ALBERT

Description: Microbial products, including lipopolysaccharide (LPS), an agonist of Toll-like receptor 4 (TLR4), regulate the lifespan of dendritic cells (DCs) by largely undefined mechanisms. Here, we identify a role for calcium-calmodulin–dependent kinase IV (CaMKIV) in this survival program. The pharmacologic inhibition of CaMKs as well as ectopic expression of kinase-inactive CaMKIV decrease the viability of monocyte-derived DCs exposed to bacterial LPS. The defect in TLR4 signaling includes a failure to accumulate the phosphorylated form of the cAMP response element-binding protein (pCREB), Bcl-2, and Bcl-xL. CaMKIV null mice have a decreased number of DCs in lymphoid tissues and fail to accumulate mature DCs in spleen on in vivo exposure to LPS. Although isolated Camk4−/− DCs are able to acquire the phenotype typical of mature cells and release normal amounts of cytokines in response to LPS, they fail to accumulate pCREB, Bcl-2, and Bcl-xL and therefore do not survive. The transgenic expression of Bcl-2 in CaMKIV null mice results in full recovery of DC survival in response to LPS. These results reveal a novel link between TLR4 and a calcium-dependent signaling cascade comprising CaMKIV-CREB-Bcl-2 that is essential for DC survival.

Description: To elucidate the basis of the potent graft versus leukemia (GvL) immune response that is initiated with donor lymphocyte infusion (DLI), we hypothesized the existence of an endogenous adjuvant that enhances antigen-specific immunity. In patients with chronic myeloid leukemia (CML) who achieved molecular remission after DLI, we previously identified a panel of CML-associated antigens that are targets of antibodies present in post-DLI sera and are predicted to bind nucleic acids. Recent studies in SLE have identified immunostimulatory nucleic-acid antibody complexes in patient sera, which can stimulate Toll-like receptors (TLRs) in plasmacytoid dendritic cells (pDCs). To identify immunostimulatory factors associated with GvL, we studied 6 patients that responded to DLI without clinically significant GvHD. Consistent with our prediction that sera from DLI responders contain an immunostimulatory activity, we measured a 3 to 50-fold increase in the expression of MIP-1α, TNF-α, IP-10 and IFN-α transcripts in normal peripheral blood mononuclear cells (PBMC) induced by exposure to 5 of 6 post-DLI sera. No increase in cytokine/chemokine expression was observed upon exposure to sera from pre DLI sera from the same patients, from 3 of 3 DLI non-responders, nor from 3 of 3 CML patients who achieved molecular remission after imatinib treatment. To identify the cell type that responds to the immunostimulatory activity in post-DLI sera, we isolated B, T, NK cells, monocytes, myeloid DCs and pDCs, and cultured them with pre- or post-DLI responder sera. In contrast to published findings that SLE sera stimulate pDCs, we found that post DLI sera induced normal monocytes to express high levels of MIP-1α, IP-10, MCP, TNF-α, IFN-α, IL-6, IL-8 and IL-12. Pretreatment of post-DLI sera with DNase, RNase, papain or pepsin resulted in marked decrease in IL-8 induction, demonstrating that this endogenous immunostimulatory factor requires both nucleic acid and protein for its adjuvant activity. Four active post-DLI sera were then used to stimulate stable HEK transfectants expressing TLR2, 3, 4, 8 or 9. IL-8 expression increased only in the TLR8 and TLR9-expressing cells lines that are known to be responsive to RNA and DNA respectively. IL-8 expression was further induced by post-DLI sera in TLR9-expressing cell lines co-transfected with CD32, suggesting that internalization by FcR may enhance delivery of nucleic acids to endosomal TLR8/9. None of the TLR transfectants could be activated by pre-DLI sera from the same responder patients, nor by post-DLI sera from non-responders. Finally, sera from responders collected between 2 weeks to several months after DLI consistently activated TLR8 and TLR9 suggesting that endogenous TLR8/9 activation may contribute to the early immunologic events that lead to an effective DLI response. Ongoing studies are focused on precisely identifying the TLR8/TLR9 agonists in DLI responders, studying the immunologic effects of this endogenous adjuvant and determining the association of our previously identified CML antigens with nucleic acids that activate TLR8 and TLR9. In summary, we demonstrate for the first time that effective tumor immunity is correlated with the presence of endogenous adjuvant-immunoglobulin complexes in patient sera, and propose that these observations form the basis for novel tumor vaccine strategies.

Description: Glucocorticoids (GCs) exert powerful anti-inflammatory effects that may relate in part to their ability to restrict the differentiation and function of dendritic cells (DCs). Although these inhibitory effects are dependent upon GCs binding to nuclear glucocorticoid receptors (GRs), fine-tuning of GR signaling is achieved by prereceptor interconversion of cortisol that binds GRs with high affinity and cortisone that does not. We show for the first time that human monocyte-derived DCs are able to generate cortisol as a consequence of up-regulated expression of the enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1). Immature DCs demonstrate selective enhancement of 11β-HSD1 reductase activity, leading to increased conversion of inactive cortisone to active cortisol. Enhancement of GC bioavailability is maintained or increased upon terminal differentiation induced by signals associated with innate immune activation. In marked contrast, maturation induced by CD40 ligation leads to a sharp reduction in cortisol generation by DCs. The differentiation of DCs from monocyte precursors is inhibited at physiologic concentrations of inactive cortisone, an effect that requires activity of the 11β-HSD1 enzyme. In conclusion, prereceptor regulation of endogenous GCs appears to be an important determinant of DC function and represents a potential target for therapeutic manipulation.

Description: The transmembrane ubiquitin ligase K5/MIR2 of Kaposi sarcoma herpesvirus (KSHV) mediates internalization and lysosomal degradation of glycoproteins involved in antigen presentation and co-stimulation. In endothelial cells (ECs), K5 additionally reduced expression of CD31/platelet–endothelial cell adhesion molecule (PECAM), an adhesion molecule regulating cell-cell interactions of ECs, platelets, monocytes, and T cells. K5 also reduced EC migration, a CD31-dependent process. Unlike other K5 substrates, both newly synthesized and pre-existing CD31 molecules were targeted by K5. K5 was transported to the cell surface and ubiquitinated pre-existing CD31, resulting in endocytosis and lysosomal degradation. In the endoplasmic reticulum, newly synthesized CD31 was degraded by proteasomes, which required binding of phosphofurin acidic cluster sorting protein-2 (PACS-2) to acidic residues in the carboxyterminal tail of K5. Thus, CD31, a novel target of K5, is efficiently removed from ECs by a dual degradation mechanism that is regulated by the subcellular sorting of the ubiquitin ligase. K5-mediated degradation of CD31 is likely to affect EC function in KS tumors.

Description: We have recently shown that the leukotriene B4 (LTB4)–BLT1 pathway is important in early effector T-cell recruitment in mouse models of inflammation. Here we characterize the phenotype and function of human peripheral blood BLT1+ T cells in health and illustrate their involvement in asthma and acute infection. In healthy individuals, BLT1+ T cells are a rare peripheral blood T-cell population enriched for the activation markers CD38 and HLA-DR. Compared with BLT1– T cells, a larger proportion of peripheral blood BLT1+ T cells express the effector cytokines IFNγ and IL-4 and inflammatory chemokine receptors, CCR1, CCR2, CCR6, and CXCR1. Consequently, in healthy individuals peripheral blood BLT1+ T cells are a rare antigen-primed T-cell subset with unique phenotypic, migratory, and functional properties. BLT1 expression on T cells is tightly regulated by inflammation and only transiently expressed after naive T-cell activation by dendritic cells. Although rare in the peripheral blood of healthy individuals, BLT1+ T cells are markedly increased in frequency in the peripheral blood in response to acute Epstein-Barr virus (EBV) infection and moderately increased in the airways of asymptomatic allergic asthmatics. Our studies provide novel insights into the LTB4-BLT1 lipid chemoattractant pathway in human T-cell responses, and how it may link innate and adaptive immunity.

Description: The number of neutrophils in the blood is tightly regulated to ensure adequate protection against microbial pathogens while minimizing damage to host tissue. Neutrophil homeostasis in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by CXCL12, through its major receptor CXCR4, plays a key role in maintaining neutrophil homeostasis. Herein, we generated mice with a myeloid lineage–restricted deletion of CXCR4 to define the mechanisms by which CXCR4 signals regulate this process. We show that CXCR4 negatively regulates neutrophil release from the bone marrow in a cell-autonomous fashion. However, CXCR4 is dispensable for neutrophil clearance from the circulation. Neutrophil mobilization responses to granulocyte colony-stimulating factor (G-CSF), CXCL2, or Listeria monocytogenes infection are absent or impaired, suggesting that disruption of CXCR4 signaling may be a common step mediating neutrophil release. Collectively, these data suggest that CXCR4 signaling maintains neutrophil homeostasis in the blood under both basal and stress granulopoiesis conditions primarily by regulating neutrophil release from the bone marrow.

Description: The number of circulating neutrophils is tightly regulated in order to effectively protect against microbial pathogens while minimizing damage to host tissue. Homeostatic control of neutrophils in the blood is achieved through a balance of neutrophil production, release from the bone marrow, and clearance from the circulation. Accumulating evidence suggests that signaling by the chemokine CXCL12, through its major receptor CXCR4, may play a key role in controlling neutrophil homeostasis. Indeed, gain-of-function mutations of CXCR4 are responsible for most cases of WHIM syndrome, a syndrome that features impaired neutrophil release from the bone marrow. Conversely, we previously reported that mice carrying a myeloid-specific deletion of CXCR4 (CXCR4f/−LysM+/Cre mice) display constitutive neutrophil release. Moreover, we provided data suggesting that neutrophil mobilization by G-CSF or Groβ are dependent on CXCR4 signaling, as neutrophil mobilization by these agents was absent in CXCR4f/−LysM+/Cre mice. These data firmly establish CXCR4 signaling as a key regulator of neutrophil release from the bone marrow under basal and stress conditions. Though controversial, there also is evidence that CXCR4 may play a role in neutrophil clearance from the blood by selectively trapping and removing aged neutrophils in the bone marrow. In this study, we examine the role of CXCR4 in neutrophil clearance using CXCR4f/−LysM+/Cre mice. Strain-matched wild type or CXCR4f/−LysM+/Cre mice were treated with a single injection of BrdU to label newly synthesized neutrophils. A similar percentage of myeloid cells in the bone marrow were labeled in wild type and CXCR4f/−LysM+/Cre mice, suggesting that the loss of CXCR4 does not affect granulocytic cell proliferation. Consistent with its role in regulating neutrophil release, the transit time for labeled neutrophils to appear in the circulation was significantly reduced in CXCR4f/−LysM+/Cre mice (45 hours) compared with wild type mice (72 hours). The half-life (t1/2 ) of neutrophils in the blood was calculated using the formula N=N0e−λt where N0 = the peak number of labeled cells, N = the number of cells at time t and λ = the decay constant. Surprisingly, no difference in the circulating neutrophil half-life was observed in CXCR4f/−LysM+/Cre mice compared to wild type mice (18.3 ± 13.6 hours vs.12.7 ± 9.5 hours respectively, P=0.43). We next performed adoptive transfer experiments to determine the site of neutrophil clearance. Specifically, an equivalent number of bone marrow neutrophils from wild type or CXCR4f/−LysM+/Cre mice were injected intravenously into recipient mice. Donor neutrophils were identified based on differential Ly5 gene expression. By 3 hours post-infusion, the majority of donor neutrophils were cleared from the blood. Compared to wild type neutrophils, CXCR4−/− neutrophils showed reduced homing to the bone marrow [number of donor neutrophils per femur: 6.7 ± 0.3 x 104 (wild type) compared to 2.6 ± 0.8 x 104 (CXCR4−/−); P

Description: The observation that a gain-of-function mutation in JAK2 (JAK2 V617F) is present in more than 90% of cases of polycythemia vera (PV) has substantially revised the diagnostic approach to this disease. However, most studies of the utility of JAK2 V617 derive from large PV populations: the majority of hematologists have relatively few PV patients in their individual practices. In order to assess the effect of JAK2 V617F analysis in practices with a small PV population, the utilization and impact of this test in 5 individual clinics in a single academic practice were analyzed. Sixteen patients (15 male) carrying a diagnosis of PV were identified. Five patients were followed in a clinic at a University medical center staffed by a single attending hematologist; one was followed by that attending physician in a clinic at the affiliated VA hospital; and 10 were followed by Hematology/Oncology Fellows in three continuity clinics at the affiliated VA hospital. In these latter clinics, the Fellow is primary provider, but reviews management with an attending physician. Median age of the overall population was 67.5 yrs; patients in the Fellow clinics were significantly older than those in the Attending clinics (80 yrs vs. 63 yrs; p 〈 0.01). Duration of disease was longer in the Fellow clinic patients than the Attending patients (8.5 yrs vs. 1.4 yrs; p=0.05). Nine patients were tested for JAK2 V617F mutation. The majority (6/9) were tested near diagnosis (median duration of erythrocytosis was 0.7 yrs for patients tested and 10 yrs for patients not tested; p = 0.02). Two of the three established patients tested did not meet PV diagnostic criteria fully. Five of the 7 not tested met WHO criteria for PV. Of the 9 patients tested, 7 had a mutation detected (5 newly diagnosed, 2 〉 1 yr duration of disease). Results of mutation testing changed the diagnosis in two cases (one where it was detected and one where it was not detected). In addition, one newly diagnosed patient who met WHO criteria did not have JAK2 V617F detected and was felt to have true “JAK2 V617F-negative” PV. JAK2 exon 12 studies were not performed. Conclusions: The use and impact of JAK2 V617F mutation testing has been reviewed in a PV population similar to what an individual or small group practice might follow. JAK2 V617F mutation testing was primarily used in the early evaluation of suspected PV, or in continuing patients whose diagnosis was not fully established. Mutation testing was infrequently used in continuing patients where the physician considered the diagnosis clearly established or where a change in diagnosis would not alter management. Negative results of mutation testing altered diagnosis if they were consistent with the patient’s clinical picture.

Description: 5083 RDW, a quantitative measure of anisocytosis, has been utilized for many years in the differential diagnosis of anemia. More recently, studies have reported that RDW varies with renal function and markers of inflammation, and that RDW is a predictor of mortality (Scand. J. Clin. Lab. Invest. 2009; 68:745-8; Arch. Pathol. Lab. Med. 2009; 133:628-32; Arch. Intern. Med. 2009; 169:588-94). However, these reports were based on studies of large populations of unselected outpatients, many of whom were hematologically normal. Utilizing a de-identified database of 32 anemic adult inpatients (17 male/15 female, 27 Caucasian) who had undergone diagnostic bone marrow examination as part of an earlier study (J. Clin. Lab. Haematol. 1999; 21:161-7), associations between RDW and clinical laboratory parameters in a more restricted patient subset were examined. RDW values for 26/32 patients were in the highest quintile observed in the National Health and Nutrition Examination Survey (RDW 〉 13.8%). Significant differences between patients in the highest quintile and other patients were observed for hemoglobin concentration (9.9 g/dL vs. 11.6 g/dL, p = 0.01), serum soluble transferrin receptor (sTfR) (30.3 nM vs. 16.7 nM, p = 0.02), and serum interleukin (IL)-1 (1.6 pg/mL vs. 0.3 pg/mL, p =0.03). No other significant differences were observed in other automated hematologic markers, in biochemical indicators of iron status, renal function, or inflammation, or in marrow aspirate iron score. When the correlation between RDW and the other parameters studied was evaluated, the only statistically significant correlations found were with C-reactive protein (CRP) (r = -0.49, p = 0.009) and sTfR (r = 0.53, p = 0.002). The significance of the correlation between sTfR and RDW was independent of other indicators of iron status, CRP, and serum IL-1. In contrast, the significance of the correlation between CRP and RDW was dependent on variation in sTfR and serum ferritin concentrations. In this group of patients with who generally had either high-normal or high RDW values, CRP and RDW were inversely correlated. In summary, associations between RDW and other laboratory or clinical parameters observed in unselected patient populations may not be applicable to complex anemic inpatients. The association between sTfR concentration and RDW appears to be independent of the relationship between sTfR concentration and patient iron status. Disclosures Means: Beckman Coulter, Inc.: Consultancy, Honoraria.

Description: The anemia of chronic disease (ACD) results from three major processes: slightly shortened red cell survival, impaired reticuloendothelial system iron mobilization, and impaired erythropoiesis. Hepcidin is an acute phase protein with specific iron regulatory properties, which, along with the anemia seen with increased hepcidin expression, have led many to consider it the major mediator of ACD. However, if hepcidin is the major factor responsible for ACD, then it should also contribute to the impaired erythropoiesis observed in this syndrome. In this study, the effects of hepcidin on erythroid colony formation in vitro are examined. At the standard recombinant human erythropoietin (rhEPO) concentration used for erythroid colony-forming unit (CFU-E) assays in vitro (1 U/mL), hepcidin had no significant effects on colony formation. However, ACD is a syndrome associated with relative reductions in EPO concentration. The effects of hepcidin 100 ng/mL on CFU-E colony formation were evaluated at lower rhEPO concentrations (0.1 _ 0.5 U/mL). Colony formation was significantly decreased in the presence of hepcidin. The regulatory pathways controlling apoptosis and proliferation of HCD57 erythroleukemia cells replicate those observed in primary human erythroid progenitors. This cell line was studied in order to more fully define the effects of hepcidin on erythropoiesis. HCD57 cells were incubated in Iscove’s modified Dulbecco’s medium with 10% fetal calf serum with 0.3 U/mL rhEPO at 37°C for 24 hrs, with or without hepcidin 100 ng/mL. No difference in cell proliferation was seen. Apoptosis in HCD57 cells is regulated by the Bad/Bcl-xL pathway. Total Bad and Bcl-xL protein expression were unchanged by exposure to hepcidin; however, the proportion of the anti-apoptotic protein pBad was decreased approximately 50%. A recent report has demonstrated that hepcidin reduces macrophage iron efflux by binding to the iron transporter ferroportin, causing its internalization and subsequent degradation. It is unlikely that the inhibition of CFU-E colony formation reported here results from this effect: although macrophages are present in the marrow cell population, and iron is required for erythropoiesis in vitro, the amount of iron-saturated transferrin contributed to the culture medium by FCS is comparable to the amount required for maximal CFU-E colony formation in serum-free medium. CONCLUSION: At reduced rhEPO concentrations, CFU-E colony formation is inhibited by hepcidin. Short-term exposure to hepcidin induces a pro-apoptotic pattern in HCD57 erythroleukemia cells. The studies reported here do not disprove the possibility that hepcidin-induced impairment of iron flow to erythroid cells contributes to ACD: rather, they suggest an additional mechanism by which hepcidin can inhibit erythropoiesis.