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

<div><p>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 <i>in vitro</i> 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 <i>ex vivo</i>. These observations strongly suggest that ECP induces both apoptosis and physiological changes in neutrophils and that these changes also take place <i>in vivo</i>. 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.</p></div

Modulation and Apoptosis of Neutrophil Granulocytes by Extracorporeal Photopheresis in the Treatment of Chronic Graft-Versus-Host Disease - Fig 2

<p><b>(A)</b><i>In vitro</i> treatment of healthy donor neutrophils with 8-methoxypsoralen and UVA induces apoptosis and decreases activation markers. Neutrophils, isolated from blood of healthy donors, were treated with 8-MOP and UVA light and cultured for 24h. Control groups were untreated neutrophils, neutrophils treated with 8-MOP and neutrophils treated with UVA light. Percentage of non-apoptotic cells was determined by the % of Annexin V and 7-AAD double-negative cells via flow cytometry 0, 4, 8 and 24h after irradiation (n = 4). Curves show mean ± standard deviation (SD). ** p≤0.01. <b>(B-D)</b> Viable (= Annexin^- / 7-AAD^-) neutrophils of the same groups (n = 4) were stained for activation markers CD11b, CD16 and CD54 at 2, 4, 8 and 24h after irradiation and protein expression was measured by flow cytometry after gating on viable cells. Curves show mean of Δ median of MFI ± SD. MFI = mean fluorescence intensity, Δ median of MFI = MFI of antibody-MFI of isotype shown.</p

Reduced activation and increased extracellular arginase-1 activity in chemoirradiated neutrophils from buffy coat of cGVHD patients.

<p>(A) Neutrophil granulocytes and mononuclear cells substituted with 8-methoxypsoralen (8-MOP) were obtained from the buffy coat of patients with cGVHD treated with a Therakos UVAR XTS device before (pre-UVA) and after (post-UVA) irradiation. (B) Neutrophils of the buffy coat before and after irradiation with UVA (as described in A) were stained with Annexin V and 7-AAD. Fold change of Annexin V^-/7-AAD^-double negative (viable) cells determined after 24h of ex vivo culture after treatment normalized to cells assessed directly after treatment (0h). Data show means of 3 patients ± standard deviation (SD). **p≤0.01. (C) Neutrophils from buffy coats of cGVHD patients (n = 3) were stained with antibodies against activation markers CD11b, CD16 and CD54 after 24h in culture. Expression was measured by flow cytometry after gating on viable cells. Data shown as difference of the median of the mean fluorescence intensity (mfi) of the specific antibody and the isotype. (D) Supernatants of neutrophils of the samples described in (A) were generated with and without stimulation with LPS for 24h and concentration of CXCL8 and CCL4 was quantified by ELISA. Mean values ± standard deviation (SD) of 3 patients in pg/ml shown. (E) Arginase activity in the same supernatants as in (D) was determined by enzymatic activity (n = 3). Values 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</p

Neutrophils account for the majority of cells treated during ECP.

<p>(A) Percentages of neutrophils, lymphocytes and monocytes of total leukocytes in buffy coats of 16 patients with chronic GVHD (cGVHD) taken from Therakos UVAR XTS ECP- device immediately after chemoirradiation prior to reinfusion. Lines show means; scatters represent individual patients. **p≤0.01, ****p≤0.0001. (B) Representative composition of a buffy coat of one patient with cGVHD.</p

<i>In vitro</i> treatment with 8-methoxypsoralen and UVA reduces pro-inflammatory effector functions in healthy donor neutrophils.

<p>(A) Neutrophil granulocytes of healthy donors were stimulated with phorbol 12-myristate13-acetate (PMA) for 0, 4, 8 and 24h after treatment with 8-methoxypsoralen and UVA light. Nitric oxide was quantified via flow cytometry by staining with DAF-FM Diacetate after gating on viable cells. Data shown from one representative donor. (B) After 24h of stimulation with LPS, release of CXCL8 and CCL4 into the supernatants was detected by ELISA. Concentrations are shown as x-fold change compared to unstimulated neutrophils. Mean changes of 6 donors ± standard deviation (SD) shown. **p≤0.01. (C) Supernatants of untreated neutrophils and neutrophils treated with 8-MOP and UVA were generated after 24h stimulation with LPS, TNFα and IFNγ (n = 6). Arginase activity was determined enzymatically. 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 = 0.04</p

Experimental design.

<p>During acquisition, patients in the conditioned group received desloratadine (US) in combination with the CS. During evocation, the drug was replaced by placebo pills. Sham-conditioned patients received the CS together with placebo pills throughout the study. During 3 subsequent days during acquisition (2–4) and evocation (17–19) patients were instructed to intake the pills together with the drink (CS) at home.</p

Sociodemographic and psychological characteristics.

<p>BDI = Becks Depression Inventory; STAI X2 = Trait anxiety form of the State-trait anxiety inventory.</p>*<p>Results of Chi² Test or univariate ANOVA.</p

Conditioned and sham-conditioned patients show reduced allergic reactions during evocation.

<p><b>A</b> After skin prick test wheal size (mm) was analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in the natural history group. <b>B</b> Symptom scores after nasal provocation were analyzed before and after acquisition as well as before and after the 1^st and 5^th evocation to the CS in patients in the conditioned, sham-conditioned as well as patients in natural history group. Data are presented as mean ±SEM. *p<0.05 **p<0.01 ***p<0.001, comparison against natural history group.</p