D receptor-ligand system in T-and B cell leukemia/lymphoma. Using real-time quantitative RT-PCR and immunoflow cytometry we show that Jurkat and Raji cells constitutively express mRNA and protein for the stress-inducible NKG2D ligands MICA/B and ULBP1 and 2, and up-regulate the expression in a cell-line specific and stress-specific manner. Furthermore, we revealed by electron microscopy, immunoflow cytometry and western blot that these ligands were expressed and secreted on exosomes, nanometer-sized microvesicles of endosomal origin. Acting as a decoy, the NKG2D ligand-bearing exosomes downregulate the in vitro NKG2D receptor-mediated cytotoxicity and thus impair NK-cell function. Interestingly, thermal and oxidative stress enhanced the exosome secretion generating more soluble NKG2D ligands that aggravated the impairment of the cytotoxic response. Taken together, our results might partly explain the clinically observed NK-cell dysfunction in patients suffering from leukemia/lymphoma. The adverse effect of thermal and oxidative stress, enhancing the release of immunosuppressive exosomes, should be considered when cytostatic and hyperthermal anti-cancer therapies are designed.

Show MeSH
Related in: MedlinePlus