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Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

Franklin C, Cesko E, Hillen U, Schilling B, Brandau S - PLoS ONE (2015)

Bottom Line: In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions.Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo.This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, University Hospital Essen, Essen, Germany; Research Division, Department of Otorhinolaryngology, University Hospital Essen, Essen, Germany.

ABSTRACT
Chronic graft-versus-host disease (cGVHD) is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

No MeSH data available.


Related in: MedlinePlus

Increased apoptosis and extracellular activity of arginase-1 in neutrophils of cGVHD patients ex vivo after ECP.Analysis was performed in neutrophils from peripheral venous blood of patients with cGVHD. (A) Peripheral venous blood samples of patients with cGVHD were taken prior to and 24h after ECP. (B) Apoptosis of neutrophils isolated from these samples was detected immediately after isolation and after 24h of culture via Annexin-V and 7-AAD staining using flow cytometry (n = 4). Data show means of Annexin-/7-AAD- (viable) cells ± standard deviation (SD). **p≤0.01. (C) Supernatants were generated of the neutrophils described in (A) +/- LPS for 24h and cytokines CXCL8 and CCL4 were quantified by ELISA. Concentrations are shown in pg/ml as mean of 4 patients ± SD. (D) Arginase-1 activity in these supernatants was determined enzymatically. Arginase activity is shown in U/L. Unit definition: 1 unit of arginase converts 1μmol of L-arginine to ornithine and urea per minute at 37°C and pH 9.5. *p≤0.05.
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pone.0134518.g005: Increased apoptosis and extracellular activity of arginase-1 in neutrophils of cGVHD patients ex vivo after ECP.Analysis was performed in neutrophils from peripheral venous blood of patients with cGVHD. (A) Peripheral venous blood samples of patients with cGVHD were taken prior to and 24h after ECP. (B) Apoptosis of neutrophils isolated from these samples was detected immediately after isolation and after 24h of culture via Annexin-V and 7-AAD staining using flow cytometry (n = 4). Data show means of Annexin-/7-AAD- (viable) cells ± standard deviation (SD). **p≤0.01. (C) Supernatants were generated of the neutrophils described in (A) +/- LPS for 24h and cytokines CXCL8 and CCL4 were quantified by ELISA. Concentrations are shown in pg/ml as mean of 4 patients ± SD. (D) Arginase-1 activity in these supernatants was determined enzymatically. Arginase activity is shown in U/L. Unit definition: 1 unit of arginase converts 1μmol of L-arginine to ornithine and urea per minute at 37°C and pH 9.5. *p≤0.05.

Mentions: If alterations in granulocytes are important for treatment of GVHD by ECP, such alterations should be found in neutrophils from venous blood taken by phlebotomy after completion of an ECP cycle. To test this hypothesis, blood samples were taken prior to and 24h after ECP cycles (Fig 5A). Neutrophils isolated from these samples are not directly treated by ECP but represent a mixture of a smaller number of ex vivo treated and reinfused leukocytes and a majority of untreated cells. Nevertheless, phenotypic and functional changes resembeling our in vitro and ex vivo data were found. Neutrophils isolated from blood samples 24h after ECP showed increased apoptosis after ex vivo culture compared to neutrophils isolated from samples taken before ECP treatment (p<0.01). At time points before 24h, no significant difference in apoptosis rates between both groups was detectable (Fig 5B). Furthermore, neutrophils isolated from samples taken after ECP show a decrease of their ability to release CCL4, but not CXCL8 (Fig 5C) and an increase of arginase-1 release (p<0.05) after LPS-stimulation (Fig 5D). Neutrophil granulocytes isolated from blood of cGVHD patients 24h after ECP are still capable of lymphocyte suppression (S2A Fig). The suppression is highest at a ratio of 2.5 MNC to 1 neutrophil (90% suppression) and lowest at a ratio of 20 MNC to 1 neutrophil (20% suppression) (S2B Fig). Neutrophils from blood of cGVHD patients after ECP are capable of suppressing both autologous and heterologous lymphocytes.


Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease.

Franklin C, Cesko E, Hillen U, Schilling B, Brandau S - PLoS ONE (2015)

Increased apoptosis and extracellular activity of arginase-1 in neutrophils of cGVHD patients ex vivo after ECP.Analysis was performed in neutrophils from peripheral venous blood of patients with cGVHD. (A) Peripheral venous blood samples of patients with cGVHD were taken prior to and 24h after ECP. (B) Apoptosis of neutrophils isolated from these samples was detected immediately after isolation and after 24h of culture via Annexin-V and 7-AAD staining using flow cytometry (n = 4). Data show means of Annexin-/7-AAD- (viable) cells ± standard deviation (SD). **p≤0.01. (C) Supernatants were generated of the neutrophils described in (A) +/- LPS for 24h and cytokines CXCL8 and CCL4 were quantified by ELISA. Concentrations are shown in pg/ml as mean of 4 patients ± SD. (D) Arginase-1 activity in these supernatants was determined enzymatically. Arginase activity is shown in U/L. Unit definition: 1 unit of arginase converts 1μmol of L-arginine to ornithine and urea per minute at 37°C and pH 9.5. *p≤0.05.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4524718&req=5

pone.0134518.g005: Increased apoptosis and extracellular activity of arginase-1 in neutrophils of cGVHD patients ex vivo after ECP.Analysis was performed in neutrophils from peripheral venous blood of patients with cGVHD. (A) Peripheral venous blood samples of patients with cGVHD were taken prior to and 24h after ECP. (B) Apoptosis of neutrophils isolated from these samples was detected immediately after isolation and after 24h of culture via Annexin-V and 7-AAD staining using flow cytometry (n = 4). Data show means of Annexin-/7-AAD- (viable) cells ± standard deviation (SD). **p≤0.01. (C) Supernatants were generated of the neutrophils described in (A) +/- LPS for 24h and cytokines CXCL8 and CCL4 were quantified by ELISA. Concentrations are shown in pg/ml as mean of 4 patients ± SD. (D) Arginase-1 activity in these supernatants was determined enzymatically. Arginase activity is shown in U/L. Unit definition: 1 unit of arginase converts 1μmol of L-arginine to ornithine and urea per minute at 37°C and pH 9.5. *p≤0.05.
Mentions: If alterations in granulocytes are important for treatment of GVHD by ECP, such alterations should be found in neutrophils from venous blood taken by phlebotomy after completion of an ECP cycle. To test this hypothesis, blood samples were taken prior to and 24h after ECP cycles (Fig 5A). Neutrophils isolated from these samples are not directly treated by ECP but represent a mixture of a smaller number of ex vivo treated and reinfused leukocytes and a majority of untreated cells. Nevertheless, phenotypic and functional changes resembeling our in vitro and ex vivo data were found. Neutrophils isolated from blood samples 24h after ECP showed increased apoptosis after ex vivo culture compared to neutrophils isolated from samples taken before ECP treatment (p<0.01). At time points before 24h, no significant difference in apoptosis rates between both groups was detectable (Fig 5B). Furthermore, neutrophils isolated from samples taken after ECP show a decrease of their ability to release CCL4, but not CXCL8 (Fig 5C) and an increase of arginase-1 release (p<0.05) after LPS-stimulation (Fig 5D). Neutrophil granulocytes isolated from blood of cGVHD patients 24h after ECP are still capable of lymphocyte suppression (S2A Fig). The suppression is highest at a ratio of 2.5 MNC to 1 neutrophil (90% suppression) and lowest at a ratio of 20 MNC to 1 neutrophil (20% suppression) (S2B Fig). Neutrophils from blood of cGVHD patients after ECP are capable of suppressing both autologous and heterologous lymphocytes.

Bottom Line: In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions.Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo.This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology, Venereology and Allergology, University Hospital Essen, Essen, Germany; Research Division, Department of Otorhinolaryngology, University Hospital Essen, Essen, Germany.

ABSTRACT
Chronic graft-versus-host disease (cGVHD) is a common side effect of allogeneic stem cell transplantation and a major cause of morbidity and mortality in affected patients. Especially skin, eyes and oral mucosa are affected. This can lead to pain and functional impairment. Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy with minimal side effects but its mode of action is still largely unknown. The objective of the present study was to examine the effects of ECP on neutrophil granulocytes in patients with cGVHD. Analysis of leukocytes from cGVHD patients obtained from the ECP device during treatment showed that neutrophil granulocytes account for the majority of cells treated during ECP. Neutrophils from healthy donors treated in vitro with 8-methoxypsoralen and UVA light as well as neutrophils from buffy coats of patients with cGVHD treated by ECP showed increased apoptosis and decreased half-life. In remaining non-apoptotic cells chemoirradiation resulted in loss of activation markers and reduced effector functions. This was accompanied by an increase in extracellular arginase-1 activity. Additional comparison of neutrophils isolated from blood of cGVHD patients before and 24h after ECP revealed a decreased half-life and reduction of effector functions of post-ECP neutrophils ex vivo. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place in vivo. This study is the first to show that ECP modulates apoptosis and inflammatory activity in neutrophil granulocytes, indicating that neutrophils may significantly contribute to the overall immunomodulatory effects attributed to this treatment.

No MeSH data available.


Related in: MedlinePlus