Extracorporeal photopheresis: a focus on apoptosis and cytokines.

Induction of apoptosis and changes to cytokine secretion patterns have been implicated in the mechanism of action of extracorporeal photopheresis (ECP). Lymphocyte apoptosis is initially detected in significant numbers prior to re-infusion and by 48 h post-ECP the majority of treated lymphocytes are apoptotic. The early apoptosis involves changes to mitochondrial function, reversal of the Bcl-2/Bax ratio and externalisation of phosphatidylserine. Apoptotic lymphocytes, observed from 20 h post-ECP, are associated with enhanced levels of CD95 and Fas-ligand. For cutaneous T cell lymphoma (CTCL), processing of the apoptotic lymphocytes, by suitable antigen presenting cells (APCs), is suggested to induce a clonal cytotoxic response which targets the malignant T cell population. Increased levels of TNFalpha and IFNgamma, observed post-ECP in monocytes and lymphocytes, respectively, are thought to further contribute to the proposed anti-tumour reaction seen in CTCL. However, down-regulation of pro-inflammatory cytokines and enhanced anti-inflammatory responses have been reported following ECP treatment. These immune responses may contribute to the tempering of the inflammatory conditions, such as graft versus host disease, which respond to ECP. Furthermore, untreated monocytes exposed to ECP-treated lymphocytes have also demonstrated a shift in monocyte cytokine-secretory pattern, toward one associated with immune tolerance. Recently, a mechanism of ECP-induced immune tolerance has been linked to the stimulation of the anti-inflammatory cytokines IL10 and TGFbeta by T regulatory cells, following the infusion of ECP-treated CD11c(+) APCs. Ultimately, the multifaceted responses, induced by ECP, may explain the diversity of clinical conditions that benefit.

Early reduction in number of T cells producing proinflammatory cytokines, observed after extracorporeal photopheresis, is not linked to apoptosis induction.

UNLABELLED: Immediately following ECP, a significant number of lymphocytes become apoptotic and the number of T cells producing TNFalpha and IFNgamma is reduced. This study sought to determine if the cytokine down-regulation was a direct consequence of apoptosis induction. METHODS: Samples were obtained from 6 graft versus host disease (GvHD) and 5 cutaneous T cell lymphoma (CTCL) patients immediately pre-ECP and from the leucocyte collection bag following 8-MOP/UVA exposure, but prior to re-infusion. Separated peripheral blood mononuclear cells (PBMC) were placed in cell culture and stimulated for 6 hours with phorbol myristate acetate (PMA), Ionomycin and Brefeldin A. Using flow cytometry, T cells were identified by CD3 expression and apoptotic T cells sub-selected by Annexin V staining. Both apoptotic and non-apoptotic T cells were evaluated for their intracellular expression of IL2, IL4, IL10, IFNgamma and TNFalpha. RESULTS: Neither patient group demonstrated a significant change in IL4 or IL10 expression post ECP. However the number of T cells expressing IL2, IFNgamma and TNFalpha was reduced in both the Annexin V-positive and -negative T cell populations (P <.05). The nonapoptotic T cells from GvHD patients demonstrated the greatest reduction in cytokine expression. CONCLUSIONS: Since proinflammatory cytokines play a major role in the pathology of GvHD, their down-regulation post-ECP may produce a direct clinical benefit. The lowest number of IL2-, IFNgamma- and TNFalpha-expressing T cells occurred within the apoptotic population; however, Annexin V-negative T cells also demonstrated a marked reduction post-ECP. However, the lack of an increase in IL4 and IL10 expression indicates that this process was not a consequence of skewing toward a Th2 cytokine profile.

Treatment of cutaneous T cell lymphoma with extracorporeal photopheresis induces Fas-ligand expression on treated T cells, but does not suppress the expression of co-stimulatory molecules on monocytes.

Following extracorporeal photopheresis (ECP), lymphocytes become apoptotic and upregulate class I MHC antigenic peptides. Conversely, ECP treated monocytes demonstrate activation markers and have an increased avidity for the phagocytosis of apoptotic T cells. Processing of apoptotic T cells by monocytes, following ECP, is thought to induce an immunomodulatory response, which targets untreated, but clonal T cells. Recently we detected apoptotic lymphocytes immediately post ECP. Although enhanced CD95 (Fas) expression has been observed 24 h post ECP, CD95 and Fas-ligand (Fas-L) expression have not been determined at this very early apoptotic stage. Exposure of monocytes to UV has previously suppressed expression of the co-stimulatory molecules required for the presentation of processed antigens to T cells. Our data demonstrate no increase in CD95 or Fas-L expression on T cells tested immediately following ECP. However, the number of T cells expressing Fas-L significantly increased 24 h post ECP (P<0.005). The expression of the co-stimulatory molecules, CD54, CD80 and CD86, remained unaltered on monocytes treated by ECP. Although the mechanism responsible for early induction of lymphocyte apoptosis remains unclear, the later apoptosis involves Fas-L expression. The maintenance of co-stimulatory molecules, on treated monocytes, indicates that they retain the ability to induce an immunomodulatory response.

Lymphocytes treated by extracorporeal photopheresis demonstrate a drop in the Bcl-2/Bax ratio: a possible mechanism involved in extracorporeal-photopheresis-induced apoptosis.

BACKGROUND: Recently, apoptosis has been identified in treated lymphocytes, prior to their re-infusion, when tested ex vivo. Previous work has demonstrated a close association between the genes p53, Bcl-2 and Bax and apoptosis induced by UV irradiation. OBJECTIVES: We wanted to establish whether the expression of the protein product of these genes was altered in lymphocytes treated with extracorporeal photopheresis (ECP) prior to re-infusion and therefore possibly implicated in the early apoptosis observed. METHOD: Lymphocytes were isolated immediately before treatment and immediately prior to re-infusion and tested for intracellular levels of p53, Bcl-2 and Bax proteins. RESULTS: No increase in p53 expression was observed at re-infusion; however, the mean fluorescent intensity ratio of the apoptotic inhibitor protein Bcl-2 to the apoptosis-inducing protein Bax dropped significantly. CONCLUSION: The early apoptosis observed in ECP-treated lymphocytes at re-infusion might be attributed to dysregulation in the expression of the apoptotic genes Bcl-2 and Bax.

Extracorporeal photopheresis in cutaneous T-cell lymphoma and graft-versus-host disease induces both immediate and progressive apoptotic processes.

BACKGROUND: Extracorporeal photopheresis (ECP) therapy is used in the treatment of many T-cell-mediated conditions including cutaneous T-cell lymphoma and graft-versus-host disease and involves the reinfusion of a patient's own white cells following exposure to 8-methoxypsoralen and ultraviolet A. ECP has been demonstrated to induce significant levels of apoptosis in treated lymphocytes. Previous work has highlighted the importance of mitochondria and the caspase cascade in the regulation and execution of apoptosis and, more recently, a functional role for CD10 has been proposed for apoptotic lymphoid cells in vivo. OBJECTIVES: To determine the effect of ECP on phosphatidylserine (PS) exposure, mitochondrial function, caspase activation and CD10 expression of treated lymphocytes. METHODS: Lymphocytes were tested pre-ECP and at several stages post-ECP for changes to PS, mitochondrial transmembrane potential (DeltaPsim), activated caspases and CD10 expression. RESULTS: Early apoptosis induced a disruption in DeltaPsim, while caspase activation was not observed until 24 h post-ECP. CD10 expression was very weak and "late" in the apoptotic process. CONCLUSIONS: The early induction of apoptosis by ECP involves mitochondrial dysfunction, while later apoptosis is associated with the activation of caspases. CD10 expression was very weak and "late", preceded by a strong PS exposure. These apoptotic processes, in vivo, would induce the immediate and progressive phagocytosis of the majority of ECP-treated lymphocytes within 48 h.

D-dimer assay for deep vein thrombosis: its role with colour Doppler sonography.

AIM: To evaluate the role of a negative D-dimer assay in the initial management of patients with clinically suspected deep venous thrombosis (DVT), using colour Doppler ultrasound as the primary diagnostic technique. MATERIALS AND METHODS: A double-blind prospective trial was performed on 143 patients with clinically suspected DVT. All patients underwent a D-dimer assay prior to anticoagulant therapy. DVT was confirmed or excluded by diagnostic colour Doppler ultrasound within 24 h of presentation. RESULTS: In nearly one-third of the cases (31.8%), Doppler ultrasound was positive. The D-dimer assay demonstrated a sensitivity of 97.7% with only one false-negative, but the specificity was low at 48.9% with 45 false-positive results. The positive predictive value for D-dimer assay was 48.8%, whilst the important negative predictive value was 98%. CONCLUSION: If D-dimer was used to screen for DVT, and patients with negative results were not imaged, then the imaging workload could be reduced by 35%. In this study one small calf vein thrombus would have been missed by adopting this practice.Bradley, M. (2000). Clinical Radiology 55, 525-527.

Extracorporeal photopheresis induces apoptosis in the lymphocytes of cutaneous T-cell lymphoma and graft-versus-host disease patients.

Extracorporeal photopheresis (ECP) is used in the treatment of T-cell-mediated disorders. However, the mechanism by which ECP achieves its effect remains illusive. Over recent years the ability of ECP to induce apoptosis has been demonstrated by cell culture experiments and retrospective histological analysis. We investigated if apoptosis could be determined in samples tested ex vivo from the UVAR:ECP system. Lymphocytes from 11 patients (six with cutaneous T-cell lymphoma, four with graft-versus-host disease, and one with scleredema) were isolated at three stages of the ECP process: immediately before ECP treatment, from the first buffy coat collected, and post UV irradiation, prior to re-infusion. Using flow cytometry each stage was tested for the early apoptotic markers; Annexin V, ApoptestTM and Carboxy-SNARF-1-AM. Comparisons of the pre-ECP and pre-infusion samples demonstrated a significant increase in apoptotic lymphocytes for all three flow cytometric techniques (P < 0.01). Increases between the pre-ECP and first buffy coat, used as a measure of the extracorporeal manipulation, were much lower. These results demonstrate that ECP directly induces significant levels of apoptosis in lymphocytes of CTCL, GvHD and scleredema patients. The apoptosis of these lymphocytes may contribute to the ECP effect.