Blockade of Fas-dependent apoptosis by soluble Fas in LGL leukemia

Altered expression of the Fas-Fas ligand apoptotic pathway leads to lymphoproliferative and autoimmune diseases. In lpr/lpr mice and children with autoimmune lymphoproliferative syndrome, defective apoptosis is due to Fas mutations. Large granular lymphocyte (LGL) leukemia is a clonal lymphoproliferative disorder associated with rheumatoid arthritis. Leukemic LGLs are resistant to Fas-dependent apoptosis despite expressing high levels of Fas. Such resistance can be overcome by activating leukemic LGLs in vitro, suggesting inhibition of Fas signaling in leukemic cells. We report that sera from patients with LGL leukemia contain high levels of soluble Fas. Ten of these 33 patients with LGL leukemia also had rheumatoid arthritis. Cloning and sequencing revealed expression of multiple Fas messenger RNA variants in leukemic LGL. These Fas variants, including 3 newly described here, encode soluble Fas molecules. Supernatants from cells transfected with these Fas variants blocked Fas-dependent apoptosis of leukemic LGLs. These results suggest that blockade of Fas-signaling by soluble Fas may be a mechanism leading to apoptosis resistance in leukemic LGLs.     

Enhanced neutrophil apoptosis in neutropenic patients with hepatosplenic schistosomiasis: evidence of serum Fas ligand

Enhanced neutrophil apoptosis has been reported in neutropenic hepatosplenic schistosomiasis. The shortening of neutrophil survival via apoptosis may explain the neutropenia that occur in these patients. However, the regulation of neutrophil apoptosis in hepatosplenic schistosomiasis has not been clearly defined. Neutrophils harvested from neutropenic patients with hepatosplenic (HS) schistosomiasis, (n=25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n=10), and age-/gender-matched healthy control subjects (n=10) were incubated with autologous serum. Neutrophils apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 (i.e. fresh neutrophils), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Fas expression was assessed in fresh neutrophils using flow cytometry. Compared with normal healthy neutrophils, and HI neutrophils, neutropenic neutrophils demonstrated greater apoptosis in the presence of autologous serum (P<0.01, 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates ( P<0.01). Moreover, anti-Fas L antibody attenuated the neutropenic serum-induced neutrophil apoptosis in normal neutrophils. Fas expression was significantly higher in the neutropenic group when compared to both HI and normal healthy controls (P<0.05). In addition, Fas expression by neutrophils was paralleled by high neutrophil apoptosis. On the other hand, neutrophil apoptosis was not correlated to the size of spleen in neutropenic group. In conclusion, the rate of neutrophil apoptosis is accelerated in patients with neutropenic hepatosplenic schistosomiasisis. These findings suggest that the enhanced neutrophil apoptosis that occurs in neutropenic HS patients is triggered by a serum factor, which is mostly a Fas ligand.     

Enhanced Neutrophil Apoptosis in Neutropenic Patients with Hepatosplenic Schistosomiasis: Evidence of Serum Fas Ligand

Enhanced neutrophil apoptosis has been reported in neutropenic hepatosplenic schistosomiasis. The shortening of neutrophil survival via apoptosis may explain the neutropenia that occur in these patients. However, the regulation of neutrophil apoptosis in hepatosplenic schistosomiasis has not been clearly defined. Neutrophils harvested from neutropenic patients with hepatosplenic (HS) schistosomiasis, (n=25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n=10), and age-/gender-matched healthy control subjects (n=10) were incubated with autologous serum. Neutrophils apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 (i.e. fresh neutrophils), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Fas expression was assessed in fresh neutrophils using flow cytometry. Compared with normal healthy neutrophils, and HI neutrophils, neutropenic neutrophils demonstrated greater apoptosis in the presence of autologous serum (P<0.01, 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates ( P<0.01). Moreover, anti-Fas L antibody attenuated the neutropenic serum-induced neutrophil apoptosis in normal neutrophils. Fas expression was significantly higher in the neutropenic group when compared to both HI and normal healthy controls (P<0.05). In addition, Fas expression by neutrophils was paralleled by high neutrophil apoptosis. On the other hand, neutrophil apoptosis was not correlated to the size of spleen in neutropenic group.

In conclusion, the rate of neutrophil apoptosis is accelerated in patients with neutropenic hepatosplenic schistosomiasisis. These findings suggest that the enhanced neutrophil apoptosis that occurs in neutropenic HS patients is triggered by a serum factor, which is mostly a Fas ligand.     

Accelerated neutrophil apoptosis in neutropenic patients with hepatosplenic schistosomiasis is induced by serum Fas ligand

Neutropenia in patients with hepatosplenic (HS) schistosomiasis may stem from enhanced neutrophil apoptosis. However, the molecular mechanism of neutrophil apoptosis has not been clearly defined. Neutrophils harvested from neutropenic patients with HS schistosomiasis (n = 25), non-neutropenic patients with hepatointestinal (HI) schistosomiasis (n = 10), and age- and sex-matched healthy control subjects (n = 10) were examined for the degree of apoptosis after incubation with autologous sera. Neutrophil apoptosis was quantified by flow cytometry through determination of propidium iodide nuclear staining and confirmed by DNA gel electrophoresis at 0 time (fresh neutrophil), 4 and 24 h culture. Neutrophils from healthy subjects were also incubated with either 10% heterologous normal or neutropenic serum, with and without anti-Fas ligand antibody. Serum Fas ligand levels were assessed in sera of patient groups and healthy controls by ELISA. Compared with normal controls and HI, HS group demonstrated greater neutrophil apoptosis in the presence of autologous serum (P < 0.01, < 0.05, respectively). Furthermore, compared with normal neutrophils exposed to heterologous normal serum, those exposed to heterologous neutropenic serum exhibited higher apoptosis rates (P < 0.01). The apoptotic effect of neutropenic sera is attenuated by anti-Fas ligand. Fas expression was significantly higher in HS group as compared to both HI and normal healthy controls (P < 0.05). Serum Fas ligand levels were significantly higher among HS group as compared to both HI and control groups (P < 0.01 for both). Neutrophil apoptosis was not correlated to the size of spleen in HS group. In conclusion, the rate of neutrophil apoptosis is accelerated in neutropenic HS schistosomiasis. These findings suggest that enhanced neutrophil apoptosis demonstrated in HS patients is triggered by soluble Fas ligand, which is mostly derived from spleen.     

Clinicopathological features of aggressive large granular lymphocyte leukaemia resemble Fas ligand transgenic mice

Fas ligand triggers cell death after interaction with its receptor Fas. Altered expression of Fas has been associated with lymphoproliferation and autoimmune disorders in both mice and man. Apoptosis of lung and liver tissue is seen in Fas ligand transgenic mice. It is not known whether constitutive expression of Fas ligand can cause a similar human disease. Four patients with aggressive large granular lymphocyte (LGL) leukaemia involving lung and liver were studied. All four patients were severely ill with pulmonary involvement. Two patients presented with hypoxia and were oxygen dependent; the other two patients had severe pulmonary hypertension. Lung biopsies showed interstitial infiltration by leukaemic LGL. The infiltrating lymphocytes expressed both Fas and Fas ligand, whereas normal pneumocytes expressed only Fas. Similar findings were observed in liver biopsies from these patients. Features mimicking the pathological changes of graft-versus-host disease were observed, including pneumocyte apoptosis. All four patients had high levels of circulating Fas ligand. Successful treatment with oral methotrexate or 2-chlorodeoxyadenosine was associated with disappearance or marked reduction of circulating Fas ligand. These results suggest that dysregulated expression of Fas ligand can lead to human disease with pathological features resembling graft-versus-host disease.     

Current concepts: large granular lymphocyte leukemia

Clonal diseases of large granular lymphocyte (LGL) disorders can arise from a CD3+ T-cell lineage or from a CD3- NK-cell lineage. CD3+ LGL leukemia is the most frequent form of LGL leukemia. T-LGL leukemia usually affects elderly people. Approximately 60% of patients are symptomatic; recurrent infections secondary to chonic neutropenia, anemia, and rheumatoid arthrititis are the main clinical manifestations. The most common phenotype is CD3+, alphabeta+, CD8+, CD57+. Clonality is detected by clonal rearrangement of the T-cell receptor gene. NK-cell LGL proliferative disorders include NK LGL leukemia which is a very aggressive disease and NK chronic lymphocytosis. Serologic findings show frequent reactivity to the BA21 epitope of HTLV-I env p21e, suggesting that a cellular or retroviral protein with homology to BA21 may be important in pathogenesis of these diseases. Clonal expansion may be facilitated by IL12 and IL15 cytokines expressed by leukemic LGL, and also by a defective Fas (CD95) apoptotic pathway. Leukemic LGL constitutively express Fas and Fas-Ligand but they are resistant to Fas-induced apotosis. Neutropenia could be due to soluble Fas-Ligand which is highly secreted in the patient's sera. Clinical and molecular remission can be obtained with oral low-dose methotrexate. Leukemic LGL express a multi-drug resistance phenotype (PgP+/LRP+) that could partly explain the chemoresistance observed in aggressive cases. It is suggested that LGL leukemia can serve as a useful model of dysregulated apoptosis as an underlying mechanism for both malignancy and autoimmune disease.     

Antigen activation and impaired Fas-induced death-inducing signaling complex formation in T-large-granular lymphocyte leukemia

Clonal T-cell expansion in patients with T-large-granular lymphocyte (LGL) leukemia occurs by an undefined mechanism that may be related to Fas apoptosis resistance. Here, we demonstrate polarized expansion of CD8(+) terminal-memory differentiation in such patients, as demonstrated by CD45RA expression and absence of CD62L expression, suggesting repeated stimulation by antigen in vivo. Elimination of antigen-stimulated T cells normally occurs through Fas-mediated apoptosis. We show that cells from LGL leukemia patients express increased levels of c-FLIP and display resistance to Fas-mediated apoptosis and abridged recruitment of proteins that comprise the death-inducing signaling complex (DISC), including the Fas-associated protein with death-domain (FADD) and caspase-8. Exposure to interleukin-2 (IL-2) for only 24 hours sensitized leukemic LGL to Fas-mediated apoptosis with enhanced formation of the DISC, and increased caspase-8 and caspase-3 activities. We observed dysregulation of c-FLIP by IL-2 in leukemic LGL, suggesting a role in Fas resistance. Our results demonstrate that expanded T cells in patients with LGL leukemia display both functional and phenotypic characteristics of prior antigen activation in vivo and display reduced capacity for Fas-mediated DISC formation.     

Assessment of neutrophil apoptosis ex vivo in hepatosplenic patients with neutropenia pre and post splenectomy

The pathophysiology of neutropenia seen in patients with schistosomiasis or hepatitis C infection that complicates the course of liver disease is poorly understood. We evaluated the neutrophil apoptosis before and after splenectomy to clarify the role of apoptosis and splenomegaly in the occurrence of neutropenia. Neutrophils were isolated from 23 hepato-splenic patients with neutropenia, 8 hepatosplenic patients with normal neutrophil counts, 7 patients who were post splenectomy, and a further ten normal control subjects. These were cultured for 24 h and the time course of neutrophil apoptosis was assessed by determination of Annexin V and propidium iodide binding by flow cytometry. Fas and Bcl2 expression were determined on fresh neutrophils using flow cytometry. Levels of tumor necrosis factor alpha, interleukin 3, and gamma interferon were evaluated using an immunosorbent assay. Neutrophil apoptosis was minimal in the fresh neutrophils, however, cultured neutrophils exhibited significantly greater apoptosis in neutropenic patients when compared to non-neutropenic patients (P=0.01 at 4 h and P<0.05 at 24 h) and control group (P<0.01 at 4 h and 24 h). After splenectomy, the percentage of neutrophil apoptosis declined to the normal control levels (P>0.05). Fas and Bcl2 expression on neutrophil were significantly higher in the neutropenic group as compared to normal controls (P<0.05, P=0.01 respectively). Serum TNF alpha, IL-3, and IFN gamma levels were not significantly different in all studied groups. In conclusion: Neutrophils from neutropenic hepatosplenic patients exhibit markedly accelerated apoptosis, which is normalized after splenectomy. Thus increased neutrophil apoptosis may in part be responsible for the occurrence of neutropenia.     

Dysregulation of CD95/CD95 Ligand-Apoptotic Pathway in CD3+ Large Granular Lymphocyte Leukemia

CD95 (Fas)-induced apoptosis plays a critical role in theelimination of activated lymphocytes and induction of peripheral tolerance. Defects in CD95/CD95L (Fas-Ligand)-apoptotic pathway have been recognized in autoimmune lymphoproliferative diseases (ALPS)and lpr or gld mice and attributed to CD95 and CD95Lgene mutations, respectively. Large granular lymphocyte (LGL) leukemia is a chronic disease characterized by a proliferation ofantigen-activated cytotoxic T lymphocytes. Autoimmune features such ashypergammaglobulinemia, rheumatoid factor, and circulating immunecomplexes are common features in LGL leukemia and ALPS. Therefore, wehypothesize that expansion of leukemic LGL may be secondary to adefective CD95 apoptotic pathway. In this study, we investigatedexpression of CD95 and CD95L in 11 patients with CD3⁺ LGLleukemia and explored the apoptotic response to agonistic CD95monoclonal antibody (MoAb). We found that leukemic LGL from eachpatient expressed constitutively high levels of CD95/CD95L, similar tothose seen in normal activated T cells. However, cells from 9 of these11 patients were totally resistant to anti-CD95-induced apoptosis.Similarly, cells were resistant to anti-CD3-MoAb-triggered cell death.Lack of anti-CD95-induced apoptosis was not due to mutations in theCD95 antigen. Leukemic LGL were not intrinsically resistant toCD95-dependent death, because LGL from all but 1 patient underwentapoptosis after phytohemagglutinin/interleukin-2 activation. Thepatient whose leukemic LGL were intrinsically resistant to CD95 had anaggressive form of LGL leukemia that was resistant to combinationchemotherapy. These findings that leukemic LGL are resistant toCD95-dependent apoptosis despite expressing high levels of CD95 aresimilar to observations made in CD95L transgenic mice. These datasuggest that LGL leukemia may be a useful model of dysregulatedapoptosis causing human malignancy and autoimmune disease.     

Assessment of Neutrophil Apoptosis Ex Vivo in Hepatosplenic Patients with Neutropenia Pre and Post Splenectomy

The pathophysiology of neutropenia seen in patients with schistosomiasis or hepatitis C infection that complicates the course of liver disease is poorly understood. We evaluated the neutrophil apoptosis before and after splenectomy to clarify the role of apoptosis and splenomegaly in the occurrence of neutropenia. Neutrophils were isolated from 23 hepato-splenic patients with neutropenia, 8 hepatosplenic patients with normal neutrophil counts, 7 patients who were post splenectomy, and a further ten normal control subjects. These were cultured for 24 h and the time course of neutrophil apoptosis was assessed by determination of Annexin V and propidium iodide binding by flow cytometry. Fas and Bcl2 expression were determined on fresh neutrophils using flow cytometry. Levels of tumor necrosis factor alpha, interleukin 3, and gamma interferon were evaluated using an immunosorbent assay.

Neutrophil apoptosis was minimal in the fresh neutrophils, however, cultured neutrophils exhibited significantly greater apoptosis in neutropenic patients when compared to non-neutropenic patients (P=0.01 at 4 h and P<0.05 at 24 h) and control group (P<0.01 at 4 h and 24 h). After splenectomy, the percentage of neutrophil apoptosis declined to the normal control levels (P>0.05). Fas and Bcl2 expression on neutrophil were significantly higher in the neutropenic group as compared to normal controls (P<0.05, P=0.01 respectively). Serum TNF alpha, IL-3, and IFN gamma levels were not significantly different in all studied groups.

In conclusion: Neutrophils from neutropenic hepatosplenic patients exhibit markedly accelerated apoptosis, which is normalized after splenectomy. Thus increased neutrophil apoptosis may in part be responsible for the occurrence of neutropenia.     

Large granular lymphocyte leukemia. Report of 38 cases and review of the literature

LGL leukemia results from a chronic, clonal proliferation of LGL. Chronic neutropenia with recurrent bacterial infection and splenomegaly are common clinical manifestations. Rheumatoid arthritis coexists in some of these patients, who thus resemble patients with Felty syndrome. Other hematologic abnormalities that may occur include pure red-cell aplasia and adult-onset cyclic neutropenia. Lymphoid infiltration of bone marrow, splenic red pulp cords, and hepatic sinusoids is characteristic; lymph node and skin involvement are rare. Multiple serologic abnormalities are frequently present, including positive tests for rheumatoid factor and/or antinuclear antibody, polyclonal hypergammaglobulinemia, and circulating immune complexes. Antineutrophil and antiplatelet antibodies are often present. Leukemic LGL exhibit phenotypic heterogeneity; the most common phenotype in our patients is CD2+, CD3+, CD8+, HNK-1+, CD16-. Despite markedly increased numbers of LGL, functional activity of the cells is usually decreased. The mechanism of cytopenias is uncertain: in pure red-cell aplasia, it appears to be due to suppressive effect on erythropoiesis by abnormal LGL, but in patients with chronic neutropenia it may be antibody-mediated. Although most patients appear to have a relatively benign clinical course, mortality from infections and progressive lymphoproliferation is substantial. Optimal therapy remains undefined. Some preliminary evidence suggests that LGL leukemia may be associated with infection with a retrovirus similar to HTLV-I. Although relatively rare, LGL leukemia is of interest because a better understanding of this disease process may contribute to our knowledge of autoimmune diseases, the immunoregulatory functions of LGL, and the mechanisms controlling normal hematopoiesis.     

Expression of Fas Antigen (CD95) on Human Leukemic Cells and Assessment of Apoptosis on Fas+ and Fas-Samples by Flowcytometry Method

Regulation of normal cell growth and turnover is balanced between cell proliferation, cell differentiation and apoptosis.A disruption of this balance is thought to be an important event leading to carcinogenesis .One of the effector molecules in apoptosis is Fas antigen . Crosslinking of Fas by its ligand (Fas L) or agonistic anti Fas antibodies induces apoptosis of cells expressing Fas on the membrane by triggering cascade of caspaces. The aim of this research was to study the percent of expression of Fas antigen on bone marrow and peripheral blood cells in 100 patients suffering from acute lymphoid and myeloid leukemia by flow cytometry method. Sample were obtained at the time of diagnosis before antileukemic therapy. Expression of Fas antigen on normal control peripheral leukocytes was also analysed. From these data, it was found that Fas antigen is expressed in all cases, but the expression level varied widely. The percentage of Fas antigen expression in all of acute lymphoid leukemia samples was below 20%, but in acute myeloid leukemia samples, 8 out of 50 cases was above 20%. In normal control samples, the mean value for monocytes was higher than granulocytes and in granulocytes higher than lymphcytes. Expression of Fas antigen in most of the leukemic cells was low and the preliminary results showed that increase in Fas antigen expression above 20% after treatment, is a favorable prognostic sign associated with increase relapse free and total survival. Thus evaluation of this antigen before, during and after treatment is recommended.     

TNF-related apoptosis-inducing ligand is involved in neutropenia of systemic lupus erythematosus

Neutropenia is a common laboratory finding in systemic lupus erythematosus (SLE). However, the molecular mechanism of SLE neutropenia has not been fully explained. In this study, we examined whether TNF-related apoptosis-inducing ligand (TRAIL) is involved in the pathogenesis of SLE neutropenia using samples from SLE patients. Serum TRAIL levels in SLE patients with neutropenia were significantly higher than those of SLE patients without neutropenia and healthy volunteers. Serum TRAIL levels showed a significant negative correlation with neutrophil counts in SLE patients. The expression of TRAIL receptor 3 was significantly lower in SLE patients with neutropenia than in patients without neutropenia or in healthy volunteers. Treatment with glucocorticoids negated the decrease of TRAIL receptor 3 expression on neutrophils of SLE patients. TRAIL may accelerate neutrophil apoptosis of neutrophils from SLE patients, and autologous T cells of SLE patients, which express TRAIL on surface, may kill autologous neutrophils. Interferon gamma and glucocorticoid modulated the expression of TRAIL on T cells of SLE patients and also modulated the expression of cellular Fas-associating protein with death domain-like interleukin-1 beta-converting enzyme (FLICE)-inhibitory protein (cFLIP), an inhibitor of death receptor signaling, in neutrophils. Thus, our results provide a novel insight into the molecular pathogenesis of SLE neutropenia.     

CD8+, CD57+ T cells from healthy elderly subjects suppress neutrophil development in vitro: implications for the neutropenia of Felty's and large granular lymphocyte syndromes

OBJECTIVE: To investigate the ability of CD8+,CD57+ large granular lymphocytes (LGL) from normal individuals and from Felty's syndrome (FS) or LGL syndrome patients to suppress allogeneic neutrophil precursor development. METHODS: Six FS patients, 5 LGL syndrome patients, and 13 elderly controls were studied. CD8+,CD57+ T cells were cocultured with cord blood-derived stem cells, and percentage inhibition was calculated. Recombinant chemokines and Fas-stimulating molecules were used in separate cultures to address possible mechanisms of suppression. Proliferation after stimulation with interleukin-2 (IL-2) and anti-CD3 was assessed. RESULTS: Significant (79%) suppression of colony-forming unit-granulocyte-macrophage (CFU-GM) by the CD8+,CD57+ subset was shown by 1 FS patient. None of the CD8+,CD57+ cells from LGL syndrome patients had any effect. Six of 13 controls studied showed >40% inhibition of CFU-GM, and all but 2 showed at least some suppression. The suppressive effect was not mediated by Fas/Fas ligand interactions or by the chemokines macrophage inhibitory protein 1alpha or IL-8. LGL from both patients and controls were largely CD28- and had reduced proliferative capacity. CONCLUSION: In a subset of FS patients, expansion of CD8+,CD57+ T cells in the bone marrow may be responsible for neutropenia by suppressing neutrophil precursors. This effect is also seen with normal LGL, which are likely to have an important function in neutrophil homeostasis.     

Cellular expressions and serum concentrations of Fas ligand and Fas receptor in patients with infectious mononucleosis

Although lymphocytosis and neutropenia are commonly associated with infectious mononucleosis (IM), the precise mechanisms involved remain unclear. Accumulated evidence has revealed that the apoptosis-mediating system, Fas receptor/Fas ligand (Fas-R/Fas-L), plays an important role in this disease. Recently, lymphocytes, monocytes, and neutrophils have been reported to constitutively express Fas-R, and the Epstein-Barr virus (EBV) has been shown to activate, in addition to B cells, peripheral blood CD8+ T cells, monocytes, and neutrophils. We elucidated cell surface expression and serum concentrations of Fas-R and Fas-L in patients with IM in an effort to more clearly define the role and contribution of apoptosis in this disease. The expression of lymphocyte surface Fas-L and Fas-R was significantly increased in patients with IM (P < .005 and P < .001, respectively), and among lymphocytes, CD4+ or CD8+ populations contained Fas-R+ as well as Fas-R- subpopulations. The constitutive Fas-R expression levels of monocytes and neutrophils were also increased in IM. Moreover, serum levels of both soluble Fas-L and Fas-R were significantly higher in patients with IM than in healthy volunteers (P < .001 and P < .0001, respectively). Positive relationships between the number of peripheral blood CD95+ lymphocytes and white blood cell count, number of lymphocytes, or number of CD4+ or CD8+ lymphocytes were observed. Our results suggest that the Fas-R/Fas-L system might play a role in eliminating EBV-infected or -activated peripheral blood cells by cell-to-cell contact or in an autocrine and/or paracrine fashion in patients with IM.     

Chronic neutropenia associated with autoimmune disease

Chronic neutropenia with autoimmune diseases is associated mainly with rheumatoid arthritis (RA), as Felty's syndrome or large granular lymphocyte (LGL) leukemia, and with systemic lupus erythematosus (SLE). Recent advances have allowed better understanding regarding the mechanism of neutropenia and improved options for treatment. Target antigens for antineutrophil antibodies have been identified for both Felty's syndrome and for SLE. The role of soluble Fas-ligand (FasL) in inducing apoptosis of neutrophils has been clarified for LGL leukemia and increased neutrophil apoptosis has been described in neutropenic patients with SLE. The role of immune complexes in affecting neutrophil traffic and function continues to be studied. Treatments of neutropenia have included methotrexate, cyclosporine A, and granulocyte colony-stimulating factor (G-CSF) as well as granulocyte-macrophage colony-stimulating factor (GM-CSF). The efficacy of both GM- and G-CSF in reversing neutropenia and decreasing the risk of infections in Felty's syndrome and SLE has been well documented. Of concern, however, have been flares of symptoms or development of leukocytoclastic vasculitis in some patients following the use of these cytokines. Recent results suggest that in these patients G-CSF should be administered at the lowest dose effective at elevating the neutrophil count above 1,000/microL.     

Fas-mediated apoptosis in acute alcoholic hepatitis

BACKGROUND/AIMS: The Fas-mediated apoptosis pathway has been implicated in liver diseases. The aim of the study was to investigate the role of this system in alcoholic hepatitis. METHODOLOGY: The expression of Fas, Fas ligand, and intracellular signaling molecules for apoptosis were determined by immunoblot analysis in fresh frozen liver samples from 19 patients with alcoholic liver disease. RESULTS: Fas and Fas ligand expression was significantly increased in the liver of patients with alcoholic hepatitis (n = 11) as compared with alcoholic liver disease patients without hepatitis (n = 8). Similarly, there were significant differences in the expression of FADD, ICE, and CPP32 in the liver between the two groups. There were significant positive correlations between the Fas ligand and the FADD, ICE, or CPP32 levels in the liver. The expression of Fas, Fas ligand, FADD, ICE, or CPP32 correlated with serum markers of hepatic injury. Plasma soluble Fas levels in patients with alcoholic hepatitis (median: 15.1 U/mL, range: 9.7-19.2 U/mL) were significantly higher than in normal controls (n = 9) (median: 2.8 U/mL, range: 1.9-3.7 U/mL; p < 0.001). There was a significant positive correlation between plasma soluble Fas levels and the hepatic expression of FADD in these patients. CONCLUSIONS: These results indicate that Fas-mediated apoptosis may play an important role in alcoholic hepatitis.     

Pathophysiologic mechanisms and management of neutropenia associated with large granular lymphocytic leukemia

Large granular lymphocyte (LGL) syndrome includes a spectrum of clonal T cell and natural killer cell chronic lymphoproliferative disorders. These conditions are thought to arise from chronic antigenic stimulation, while the long-term survival of the abnormal LGLs appears to be sustained by resistance to apoptosis and/or impaired survival signaling. T-cell LGL (T-LGL) leukemia is the most common LGL disorder in the Western world. Despite its indolent course, the disease is often associated with neutropenia, the pathogenesis of which is multifactorial, comprising both humoral and cytotoxic mechanisms. This article addresses the pathogenesis of T-LGL leukemia and natural killer cell chronic lymphoproliferative disorder, as well as that of T-LGL leukemia-associated neutropenia. Furthermore, as symptomatic neutropenia represents an indication for initiating treatment, available therapeutic options are also discussed.     

Successful treatment of neutropenia in T-LGL leukemia (Tγ-lymphocytosis) with granulocyte colony-stimulating factor

Summary Severe neutropenia is a common feature in patients with T-large granular lymphocytic (LGL) leukemia. Neutropenia often causes severe infections and septicemia, thus representing a major cause of morbidity and mortality in this disease. We have treated two outpatients with T-LGL leukemia who had severe neutropenia (neutrophils <0.2×10⁹/l) successfully with G-CSF (5g/kg daily, s.c). After 10 days of treatment the neutrophil count was within the normal range and a severe oral infection healed rapidly. We conclude that G-CSF therapy is able to normalize the neutrophil count in T-LGL leukemia within a few days and that it can be used to treat severe infections in these patients even on an outpatient basis.