Decreased neutrophil function in newly diagnosed multiple myeloma patients is restored with lenalidomide therapy.

OBJECTIVES: Bacterial infections are common and a major cause of morbidity and mortality in multiple myeloma (MM). We have investigated the function of polymorphonuclear leukocyte (PMN), the immune system's first line of defense against bacteria, in peripheral blood (PB) and bone marrow (BM) samples from patients with newly diagnosed MM (NDMM), smoldering MM (SMM), monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. METHODS: Phagocytosis and oxidative burst in PMN cells from patients and healthy donors were investigated using PhagoTest and PhagoBurst assay. RESULTS: PMN from NDMM, SMM, and MGUS patients had reduced phagocytosis and oxidative burst ability compared with healthy controls. The dysfunction was most prominent in BM samples from MM, SMM, and MGUS patients. Importantly the reduced phagocytosis in MM patients was restored in patients on lenalidomide therapy. Consistently the ability of Escherichia coli stimulated oxidative burst in BM was reduced for the MM, SMM, and MGUS cohort in contrast to the healthy controls and the patients on lenalidomide treatment. CONCLUSION: Our results show that MM patients have neutrophil dysfunction that could contribute to susceptibility for bacterial infections and that lenalidomide therapy was associated with restored PMN function.

Disease Activity and Tendency to Relapse in ANCA-Associated Vasculitis Are Reflected in Neutrophil and Intermediate Monocyte Frequencies.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune diseases with inflammation affecting small blood vessels and includes granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). In this study, we investigated granulocyte and monocyte subsets in a large cohort of AAV patients with emphasis on disease activity and tendency to relapse. A cohort of 105 patients with GPA or MPA and 126 healthy controls (HCs) were included. Clinical and laboratory data were collected for all patients, including disease activity, tendency to relapse, and pharmacological treatment. Using flow cytometry, circulating eosinophils, basophils, neutrophils, and monocytes were assessed. The monocytes were subdivided into classical (CD14++CD16-), intermediate (CD14++CD16+), and nonclassical (CD14-CD16+) monocytes. Mature (CD16high) or newly released (CD16dim) neutrophils were defined, as well as the frequency of CD177+ neutrophils. AAV patients displayed increased frequencies of intermediate monocytes, mature and newly released neutrophils, and an expanded population of CD177+ neutrophils compared to HC. MPA patients differed from GPA patients in terms of lower frequency of classical monocytes. No differences in cell frequencies regarding ANCA phenotype were observed. Paired data from 23 patients demonstrated that active disease was associated with an increased frequency of mature neutrophils and a decreased frequency of monocytes, in particular intermediate monocytes. Moreover, GPA patients with a tendency to relapse displayed an increased frequency of mature neutrophils with increased expression of CD177+. Relapsing MPA patients, on the other hand, showed decreased frequency of intermediate monocytes. Finally, rituximab treatment was associated with increased frequencies of classical and intermediate monocytes. In conclusion, AAV patients exhibit a skewing of different neutrophil and monocyte subpopulations that are associated with disease subtypes, disease activity, rituximab treatment, and propensity to relapse. These changes may contribute to the inflammatory process and could potentially be used as biomarkers for relapse prediction.

Suppression of T-Cell Proliferation by Normal Density Granulocytes Led to CD183 Downregulation and Cytokine Inhibition in T-Cells.

Normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study, we tested the hypothesis that NDGs from healthy donors preferentially inhibit T helper 1 (Th1) cells and investigated the myeloid-derived suppressive effect in different T-cell populations. We found that NDG-induced suppression of T-cell proliferation was contact dependent, mediated by integrin CD11b, and dependent on NDG-production of reactive oxygen species (ROS). The suppression was rapid and occurred within the first few hours of coculture. The suppression did not influence the CD8+/CD4+ ratio indicating an equal sensitivity in these populations. We further analyzed the CD4+ T helper subsets and found that NDGs induced a loss of Th1 surface marker, CD183, that was unrelated to ligand-binding to CD183. In addition, we analyzed the Th1, Th2, and Th17 cytokine production and found that all cytokine groups were suppressed when T-cells were incubated with NDGs. We therefore concluded that NDGs do not preferentially suppress Th1-cells. Instead, NDGs generally suppress Th cells and cytotoxic T-cells but specifically downregulate the Th1 marker CD183.

Methotrexate Treatment Suppresses Monocytes in Nonresponders to Pneumococcal Conjugate Vaccine in Rheumatoid Arthritis Patients.

Patients with rheumatoid arthritis (RA) have an increased risk of infections; therefore, immunization against vaccine-preventable diseases is important. Methotrexate (MTX) impairs the antibody response to pneumococcal conjugate vaccine (PCV) in patients with arthritis, and the underlying mechanism is largely unknown. Here, we investigate the potential role of the innate immune system in the faltering antibody response following PCV vaccination in RA patients treated with MTX. Phenotypes of circulating granulocytes and monocytes were analyzed in 11 RA patients treated with MTX, 13 RA patients without disease-modifying antirheumatic drug treatment (0DMARD), and 13 healthy controls (HC). Peripheral blood samples were collected before and 7 days after vaccination. In addition, the MTX group was sampled before initiating treatment. Frequencies of granulocyte and monocyte subsets were determined using flow cytometry. Serotype-specific IgG were quantified using a multiplex bead assay, pre- and 4-6 weeks after vaccination. At baseline, no differences in granulocyte and monocyte frequencies were observed between the groups. Within the MTX group, the frequency of basophils increased during treatment and was higher compared to the HC and 0DMARD groups at the prevaccination time point. MTX patients were categorized into responders and nonresponders according to the antibody response. Before initiation of MTX, there were no differences in granulocyte and monocyte frequencies between the two subgroups. However, following 6-12 weeks of MTX treatment, both the frequency and concentration of monocytes were lower in PCV nonresponders compared to responders, and the difference in monocyte frequency remained after vaccination. In conclusion, the suppressive effect of MTX on monocyte concentration and frequency could act as a biomarker to identify nonresponders to PCV vaccination.

Bone Marrow Neutrophils of Multiple Myeloma Patients Exhibit Myeloid-Derived Suppressor Cell Activity.

Activated normal density granulocytes (NDGs) can suppress T-cell responses in a similar way as myeloid-derived suppressor cells (MDSCs). In this study, we tested the hypothesis that NDGs from blood and bone marrow of multiple myeloma (MM) patients have the ability to suppress T-cells, as MDSC. MM is an incurable plasma cell malignancy of the bone marrow. Like most malignancies, myeloma cells alter its microenvironment to promote tumor growth, including inhibition of the immune system. We found that MM NDG from the bone marrow suppressed proliferation of T-cells, in contrast to healthy donors. The inhibitory effect could not be explained by changed levels of mature or immature NDG in the bone marrow. Moreover, NDG isolated from the blood of both myeloma patients and healthy individuals could inhibit T-cell proliferation and IFN-γ production. On the contrary to previous studies, blood NDGs did not have to be preactivated to mediate suppressive effects. Instead, they became activated during coculture, indicating that contact with activated T-cells is important for their ability to regulate T-cells. The inhibitory effect was dependent on the production of reactive oxygen species and could be reverted by the addition of its inhibitor, catalase. Our findings suggest that blood NDGs from MM patients are suppressive, but no more than NDGs from healthy donors. However, only bone marrow NDG from MM patients exhibited MDSC function. This MDSC-like suppression mediated by bone marrow NDG could be important for the growth of malignant plasma cells in MM patients.

Methotrexate reduces circulating Th17 cells and impairs plasmablast and memory B cell expansions following pneumococcal conjugate immunization in RA patients.

Methotrexate (MTX) impairs antibody response after pneumococcal vaccination. We aimed to investigate differences in phenotypes of circulating B and T cells after pneumococcal conjugate vaccine (PCV) in rheumatoid arthritis (RA) patients on MTX (MTX group), RA without disease-modifying drugs (0DMARD), and controls (HC). MTX group (n = 11), 0DMARD (n = 12) and HC (n = 13) were studied. Blood samples were collected: before MTX, ≥ 4 weeks on stable MTX dose (prevaccination), and 7 days postvaccination (MTX group), and pre- and 7 days postvaccination (0DMARD and HC). Phenotypes of B- and T cell subsets were determined using flow cytometry. Serotype-specific IgG were quantified using multiplex bead assay, pre- and 4-6 weeks postvaccination. Concentrations of plasmablasts and switched memory B cells increased after PCV in HC (both p = 0.03) and the 0DMARD group (p = 0.01 and p = 0.02), but not in the MTX group. Postimmunization plasmablasts were lower in MTX group, compared to the 0DMARD group and HC (p = 0.002 and p < 0.001). Th17 cells decreased after MTX start (p = 0.02), and increased in HC after immunization (p = 0.01). Postimmunization plasmablasts correlated with mean antibody response ratio in all RA patients (R = 0.57, p = 0.035). Methotrexate reduced Th17 cells and blocked activation of plasmablasts and switched memory B cells following polysaccharide-protein conjugate antigen challenge in RA.

Increased Frequencies of Switched Memory B Cells and Plasmablasts in Peripheral Blood from Patients with ANCA-Associated Vasculitis.

B cells are thought to play a central role in the pathogenesis of antineutrophil cytoplasmic antibody- (ANCA-) associated vasculitis (AAV). ANCAs have been proposed to cause vasculitis by activating primed neutrophils to damage small blood vessels. We studied a cohort of AAV patients of which a majority were in remission and diagnosed with granulomatosis with polyangiitis (GPA). Using flow cytometry, the frequencies of CD19+ B cells and subsets in peripheral blood from 106 patients with AAV and 134 healthy controls were assessed. B cells were divided into naive, preswitch memory, switched memory, and exhausted memory cells. Naive and switched memory cells were further subdivided into transitional cells and plasmablasts, respectively. In addition, serum concentrations of immunoglobulin A, G, and M were measured and clinical data were retrieved. AAV patients displayed, in relation to healthy controls, a decreased frequency of B cells of lymphocytes (5.1% vs. 8.3%) and total B cell number. For the subsets, a decrease in percentage of transitional B cells (0.7% vs. 4.4%) and expansions of switched memory B cells (22.3% vs. 16.5%) and plasmablasts (0.9% vs. 0.3%) were seen. A higher proportion of B cells was activated (CD95+) in patients (20.6% vs. 10.3%), and immunoglobulin levels were largely unaltered. No differences in B cell frequencies between patients in active disease and remission were observed. Patients in remission with a tendency to relapse had, compared to nonrelapsing patients, decreased frequencies of B cells (3.5% vs. 6.5%) and transitional B cells (0.1% vs. 1.1%) and an increased frequency of activated exhausted memory B cells (30.8% vs. 22.3%). AAV patients exhibit specific changes in frequencies of CD19+ B cells and their subsets in peripheral blood. These alterations could contribute to the autoantibody-driven inflammatory process in AAV.

Neutrophils from ANCA-associated vasculitis patients show an increased capacity to activate the complement system via the alternative pathway after ANCA stimulation.

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV), including granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA), are autoimmune conditions associated with small vessel inflammation. Earlier studies indicate that complement activation via the alternative pathway plays a major role in the pathogenesis. In this study we have investigated if ANCA-activation of neutrophils from AAV patients leads to activation of the alternative complement pathway. C5a-primed neutrophils (PMN) from 10 AAV patients and 10 healthy controls (HC) were stimulated with PMA or IgG purified from PR3-ANCA positive patients (ANCA IgG). The supernatants were analyzed for release of complement proteins and markers of different granules by ELISA, and release of microparticles (MP) by flow cytometry. The ability of the supernatants to activate the alternative complement pathway was determined by incubation with normal serum and C3bBbP and C5a were measured by ELISA. MP were analyzed by flow cytometry and removed by centrifugation. The supernatants from the AAV patients' neutrophils produced significantly more C3bBbP compared with HCs (p = 0.0001). C3bBbP levels correlated with the number of MP. After removal of MP from the supernatants, alternative pathway activation was significantly lower. This study shows that primed and ANCA-stimulated neutrophils from AAV patients have a greater ability to activate the alternative complement pathway compared to primed neutrophils from healthy controls. This finding emphasizes the role of complement in the pathogenesis of AAV - underlining the therapeutic potential of C5a and other complement blockade.

Eosinophils in anti-neutrophil cytoplasmic antibody associated vasculitis.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) are characterized by autoimmune small vessel inflammation. Eosinophils are multifunctional cells with both pro-inflammatory and immunoregulatory properties. Tissue activated eosinophils secrete cyto- and chemokines and form extracellular traps (EETs), they release free granules and produce reactive oxygen species. The role of eosinophils is well established in eosinophilic granulomatosis with polyangiitis (EGPA) but very little is known about their role in granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA). METHODS: The expression of surface markers CD11c, CD11b, CD16, CD35, CD62L, CD64, CD88, Siglec-8 and CD193 and reactive oxygen species production by peripheral blood eosinophils were studied using flow cytometry. Fluorescence microscopy was used to visualize the release of eosinophil extracellular DNA traps (EETs). 98 GPA and MPA patients and 121 healthy controls were included in the study. RESULTS: Both GPA and MPA patients had decreased frequency of eosinophils in peripheral blood compared with healthy controls (p < 0.0001), which could not solely be explained by corticosteroid treatment. The patient's eosinophils showed increased surface expression of the Fc receptors CD16 (p < 0.0001) and CD64 (p = 0.0035) as well as CCR3 (CD193) (p = 0.0022). Decreased expression was found of the complement receptors CD35 (p = 0.0022), CD88 (p < 0,0001) as well as CD11c (p < 0,0001), CD11b (p = 0.0061) and Siglec-8 (p = 0,0015). Moreover, GPA and MPA eosinophils, showed decreased capacity to produce ROS (p < 0.0001). ANCA stimulation of eosinophils from GPA and MPA patients after C5a priming enhanced EETosis (p = 0,0088). CONCLUSIONS: The percentage of eosinophils were decreased in peripheral blood in GPA and MPA patients and showed altered surface marker expression and function. The enhanced EETosis after ANCA stimulation, suggests that eosinophil can contribute to the autoantibody driven inflammatory process.

Phenotypic Characterization of Circulating CD4+ T Cells in ANCA-Associated Vasculitis.

T cell-mediated immune responses are thought to play an important role in the pathogenesis of anti-neutrophil cytoplasmic antibody- (ANCA-) associated vasculitides (AAV). CD4+ T cells can be divided into subsets depending on their expression of chemokine receptors. In this study, different CD4+ T cell populations in patients with AAV were analysed and compared to healthy blood donors as well as therapy controls. 18 patients with active AAV, 46 in remission, 21 healthy controls (HBD), and 15 therapy controls (TC) were enrolled. CD4+ T cells were divided into Th1, Th2, and Th17 cells and further subdivided into naïve, central memory, effector memory, and effector cells. Regulatory T cells were also analysed. Concentrations of cytokines and chemokines produced by the respective CD4+ T cell subset in plasma from 33 of the patients were measured by ELISA and compared to HBD. Clinical data were collected on all patients. CCL20 concentrations and percentages of Th17 cells (p = 0.019) were elevated in AAV patients compared to HBD. AAV patients had lower percentages of naïve CD4+ T cells (p = 0.0016) and a corresponding increase in proportion of effector memory CD4+ T cells when comparing to HBD (p = 0.027). Therapy controls showed similar results as AAV patients. In this study, we found that CD4+ T cell phenotype distribution is altered in AAV patients, in line with previously published work. However, no differences were found between AAV patients and TC, stressing the importance of treatment impact on this kind of studies.

Bone marrow eosinophils in plasma cell disorders.

In experimental studies, eosinophils have been shown to promote the survival, proliferation, and retention of plasma cells in the bone marrow (BM). The clinical significance of eosinophils in plasma cell disorders (PCDs) in humans is largely unknown. This study focuses on the frequency and phenotype of eosinophils in the BM and peripheral blood (PB) in patients with untreated PCD compared with healthy controls. The number of eosinophils per se did not correlate with the number of BM plasma cells or disease stage. The expression of chemokine receptor 4, which is important in the homing capacity to bone marrow stromal cells, was significantly higher in patient eosinophils and increased with disease stage. BM eosinophils from patients, especially from those with manifest disease, were more activated. Another finding in this study was that eosinophils in PB and BM from both patients and healthy controls expressed CD80 (B7-1). We discuss probable immunomodulatory consequences of surface expression of CD80 by eosinophils in conditions with marked T-cell exhaustion (e.g., multiple myeloma). Finally, we found that patients treated with corticosteroids had low levels of circulating eosinophils but preserved levels of eosinophils in the BM.

Monocyte Chemoattractant Protein-1 in Antineutrophil Cytoplasmic Autoantibody-Associated Vasculitis: Biomarker Potential and Association with Polymorphisms in the MCP-1 and the CC Chemokine Receptor-2 Gene.

Antineutrophil cytoplasmic autoantibody- (ANCA-) associated vasculitis (AAV) are relapsing-remitting disorders with unpredictable prognosis. There is a need of biomarkers for distinguishing which patients will have a more severe outcome and also for predicting relapses in disease activity. This study confirms the previous results of urinary MCP-1 (uMCP-1) as a prognostic marker and explores its potential as a marker of disease activity. Method. 114 patients with AAV were followed regularly between 2002 and 2011 at Skåne University Hospital. Urine samples, blood samples, and clinical status were registered. The urine samples were analyzed in an in-house-developed ELISA. PCR-RLFP was used to analyze the MCP-1 and CCR2 genes. Results. Patients with severe prognosis had significantly higher levels of uMCP-1 compared to patients with nonsevere prognosis and healthy controls. Patients with renal damage had higher levels compared to patients who did not have renal damage. There was also a tendency of higher uMCP-1 levels in active disease as compared to remission. AA in the -2518 position in the MCP-1 gene was associated with a more severe outcome compared to the A/G or the G/G genotype. The A/A genotype were also associated with higher levels of uMCP-1. No significant associations were seen for the CCR2-V64I. Conclusion. This study confirmed the connection between high uMCP-1 levels and poor prognosis and also disease activity. It also suggests an association of the A/A genotype at position -2518 in the MCP-1 gene and poor prognosis in AAV. uMCP-1 is clearly a candidate biomarker of potential clinical value. The A/A genotype association needs further evaluation.

Impaired phagocytosis and reactive oxygen species production in phagocytes is associated with systemic vasculitis.

BACKGROUND: Anti-neutrophil cytoplasmic antibodies associated vasculitides (AAV) is a group of autoimmune diseases, characterized by small vessel inflammation. Phagocytes such as neutrophils and monocytes are the main effector cells found around the inflamed vessel wall. Therefore, we wanted to investigate aspects of function and activation of these cells in patients with AAV. METHODS: Flow cytometry was used to evaluate: the expression of activation markers (CD11c, CD62L, CD177 and C5aR); the number of recently released neutrophils from bone marrow, defined as CD10(-)D16(low) cells in peripheral blood; and the capacity of peripheral blood monocytes and polymorphonuclear leukocytes (PMN) to produce reactive oxygen species and to phagocytose opsonized bacteria. RESULTS: AAV patients (n = 104) showed an increase of CD10(-)CD16(low) neutrophils and total PMN in peripheral blood, suggesting a combination of increased bone marrow release and prolonged survival. An increased percentage of AAV PMN expressed CD177 but no other signs of activation were seen. A decreased production of reactive oxygen species was observed in AAV phagocytes, which was associated with disease activity. Moreover, granulocytes from patients with microscopic polyangiitis showed lower oxidative burst capacity compared to patients with granulomatosis with polyangiitis or eosinophilic granulomatosis with polyangiitis. In addition, decreased phagocytosis capacity was seen in PMN and monocytes. CONCLUSION: Our results indicate that phagocytes from AAV patients have impaired function, are easily mobilized from bone marrow but are not particularly activated. The association between low reactive oxygen species formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.

Phagocyte function decreases after high-dose treatment with melphalan and autologous stem cell transplantation in patients with multiple myeloma.

High-dose melphalan with autologous hematopoietic stem cell transplantation (ASCT) is the standard of care for younger patients with newly diagnosed multiple myeloma and is aimed at achieving as deep and complete a response as possible after various combinations of induction therapy. However, it is frequently associated with infectious complications. This study investigated the effects of high-dose treatment with autologous stem cell support on patients' innate immunity, with a focus on subpopulations and functioning of recently released polymorphonuclear leukocytes (PMNs) and monocytes in peripheral blood. Flow cytometry-based analysis was used to measure the degree of PMN maturation and activation, before and after ASCT and compared with healthy controls. After high-dose treatment and ASCT, a smaller proportion of patients' PMNs had the capacity for oxidative burst. Moreover, patients' PMNs, both before and after ASCT, had a reduced capacity for phagocytosis. Eosinophils, which recently have been suggested to play a role in promoting malignant plasma cell proliferation, were markedly reduced after ASCT, with slow regeneration. HLA-DR expression by monocytes was significantly depressed after ASCT, a characteristic often attributed to monocytic myeloid-derived suppressor cells. Our results suggest that several aspects of phagocytic function are impaired for at least 20 days after ASCT.

Low production of reactive oxygen species in granulocytes is associated with organ damage in systemic lupus erythematosus.

INTRODUCTION: Polymorphonuclear leukocytes (PMN) are main effector cells in the acute immune response. While the specific role of PMN in systemic lupus erythematosus (SLE) and autoimmunity is still unclear, their importance in chronic inflammation is gaining more attention. Here we investigate aspects of function, bone marrow release and activation of PMN in patients with SLE. METHODS: The following PMN functions and subsets were evaluated using flow cytometry; (a) production of reactive oxygen species (ROS) after ex vivo stimulation with phorbol 12-myristate 13-acetate (PMA) or Escherichia coli (E. coli); (b) capacity to phagocytose antibody-coated necrotic cell material; (c) PMN recently released from bone marrow, defined as percentage of CD10(-)D16(low) in peripheral blood, and (d) PMN activation markers; CD11b, CD62L and C5aR. RESULTS: SLE patients (n = 92) showed lower ROS production compared with healthy controls (n = 38) after activation ex vivo. The ROS production was not associated with corticosteroid dose or other immunotherapies. PMA induced ROS production was significantly reduced in patients with severe disease. In contrast, neither ROS levels after E. coli activation, nor the capacity to phagocytose were associated with disease severity. This suggests that decreased ROS production after PMA activation is a sign of changed PMN behaviour rather than generally impaired functions. The CD10(-)CD16(low) phenotype constitute 2% of PMN in peripheral blood of SLE patients compared with 6.4% in controls, indicating a decreased release of PMN from the bone marrow in SLE. A decreased expression of C5aR on PMN was observed in SLE patients, pointing towards in vivo activation. CONCLUSIONS: Our results indicate that PMN from SLE patients have altered function, are partly activated and are released abnormally from bone marrow. The association between low ROS formation in PMN and disease severity is consistent with findings in other autoimmune diseases and might be considered as a risk factor.

A candidate gene approach to ANCA-associated vasculitis reveals links to the C3 and CTLA-4 genes but not to the IL1-Ra and Fcγ-RIIa genes.

BACKGROUND/AIMS: The aim of the study is to search for associations between Antineutrophil cytoplasm antibody (ANCA)-associated vasculitis (AAV) and polymorphisms in the genes of four key molecules possibly involved in different pathogenic pathways; complement C3, CTLA-4, Fcγ-RIIa and IL1-Ra. PATIENTS AND METHODS: Patients with AAV (n=105) subgrouped as microscopic polyangiitis or granulomatosis with polyangiitis (Wegener's granulomatosis) and myeloperoxidase (MPO) or proteinase 3 (PR3) ANCA positive were compared to a control group of 200 blood donors. Polymorphisms in the genes were analysed with PCR amplification of DNA. RESULTS: The diagnosis of AAV was confirmed in the 105 cases. The gene frequency of C3F was 0.27 in the PR3-ANCA subgroup (p=0.041) compared to 0,19 in the control group. The number of patients homozygous for the shortest 86 bp allele of CTLA-4 was significantly decreased in the whole group of patients (p=0.049). No differences were evident in the Fcγ-RIIa and IL1-Ra polymorphisms when compared to controls, neither in the whole group of patients, nor in any of the sub-groups. CONCLUSION: The aberrant gene frequency of the C3F allele among PR3-ANCA positive patients and the findings with the CTLA-4 polymorphism indicates that complement may be involved in pathogenesis and that T-cell activation also is of importance in these diseases.

Decreased neutrophil apoptosis in quiescent ANCA-associated systemic vasculitis.

BACKGROUND: ANCA-Associated Systemic Vasculitis (AASV) is characterized by leukocytoclasis, accumulation of unscavenged apoptotic and necrotic neutrophils in perivascular tissues. Dysregulation of neutrophil cell death may contribute directly to the pathogenesis of AASV. METHODS: Neutrophils from Healthy Blood Donors (HBD), patients with AASV most in complete remission, Polycythemia Vera (PV), Systemic Lupus Erythematosus (SLE), Rheumatoid Arthritis (RA) and renal transplant recipients (TP) were incubated in vitro, and the rate of spontaneous apoptosis was measured by FACS. Plasma levels of cytokines and sFAS were measured with cytometric bead array and ELISA. Expression of pro/anti-apoptotic factors, transcription factors C/EBP-α, C/EBP-ß and PU.1 and inhibitors of survival/JAK2-pathway were measured by real-time-PCR. RESULTS: AASV, PV and RA neutrophils had a significantly lower rate of apoptosis compared to HBD neutrophils (AASV 50 ± 14% vs. HBD 64 ± 11%, p<0.0001). In RA but not in AASV and PV, low apoptosis rate correlated with increased plasma levels of GM-CSF and high mRNA levels of anti-apoptotic factors Bcl-2A1 and Mcl-1. AASV patients had normal levels of G-CSF, GM-CSF and IL-3. Both C/EBP-α, C/EBP-ß were significantly higher in neutrophils from AASV patients than HBD. Levels of sFAS were significantly higher in AASV compared to HBD. CONCLUSION: Neutrophil apoptosis rates in vitro are decreased in AASV, RA and PV but mechanisms seem to differ. Increased mRNA levels of granulopoiesis-associated transcription factors and increased levels of sFAS in plasma were observed in AASV. Additional studies are required to define the mechanisms behind the decreased apoptosis rates, and possible connections with accumulation of dying neutrophils in regions of vascular lesions in AASV patients.

Dendritic cells exposed to estrogen in vitro exhibit therapeutic effects in ongoing experimental allergic encephalomyelitis.

Immunomodulatory effects of estrogen have been demonstrated by clinical and experimental observations, but the mechanisms by which estrogen exhibits the effects remain to be defined. One possible mechanism by which estrogen inhibits the development of experimental allergic encephalomyelitis (EAE), a commonly used model of multiple sclerosis (MS) in humans, is over the functions of dendritic cells (DC). Here, we describe that splenic DC from Lewis rats obtained on day 12 post-immunization (p.i.) with myelin basic protein (MBP) encephalitogenic peptide 68-86+Freund's complete adjuvant (FCA), after being exposed in vitro 17beta-estradiol, exhibited therapeutic effects on acute EAE when injected subcutaneously on day 5 p.i. Blood mononuclear cells (MNC) were isolated from thus treated rats on day 12 p.i. Administration of estrogen-exposed DC prevented the expansion of CD4+ T cells and increased proportions of regulatory T cells producing IL-10 and CD4+CD28- suppressor T cells, accompanied with increased IL-10 and IFN-gamma, and reduced TNF-alpha production. Infiltrates of CD68+ macrophages within the central nervous system and MBP 68-86-induced T cell proliferation were inhibited in rats injected with estrogen-exposed DC compared to rats injected with naive DC. Estrogen up-regulated the expression of indoleamine 2,3-dioxygenase, which promotes tolerogenic properties of DC. The results suggest that in vitro exposure of DC to estrogen modulates DC functions and results in a therapeutic effect of DC.

Circulating CD4+CD25+ T regulatory cells are not altered in multiple sclerosis and unaffected by disease-modulating drugs.

Experimental animal models for autoimmunity have demonstrated the existence and crucial role of CD4(+)CD25(+) T regulatory (Tr) cells in suppressing autoreactive T cells and promoting peripheral tolerance. Recent in vitro functional studies showed that Tr cells are enriched in the CD25(high) cell population among CD4(+) T cells, and that they totally inhibit proliferation and cytokine secretion by CD4(+) T cells. It is not yet known if circulating Tr cells are involved in multiple sclerosis (MS). This study was done firstly to determine whether alterations of the CD4 (+) CD25(high) T cells occur in MS, examining their frequencies. As it was reported that the suppressive activity of CD4(+)CD25(+) Tr cells is mainly through cell surface contact pathway, we secondly analyzed the expression of the functionally important cell surface molecules of CD4(+)CD25(high) Tr cells. Two- or three-colour flow cytometry was used to identify and quantify CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells among blood CD4(+) T cells in MS patients without treatment vs. patients treated with either interferon-beta (IFN-beta) or glatiramer acetate (GA) or IFN-beta + GA in combination vs. healthy controls (HC). Expression of functionally important surface molecules CD45RO, CD69, CD95, HLA-DR, and intracellular CTLA-4 and IL-10 production by CD4(+)CD25(high) Tr cells were investigated. CD4(+)CD25(+) T cells constituted around 6% of CD4(+)T cells in all MS patient groups, and 7% in HC. There were also no changes in the proportions of CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells in a longitudinal follow-up of MS patients before and during IFN-beta treatment. Frequencies of circulating CD4(+)CD25(high)Tr cells among CD4(+) T cells were also similar and their surface or intracellular molecular expression did not vary in MS patients, irrespective of treatment, compared to HC. This study suggests that levels of circulating CD4(+)CD25(+) Tr cells and CD4(+)CD25(high) Tr cells are not altered in MS, and are unaffected by substances currently used to modulate the disease.